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Neuroscience 146 (2007) 1743–1757

0d

EUROTOXICITY MECHANISMS OF THIOETHER

CSTASY METABOLITES

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. P. CAPELA,a* C. MACEDO,b P. S. BRANCO,b

. M. FERREIRA,b A. M. LOBO,b E. FERNANDES,c

. REMIÃO,a M. L. BASTOS,a U. DIRNAGL,d A. MEISELd

ND F. CARVALHOa*

REQUIMTE (Rede de Química e Tecnologia), Toxicology Depart-ent, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha,64, 4099-030 Porto, Portugal

REQUIMTE/CQFB, (Centro de Química Fina e Biotecnologia),hemistry Department, Faculty of Science and Technology, Universityf Nova de Lisboa, 2829-516 Caparica, Portugal

REQUIMTE, Physical Chemistry Department, Faculty of Pharmacy,niversity of Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal

Department of Experimental Neurology and Center for Stroke Re-earch, Charité-Universitätsmedizin, Charitéplatz 1 D-10117 Berlin,ermany

bstract—3,4-Methylenedioxymethamphetamine (MDMA orecstasy”), is a widely abused, psychoactive recreationalrug that is known to induce neurotoxic effects. Human andat hepatic metabolism of MDMA involves N-demethylation to,4-methylenedioxyamphetamine (MDA), which is also a drugf abuse. MDMA and MDA are O-demethylenated to N-methyl--methyldopamine (N-Me-�-MeDA) and �-methyldopamine

�-MeDA), respectively, which are both catechols that canndergo oxidation to the corresponding ortho-quinones.rtho-quinones may be conjugated with glutathione (GSH) to

orm glutathionyl adducts, which can be transported into therain and metabolized to the correspondent N-acetylcysteineNAC) adducts. In this study we evaluated the neurotoxicityf nine MDMA metabolites, obtained by synthesis: N-Me-�-eDA, �-MeDA and their correspondent GSH and NAC ad-ucts. The studies were conducted in rat cortical neuronalultures, for a 6 h of exposure period, under normal (36.5 °C)nd hyperthermic (40 °C) conditions. Our findings show thathioether MDMA metabolites are strong neurotoxins, signifi-antly more than their correspondent parent catechols. Onhe other hand, N-Me-�-MeDA and �-MeDA are more neuro-

Corresponding author. Fax: �351–222003977.-mail address: joaocapela@ff.up.pt (J. P. Capela), felixdc@ff.up.pt

F. Carvalho).bbreviations: Ac-DEVD-pNA, acetyl-Asp-Glu-Val-Asp p-nitroanilide;SA, bovine serum albumin; DA, dopamine; DHR-123, dihydrorho-amine 123; DIV, day in vitro; GSSG, oxidized glutathione; GSH,lutathione; MDA, 3,4-methylenedioxyamphetamine; MDMA, 3,4-ethylenedioxymethamphetamine or “ecstasy”; MTT, 3-(4,5-

imethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide; NAC, N-acetyl-ysteine; NBT, nitro blue tetrazolium; N-Me-�-MeDA, N-methyl-�-ethyldopamine; PBS, phosphate-buffered saline; PMSF,

henylmethylsulfonyl fluoride; RNS, reactive nitrogen species; ROS,eactive oxygen species; �-MeDA, �-methyldopamine; 2-(GSH)-�-eDA, 2-(glutathion-S-yl)-�-methyldopamine; 2, 5-(GSH)-�-MeDA,,5-bis(glutathion-S-yl)-�-methyldopamine; 5-(GSH)-N-Me-�-eDA, 5-(glutathion-S-yl)-N-methyl-�-methyldopamine; 5-(GSH)--MeDA, 5-(glutathion-S-yl)-�-methyldopamine; 5-(NAC)-N-Me-�-eDA, 5-(N-acetylcystein-S-yl)-N-methyl-�-methyldopamine;

t-(NAC)-�-MeDA, 5-(N-acetylcystein-S-yl)-�-methyldopamine; 5-OH--MeDA, 1-(3=,4=,5=-trihydroxyphenyl)-2-aminopropane.

306-4522/07$30.00�0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2007.03.028

1743

oxic than MDMA. GSH and NAC conjugates of N-Me-�-MeDAnd �-MeDA induced a concentration dependent delayed neu-onal death, accompanied by activation of caspase 3, whichccurred earlier in hyperthermic conditions. Furthermore,hioether MDMA metabolites time-dependently increased theroduction of reactive species, concentration-dependently de-leted intracellular GSH and increased protein bound quinones.inally, thioether MDMA metabolites induced neuronal deathnd oxidative stress was prevented by NAC, an antioxidant andSH precursor. This study provides new insights into the neu-

otoxicity mechanisms of thioether MDMA metabolites andighlights their importance in “ecstasy” neurotoxicity. © 2007

BRO. Published by Elsevier Ltd. All rights reserved.

ey words: cortical neurons, MDMA, thioether MDMA-metab-lites, NAC, neurotoxicity.

llicit use of 3,4-methylenedioxymethamphetamine (MDMAr “ecstasy”) is reaching epidemic proportions (Parrott,005). It is widely acknowledged that MDMA produces ancute and rapid enhancement in the release of both sero-onin (5-HT) and dopamine (DA) from nerve endings in therain of experimental animals. Neurotoxic damage on-HT nerve endings in the forebrain has been demon-trated both biochemically and histologically, and lasts foronths in rats and years in primates (Schmidt, 1987; Ali etl., 1993; Hatzidimitriou et al., 1999; Green et al., 2003).urthermore, studies in “ecstasy” users indicate thatDMA-induced neurodegeneration may also occur in hu-ans (McCann et al., 1998; Reneman et al., 2001).

The loss of serotonergic terminals in MDMA-adminis-rated rats is by far the most studied neurotoxic event.owever, some studies emphasize that MDMA-inducedeurotoxicity is not only limited to serotonergic neurons,ith a broader actual neurodegeneration occurring in therains of MDMA-treated animals. Indeed, studies that an-lyzed the localization of MDMA-induced neuronal degen-ration throughout the entire rat brain, have reported neu-onal degeneration in different brain areas, such as, thearietal cortex, the insular/perirhinal cortex, the ventromedial/entrolateral thalamus, and the tenia tecta (Commins et al.,987; Jensen et al., 1993; Schmued, 2003; Armstrong andoguchi, 2004; Meyer et al., 2004). Accordingly, in vitro stud-

es demonstrated that MDMA, and related amphetamines,ould induce neuronal apoptosis in cortical and cerebellarranule neurons (Stumm et al., 1999; Jimenez et al., 2004;apela et al., 2006b). These works demonstrated an apopto-

ic neuronal death characterized by endonucleosomal DNAleavage and differential expression of antiapoptotic and pro-poptotic proteins (bcl-XL/S variants) accompanied by activa-

ion of caspase 3 in neuronal cultures.ved.

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–17571744

Another considerable aspect in MDMA-induced neuro-oxicity is the hyperthermia elicited by MDMA. This repre-ents a clinically relevant aspect in MDMA abusers, sinceody temperatures may reach up to 43 °C (Henry, 1992).isuse of MDMA in crowded conditions with a high ambi-nt temperature, physical activity, and dehydration, i.e.nder the conditions that MDMA is used at “rave” parties,ay all contribute to increase the hyperthermic response

nduced by MDMA and thereby promote toxic eventsGreen et al., 2003; Crean et al., 2006).

Several studies failed to demonstrate serotonergiceurotoxicity when MDMA or 3,4-methylenedioxyamphet-mine (MDA) were injected directly into the brain (Parisnd Cunningham, 1992; Esteban et al., 2001). Since theyould not reproduce the serotonergic neurotoxicity seen afterhe peripheral administration of the drugs, it was postulatedhat systemic metabolism, leading to toxic metabolites, coulde important for the occurrence of neurotoxic events (de laorre and Farre, 2004; Monks et al., 2004).

In humans, as well as in rats, MDMA is mainly clearedy hepatic metabolism (de la Torre and Farre, 2004).DMA metabolism involves N-demethylation to MDA,hich is also a well known drug of abuse. MDMA and MDAre O-demethylenated to N-methyl-�-methyldopamine (N-e-�-MeDA) and �-methyldopamine (�-MeDA), respec-

ively (Lim and Foltz, 1988; Pizarro et al., 2004), both ofhich are catechols and can be O-methylated to form-hydroxy-3-methoxymethamphetamine (3-O-Me-N-Me-�-eDA, HMMA) or 4-hydroxy-3-methoxyamphetamine (3--Me-�-MeDA, HMA), respectively (Monks et al., 2004).lternatively, N-Me-�-MeDA and �-MeDA can undergoxidation to the corresponding ortho-quinones. These qui-ones are highly redox active molecules that can undergoedox-cycling, which originates semiquinone radicals andeads to the generation of reactive oxygen species (ROS)nd reactive nitrogen species (RNS) (Monks and Lau,997). As the reactive ortho-quinone intermediates areichael acceptors, cellular damage can occur throughlkylation of crucial cellular proteins and/or DNA (Monksnd Lau, 1997). Ortho-quinones can form adducts withlutathione (GSH) and other thiol-containing compoundsHiramatsu et al., 1990). These GSH conjugates remainedox active and can undergo the addition of a secondolecule of GSH, yielding a 2,5-bis-glutathionyl conjugate.he systemic formation of GSH conjugates of �-MeDA and-Me-�-MeDA, is followed by uptake into and further me-

abolism in the brain, as demonstrated by the fact that GSHnd N-acetylcysteine (NAC) conjugates of N-Me-�-MeDAre present in the striatum of rats administered MDMA by.c. injection (Jones et al., 2005). In fact, there is a carrierapable of transferring GSH and also GSH conjugatescross the blood–brain barrier into the brain (Patel et al.,993; Miller et al., 1996; Kannan et al., 1999).

Experiments conducted in rats have shown that theirect injection of thioether MDMA metabolites into therain produced prolonged depletions in 5-HT and neurobe-avioral changes similar to those obtained after in vivoeripheral administration of MDA and MDMA (Miller et al.,

996, 1997; Bai et al., 1999; Easton et al., 2003). Addi- f

ionally, we have recently shown that the catechol MDMA-etabolites, �-MeDA and N-Me-�-MeDA are more toxic

han MDMA in cortical neurons and that 5-(GSH)-�-MeDA5-(glutathion-S-yl)-�-methyldopamine], a thioether metab-lite, is more neurotoxic than the parent catechol (Capelat al., 2006a). However, the underlying mechanisms of theeurotoxicity induced by thioether MDMA metabolites re-ain to be fully elucidated. Therefore, our study aimed tovaluate the neurotoxicity mechanisms of thioether MDMAetabolites and their toxicity profile in rat cortical neuronsnder normal and hyperthermic temperatures.

EXPERIMENTAL PROCEDURES

aterials

aterials for cell cultures were obtained from the following sou-ces: Neurobasal medium and supplement B27 from InvitrogenPaisley, UK); Modified Eagle’s medium, phosphate buffered sa-ine (PBS), Hepes buffer, trypsin/EDTA, penicillin/streptomycin,-glutamine, collagen-G and poly-L-lysin from Biochrom (Berlin,ermany). 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazoliumromide (MTT), nitro blue tetrazolium (NBT), potassium glycinate,henylmethylsulfonyl fluoride (PMSF), NAC, reduced (GSH) andxidized glutathione (GSSG), glutathione reductase (GR) (EC.6.4.2), 2-vinylpyridine, �-nicotinamide adenine dinucleotidehosphate reduced form (�-NADPH), 5,5-dithio-bis(2-nitrobenzoiccid) (DTNB), mushroom tyrosinase (4.400 units/mg), peptideubstrate for the caspase 3 assay acetyl-Asp-Glu-Val-Asp p-ni-roanilide (Ac-DEVD-pNA) were obtained from Sigma-Aldrich (St.ouis, MO, USA). MDMA (HCl salt) was extracted and purifiedrom high purity MDMA tablets that were kindly provided by theortuguese Criminal Police Department. The obtained salt wasure and fully characterized by NMR and mass spectrometry meth-dologies. 1H and 13C NMR spectra were recorded on a BrukerRheinstetten, Germany) Avance 300 spectrometer (at 300.13 and5.47 MHz, respectively) and mass spectra were acquired with a-TOF 2 (Micromass, Manchester, UK). The metabolites N-Me-�-eDA, �-MeDA, 5-(GSH)-�-MeDA, 2-(glutathion-S-yl)-�-methyldo-amine hydrobromide [2-(GSH)-�-MeDA], 2,5-bis(glutathion-S-yl)-�-ethyldopamine hydrobromide [2,5-(GSH)-�-MeDA], 5-(gluta-

hion-S-yl)-N-methyl-�-methyldopamine hydrobromide [5-(GSH)--Me-�-MeDA], 5-(N-acetylcystein-S-yl)-�-methyldopamineydrobromide [5-(NAC)-�-MeDA], 5-(N-acetylcystein-S-yl)-N-ethyl-�-methyldopamine hydrobromide [5-(NAC)-N-Me-�-eDA], and 1-(3=,4=,5=-trihydroxyphenyl)-2-aminopropane (5-OH--MeDA) were synthesized and fully characterized by NMR andass spectrometry methodologies by REQUIMTE/CQFB, Depar-

amento de Química, FCT, Universidade Nova de Lisboa, accord-ng to previously published methods (Capela et al., 2006a;

acedo et al., 2007). All other chemicals were purchased fromigma-Aldrich of the highest grade commercially available.

ell culture

rimary neuronal cultures of cerebral cortex were obtained frommbryos (E-18) of Wistar rats. Animal experiments were licensedy the Portuguese General Directorate of Veterinary Medicine.ousing and experimental treatment of animals were in accor-ance with the Institute for Laboratory Animal Research Guide forhe Care and Use of Laboratory Animals (1996). All proceduresere taken to minimize the number of animals used and theiruffering. The experiments complied with the current laws ofortugal. Cultures were prepared according to previously pub-

ished methods (Capela et al., 2006a,b). Briefly, cerebral cortexas dissected, meninges were removed and tissue was incubated

or 15 min in trypsin/EDTA (0.05/0.02% w/v in PBS) at 37 °C; the

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–1757 1745

ultures were rinsed twice with PBS and once with dissociationedium (modified Eagle’s medium with 10% fetal calf serum,0 mM Hepes, 44 mM glucose, 100 U penicillin plus streptomycin/l, 2 mM L-glutamine, 100 IE insulin/l), dissociated by Pasteuripette in dissociation medium, pelleted by centrifugation (210�gor 2 min), re-dissociated in starter medium (Neurobasal mediumith supplemental B27, 100 U penicillin�streptomycin/ml, 0.5 mM

-glutamine, 25 �M glutamate), and plated in 48-well plates in aensity of 1.5�105 cells/well. Wells were pre-treated by incuba-ion with poly-L-lysine (0.25% w/v in PBS), overnight at 4 °C, theninsed with PBS, followed by incubation with coating mediumdissociation medium with 0.03 w/v collagen G), for 1 h at 37 °Cnd then they were rinsed twice with PBS. Afterward cells wereeeded in starter medium. Cultures were kept at 36.5 °C and 5%O2 and fed at the 4th day in vitro (DIV) with cultivating medium

starter medium without glutamate) by replacing one-half of theedium. The cultures were used for experiments after the 8thIV, containing �10% astroglial cells. Since these neuronal cul-

ures are serum free, microglia are virtually absent in the culturest the day of the experiments.

xperimental protocol

revious published experiments established 40 °C as the temper-ture for the hyperthermia experiments (Capela et al., 2006b).ultures were treated after the 8th DIV with MDMA and metabo-

ites in a concentration range 100–400 �M, single applicationithout feeding. Incubation was made for a short time period of 6 hr for a long term of 24 h and cultures were placed under normalemperature (36.5 °C) or hyperthermic temperature (40 °C). Inxperiments using the protective agent NAC, 1 mM concentrationas applied to the culture 1 h before the MDMA metabolites. Theoncentration of NAC was chosen after screening experiments tovaluate the protection (Capela et al., 2006a). Protection experi-ents with NAC were performed at the normothermic temperature

36.5 °C). Drugs were diluted in medium or purified water. Controlseceived an equivalent amount of vehicle. Cultured cells were as-essed morphologically by phase contrast microscopy and viabilityy life–death assays at two different time points, 6 h and 24 h.

Thioether MDMA metabolites concentrations were selectedccording to their ability to produce toxicity (Capela et al., 2006a)nd to the work of Jones et al. (2005). This work measured brainoncentrations of approximately 50 �M for 5-(GSH)-N-Me-�-eDA and 2,5-(GSH)-N-Me-�-MeDA, after a single s.c. MDMAose of 20 mg/kg. In fact, a multiple dosage regimen is often used

n animal studies, to match the pattern of MDMA use by abusers,uggesting that these metabolites may accumulate in the brain fol-

owing multiple drug administration (Green et al., 2003; Jones et al.,005). It seems then reasonable to believe that the metabolites used

n this study may achieve neurotoxic concentrations in vivo.

ife–death assay

ell damage was assessed quantitatively using the MTT assay,hich measures cellular metabolic activity (Capela et al., 2006a).fter adding 500 �g MTT/ml to each well, and incubating at6.5 °C for 35 min, the reaction was stopped by 10% sodiumodecyl sulfate (SDS) in 0.01 M hydrochloric acid (HCl), whichas followed by overnight incubation at 36.5 °C and photometricetection of formazan at 550 nm.

aspase 3 assay

aspase 3 assay was based on a colorimetric assay previouslyescribed (Jimenez et al., 2004). The hydrolysis of the peptideubstrate Ac-DEVD-pNA by caspase 3, results in the release ofhe p-nitroaniline (pNA) moiety, which has a high absorbance at05 nm. Approximately 3�106 cells were lysed in lysis buffer

50 mM Hepes, 1 mM DTT, 0.1 mM EDTA, 0.1% CHAPS (pH 7.4)] p

or 10 min on ice (no protease inhibitors). Cell lysate was added tossay buffer [100 mM NaCl, 50 mM Hepes, 10 mM DTT, 1 mMDTA, 10% glycerol, 0.1% CHAPS (pH 7.5)] containing 16 �M of

he colorimetric caspase 3 substrate. After incubation at 37 °C forh, absorbance was measured in a microplate reader at 405 nm.rotein concentrations were determined using the Bradford assay.

ree radicals measurements

etection and quantification of intracellular reactive species, in-luding RNS and ROS were performed by the dihydrorhodamine23 (DHR-123) assay. DHR-123 is non-fluorescent and whennters the cells is oxidized to the fluorescent rhodamine 123,pecially by peroxynitrite (ONOO�) and hydroxyl radical (HO · )Gomes et al., 2005). Cultures were pre-incubated with 100 �MHR-123 for 30 min. At the end of the incubation period, cellsere washed two times with PBS and added with new medium.fterward, neurons were treated with 400 �M of the metabolites-(GSH)-N-Me-�-MeDA, 5-(NAC)-N-Me-�-MeDA or 5-(NAC)-�-eDA. When used as a protective agent, NAC (1 mM) wasre-incubated with the cells 1 h before adding MDMA metabolites.hodamine 123 fluorescence was subsequently quantified duringh using a fluorescence plate reader (BioTek Instruments, Wi-

ooski, VT, USA) (baseline: 485 nm excitation and 528 nm emis-ion).

SH measurement

easurements of neuronal intracellular GSH levels were carriedut according to a previously published method (Carvalho et al.,004). Briefly, approximately 1.5�106 cultured cells were scrapednd proteins precipitated with perchloric acid (final concentration%) for 15 min at 4 °C, then centrifuged and the supernatant waseutralized with an equimolar solution of KHCO3. GSH contentsere measured by the rate of colorimetric change of 600 �M,5=-dithiobis(nitrobenzoic acid) at 412 nm in the presence of 1.4

of GSH reductase and 0.22 mM NADPH, using a microplateeader. Although GSSG was also tested, using 2-vinylpiridine tolock free SH groups, intracellular GSSG levels were found to beelow the limit of detection of the method (approximately.25 nmol/ml). Thus, only total GSH data are presented. Theerchloric acid pellet was suspended in 1 M NaOH and the proteineasured by the Lowry method, using bovine serum albumin

BSA) as the standard. GSH contents were normalized to the totalrotein.

TP measurement

TP was measured using a bioluminescence test based in the facthat luciferase catalyzes the formation of light from ATP anduciferin. The emitted light intensity is linearly related to the ATPoncentration and it was measured using a 96-well Microplateuminometer (BioTek Instruments). Briefly, approximately 1.5�06 cultured cells were scraped and precipitated with perchloriccid (final concentration of 5%), for 15 min at 4 °C, and centri-uged. The supernatant was then neutralized with an equimolarolution of KHCO3. After centrifugation, this sample was added to96-well opaque microplate containing the luciferin–luciferase

ssay solution [final concentrations: luciferin 0.15 mM, lucif-rase from Photinus pyralis (American firefly) 30,000 light units,lycine 50 mM, 10 mM MgSO4, Tris 1 mM, EDTA 0.55 mM, BSA% (pH 7.6)]. An ATP calibration curve was performed (ATPtandard stocks and samples in HClO4 5% were kept at �80 °Cntil the assay). The perchloric acid pellet was suspended in 1 NaOH and the protein measured by the Lowry method, using BSAs the standard. ATP contents were normalized to the total

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–17571746

easurement of protein-bound quinones:uinoproteins

or the detection of protein-bound quinones, the NBT/glycinateolorimetric assay was performed, based on a previously de-cribed method (Miyazaki et al., 2006). Briefly, approximately�106 cultured cells were scraped and centrifuged (5000 r.p.m.

or 5 min). The pellet was lysed in ice cold RIPA buffer, supple-ented with 5 mM of PMSF. The samples where sonicated and

he whole protein content was quantified by the Bradford method,sing BSA as a protein standard. One hundred micrograms of the

ysates in RIPA buffer was added to 240 �l of a 2 M potassiumlycinate (pH 10) solution. The protein containing solution wasdded to 500 �l of NBT reagent (0.24 mM NBT in 2 M potassiumlycinate, pH 10). The reaction was performed for 3 h at room

ig. 1. Neurotoxic effect of MDMA and its metabolites in cortical nerowing concentrations (100, 200 and 400 �M) of MDMA (A) and of-Me-�-MeDA (D), �-MeDA (E), 5-(GSH)-�-MeDA (F) and 5-(NAC)-�-ata are presented as percentage of control vehicle-treated cultures, w

xperiments). Multiple comparisons by the Student-Newman-Keuls post hoc teoncentration vs. control; �� P�0.01 higher concentration vs. lower concentra

emperature in the dark. The absorbance of blue–purple coloreveloped in the reaction mixture was measured at 530 nm.

tatistical analysis

esults are presented as mean�S.E.M. To avoid possible varia-ions of the cell cultures depending on the quality of dissection andeeding procedures, data were pooled from two to three indepen-ent experiments. Non-parametric tests were used, unless other-ise noticed. Kruskal-Wallis test (one-way ANOVA on ranks) wassed to compare means of different treatment groups, followed byhe Student-Newman-Keuls post hoc test, if a significant P-valuead been obtained. For pairwise comparisons, Mann-Whitneyank sum test was used. In particular, for the ROS measurementsncluded in Fig. 8 statistical analysis was conducted by means of

two-way ANOVA with repeated measurements test, followed by

luated by the MTT test. Neuronal cultures were exposed for 6 h toetabolites N-Me-�-MeDA (B), 5-(GSH)-N-Me-�-MeDA (C), 5-(NAC)-), under normothermic (36.5 °C) and hyperthermic (40 °C) conditions.ue was set to 100% (n�12–18 per condition out of three independent

urons evaMDMA mMeDA (Ghich val

st were performed after the Kruskal-Wallis test (* P�0.05, ** P�0.01tion; ## P�0.01 concentration at 36.5 °C vs. concentration at 40 °C).

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–1757 1747

he Bonferoni post hoc test. Details of the statistical analysis areescribed in each figure legend. Statistical significance was ac-epted at P values less than 0.05.

RESULTS

hioether MDMA metabolites induced neurotoxicityn cortical neuronal cultures is concentration-,emperature- and time-dependent. Thioether MDMAetabolites are strong neurotoxins relatively to theirarent catechols and MDMA

euronal viability assessed by the MTT test at the end ofh incubation period revealed toxicity for the GSH andAC conjugates: 5-(GSH)-N-Me-�-MeDA (Fig. 1C),-(NAC)-N-Me-�-MeDA (Fig. 1D), 5-(GSH)-�-MeDA (Fig.F) and 5-(NAC)-�-MeDA (Fig. 1G). At this time-pointhere was a concentration-dependent-induced toxicity atoth normothermia and hyperthermia for these com-ounds. The metabolite 5-(GSH)-N-Me-�-MeDA (Fig. 1D)howed the highest degree of neurotoxicity among thehioether MDMA metabolites tested. The hyperthermicondition did not change cell viability in control cells com-aratively to the normothermic condition. On the otherand, the observed toxicity was potentiated at 40 °C for thehioether MDMA metabolites (Fig. 1). At 6 h, MDMA cate-hol metabolites, N-Me-�-MeDA (Fig. 1B) and �-MeDAFig. 1E), showed no toxic effects in cortical neurons, whileDMA (Fig. 1A) showed minor toxicity.

Culture incubation with MDMA, �-MeDA, 5-(GSH)-�-eDA, 5-(NAC)-�-MeDA, N-Me-�-MeDA, 5-(GSH)-N-Me--MeDA and 5-(NAC)-N-Me-�-MeDA for a longer time-eriod (24 h), under normothermic conditions, revealed an

ncrease in the neurotoxicity and also a concentration-

ig. 2. Neurotoxic effect of MDMA and its metabolites in cortical neurowing concentrations (100, 200 and 400 �M) of MDMA, N-Me-�-MeDA, 5-(GSH)-�-MeDA and 5-(NAC)-�-MeDA (B), under normo

ehicle-treated cultures, which value was set to 100% (n�18–24 per conditireatment groups were compared using the Kruskal-Wallis test, P�0.001.

ependent toxicity for all the tested compounds (Fig. 2).nce again, the neurotoxicity elicited by the thioetherDMA metabolites showed to be stronger than their cor-

espondent catechol parent compounds. Meanwhile, theoxicity induced by the catechols N-Me-�-MeDA and-MeDA was higher than MDMA.

Cortical cultures were also incubated with other MDMAetabolites: 2,5-(GSH)-�-MeDA (Fig. 3A), 2-(GSH)-�-MeDA

Fig. 3B) and 5-OH-�-MeDA (Fig. 3C) for a time-period of 6 h.he GSH bisconjugated metabolite, 2,5-(GSH)-�-MeDA,as shown to be toxic only at the higher concentration

400 �M), under hyperthermia. The metabolites 2-(GSH)-�-eDA and 5-OH-�-MeDA may represent minor metabolicathways regarding the relevance for brain toxicology.-(GSH)-�-MeDA formation should be low, since the additionf a first molecule is chemically favored in position 5 of theromatic ring, yielding 5-(GSH)-�-MeDA (Hiramatsu et al.,990; Monks et al., 2004). 5-OH-�-MeDA is a tri-hydroxylatedDMA metabolite and displays a similar reactivity and chem-

cal structure with other tri-hydroxylated metabolites, thatave been identified in the brain and liver of MDMA treatedats (Elayan et al., 1993). On the other hand, these metabo-ites are important to determine the structure–activity relation-hip concerning the toxicity. 2-(GSH)-�-MeDA also revealedeurotoxicity at the higher concentration tested (400 �M),nd the neurotoxicity was potentiated under hyperthermia.hus, the toxicity of the metabolites 2-(GSH)-�-MeDA and-(GSH)-�-MeDA was higher than that of the 2,5-bis-(GSH)onjugated adduct. Meanwhile, the highly reactive metabo-

ite, 5-OH-�-MeDA, was a strong neurotoxin and there was aoncentration-dependent neurotoxicity with no difference be-ween the two exposure temperatures.

luated by the MTT test. Neuronal cultures were exposed for 24 h to5-(GSH)-N-Me-�-MeDA, 5-(NAC)-N-Me-�-MeDA (A) or MDMA and36.5 °C) conditions. Data are presented as percentage of control

rons eva-MeDA,thermic (

on out of three independent experiments). The means for different

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–17571748

Taken altogether, thioether MDMA metabolites werehown to be neurotoxic in a concentration-, temperature-nd time-dependent manner. These metabolites may beonsidered strong neurotoxins, when compared with theirarent catechols and MDMA.

AC prevented the neuronal death inducedy thioether MDMA metabolites

he pre-incubation of cells with NAC (1 mM), a precursorf GSH and an anti-oxidant, 1 h prior to cultures stimulationith the metabolites: 5-(GSH)-N-Me-�-MeDA (Fig. 4A–B),-(GSH)-�-MeDA (Fig. 4C–D), 5-(NAC)-N-Me-�-MeDAFig. 4E–F) and 5-(NAC)-�-MeDA (Fig. 4G–H) providedrotection against the metabolites-induced neurotoxicity inortical neurons for 6 and 24 h time-periods, as revealedy the MTT test. Remarkably, the protection afforded byAC against the neurotoxicity of the thioether MDMA me-

abolites was complete at the 6 h time of exposure. After

ig. 3. Neurotoxic effect of the MDMA metabolites, 2,5-(GSH)-�-eDA (A), 2-(GSH)-�-MeDA (B) and 5-OH-�-MeDA (C) in corticaleurons after exposure to a time-period of 6 h. After treatment, cul-ures were incubated under normothermic (36.5 °C) and hyperthermic40 °C) conditions and cell death was estimated after 6 h by the MTTest. Data are presented as percentage of control vehicle-treatedultures, which value was set to 100% (n�12–18 per condition out ofwo to three independent experiments). Multiple comparisons by thetudent-Newman-Keuls post hoc test were performed after theruskal-Wallis test (* P�0.05, ** P�0.01 concentration vs. control;� P�0.01 higher concentration vs. lower concentration; ## P�0.01oncentration at 36.5 °C vs. concentration at 40 °C).

4 h time-period of exposure there was full protection until a

he 200 �M concentration, while for the 400 �M concen-ration there was only minor protection.

Phase-contrast microscopy revealed a delayed type ofeuronal death, following treatment with MDMA metabo-

ites, with typical apoptotic features (Fig. 5C). Neuronsxposed to 400 �M 5-(GSH)-N-Me-�-MeDA, for a 6 heriod, showed signs of neurite disintegration, nuclearragmentation, cytoplasmic shrinkage, loss of membranentegrity and neuritic processes. As it can be seen in Fig.D the pre-incubation with NAC 1 mM before the GSHonjugated metabolite treatment prevented these toxicrocesses.

hioether MDMA metabolites induced a delayedeuronal death that is accompanied by activationf caspase 3

o assess whether the delayed neuronal death involvedaspase 3 activation, we measured caspase 3 activityollowing the exposure of cortical neurons to the more toxicDMA thioether metabolites, the NAC MDMA metabolites-(NAC)-�-MeDA and 5-(NAC)-N-Me-�-MeDA (Fig. 6).aspase 3 was activated in cortical neurons 24 h after the

ncubation with 200 �M of these two NAC metabolites.reliminary experiments also showed an increase inaspase 3 activity, in the same conditions, for 5-(GSH)-N-e-�-MeDA and 5-(GSH)-�-MeDA (data not shown).

hioether MDMA metabolites induce the formationf reactive species in neurons in a time-dependentanner: an effect abolished by NAC

etection of intracellular reactive species, including RNSnd ROS, was performed by using DHR-123 as a fluores-ence probe (Fig. 7). Incubation of cortical neurons with00 �M of the metabolites 5-(GSH)-N-Me-�-MeDA (Fig.A), 5-(NAC)-N-Me-�-MeDA (Fig. 7B) and 5-(NAC)-�-eDA (Fig. 7C) during 6 h increased, in a time-dependentanner, the formation of ROS/RNS in the cells. As earlys 2 h after the incubation with 5-(GSH)-N-Me-�-MeDA itas possible to verify a significant increase of reactivepecies production relatively to the control. In the case ofhis thioether metabolite the increase in reactive speciesroduction reached more than 2.5 times that of the control.his was the metabolite for which the generation of reac-

ive species was higher among the three herein showed.eanwhile, reactive species production by the metabolites-(NAC)-N-Me-�-MeDA and 5-(NAC)-�-MeDA, during 6 h of

ncubation, almost doubled relatively to the control. Impor-antly, experiments showed that neurons exposure toDMA, N-Me-�-MeDA, �-MeDA and to 5-(GSH)-�-MeDAuring the same period did not elicit any significant change

n the fluorescence comparatively to the control (data nothown).

NAC (1 mM) pre-treatment with any of the thioetherDMA metabolites prevented reactive species production.he levels of fluorescence in the NAC plus metabolite

reatment remained without change during the 6 h of ex-osure and were equivalent to the ones found with NAC

lone.

TGm

Nm

Mctl

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–1757 1749

hioether MDMA metabolites deplete intracellularSH levels in neurons in a concentration-dependentanner, an effect blocked by NAC

euronal cultures’ redox status was also evaluated by the

Control 100μM 200μM 400μM0

25

50

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100 24h+ NA

##

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** **

##

##++

% o

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Control 100μM 200μM 400μM0

25

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####

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Control 100μM 200μM 400μM0

25

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## ##

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**++**

++

SG

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OH

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Control 100μM 200μM 400μM0

25

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# ##

% o

f C

on

tro

ls

**++

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SNAC

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OH

5-(NAC)-N-Me-α-MeDA

ig. 4. Effect of NAC pre-treatment in the neurotoxicity of thioether MDoncentrations (100, 200 and 400 �M) of thioether MDMA metaboliteeDA (E, F) and 5-(NAC)-�-MeDA (G, H), either with or without NAC 14 h by the MTT test. Data are presented as percentage of control vehree independent experiments). Multiple comparisons by the Stude* P�0.05, ** P�0.01 concentration vs. control; �� P�0.01 higher concentration plus NAC 1 mM).

easurement of intracellular GSH levels. MDMA, N-Me-�- t

eDA and the respective GSH and NAC adducts of thisatechol, which had shown higher neurotoxicity, wereested in their ability for interference with neuronal GSHevels. The exposure of neuronal cultures to MDMA or to

Control 100μM 200μM 400μM0

25

50

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100

##

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**++

**

**

Control 100μM 200μM 400μM0

25

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100 24h+ NAC

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**++

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Control 100μM 200μM 400μM0

25

50

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+ NAC

##

% o

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SG

NH2OH

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5-(GSH)-α-MeDA

Control 100μM 200μM 400μM0

25

50

75

100 6h+ NAC

## ##

**++

**++

SNAC

NH2OH

OH

5-(NAC)-α-MeDA

bolites to cortical neurons. Neuronal cultures were exposed to growingH)-N-Me-�-MeDA (A, B), 5-(GSH)-�-MeDA (C, D), 5-(NAC)-N-Me-�--treatment, under normothermia. Cell death was estimated after 6 andted cultures, which value was set to 100% (n�18 per condition out ofan-Keuls post hoc test were performed after the Kruskal-Wallis testtion vs. lower concentration; # P�0.05, ## P�0.01 concentration vs.

C

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MA metas, 5-(GSmM pre

hicle-treant-Newmoncentra

he catechol N-Me-�-MeDA did not induce any significant

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–17571750

hange in the intracellular GSH levels, at any of the testedoncentrations and incubation temperatures, during theime period of 6 h (Fig. 8A–B). Incubation of cortical neu-ons with growing concentrations of the metabolites,-(GSH)-N-Me-�-MeDA (Fig. 8C) and 5-(NAC)-N-Me-�-eDA (Fig. 8D), during 6 h, either at normothermia oryperthermia, depleted the intracellular GSH levels in aoncentration-dependent manner. Surprisingly, the lowerhioether metabolite concentration of 100 �M did not in-uce neuronal death after 6 h of exposure, but was able toromote a significant depletion of intracellular GSH. Forhe MDMA metabolite 5-(GSH)-N-Me-�-MeDA, the deple-ion of GSH was more significant when cultures werelaced for 6 h under hyperthermic temperatures. The effectas not accompanied by the parallel increase in the levelsf GSSG. The levels of GSSG in the treated cells remainedelow the detection levels of the method (data not shown).

ig. 5. Phase-contrast microphotographs from cortical neurons, after 6f 5-(GSH)-N-Me-�-MeDA (C) and treatment with 400 �M of 5-(GSH)

ig. 6. Caspase 3 activity in cortical cultures, 24 h after the incubationith 200 �M of the NAC MDMA metabolites 5-(NAC)-�-MeDAnd 5-(NAC)-N-Me-�-MeDA. Multiple comparisons by the Student-

iewman-Keuls post hoc test were performed after the Kruskal-Wallis

est (** P�0.01 concentration vs. control).

nterestingly, neuronal cultures exposure, for 6 h, to theigh temperature condition (40 °C) led by itself to an in-rease in the intracellular GSH levels relative to controlsxposed at normothermia (36.5 °C) (Fig. 8A–D).

NAC (1 mM) pre-treatment blocked the depletion ofntracellular GSH induced by the thioether MDMA metab-lites 5-(GSH)-N-Me-�-MeDA (Fig. 8E) and 5-(NAC)-N-e-�-MeDA (Fig. 8F). In fact, the treatment with NAC per

e significantly increased the intracellular GSH levels rel-tively to controls, when neuronal cultures were placed forh under the normothermic temperature.

hioether MDMA metabolites increase protein bounduinone products in neurons in a concentration-ependent manner, an effect not prevented by NAC

he presence of protein bound quinone products (quino-roteins) in cortical neurons was evaluated after 6 h expo-ure to thioether MDMA metabolites under normal temper-ture. Neuronal cultures exposure to 5-(GSH)-N-Me-�-eDA, 5-(NAC)-N-Me-�-MeDA or 5-(NAC)-�-MeDA led tosignificant increase in the formation of quinoproteins

Table 1). The measurements also showed that 5-(GSH)--Me-�-MeDA (Table 1A) induced the production of qui-oproteins in a concentration dependent manner. Interest-

ngly, pre-treatment with NAC 1 mM did not prevent theormation of quinoproteins in neurons exposed to thioetherDMA metabolites (Table 1 B).

ffect of thioether MDMA metabolitesn intracellular ATP levels

ortical neuronal cultures exposure to MDMA, for 6 h, did not

ure: control (A), treatment with NAC 1 mM (B), treatment with 400 �M-MeDA plus NAC 1 mM (D) (magnification 400�).

nduce any significant change in the intracellular ATP levels,

ept9oAmp9t

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–1757 1751

ither at normothermic or hyperthermic temperatures of ex-osure (Fig. 9A). The same results were obtained for the

hioether MDMA metabolite 5-(NAC)-N-Me-�-MeDA (Fig.C) where after 6 h of exposure, either under normothermiar hyperthermia, there were no changes in the intracellularTP levels. Interestingly, the 400 �M concentration of theetabolite 5-(GSH)-N-Me-�-MeDA induced a dramatic de-letion of ATP levels, at both incubation temperatures (Fig.B). This reduction was fully reversed by NAC (1 mM) pre-reatment under normothermic conditions (Fig. 9D).

DISCUSSIONhe key findings of our study were: (1) thioether MDMA

A

B

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ig. 7. Thioether MDMA metabolites’ effect in the formation of reacti00 �M of the metabolites 5-(GSH)-N-Me-�-MeDA (A), 5-(NAC)-N-Mre-treatment, under normothermia. The fluorescence of rhodamine 12

n control cultures, which value was set to 100% (n�12–18 per conditonferroni post hoc test were performed after the two-way ANOVA withs. control; ## P�0.01, ### P�0.001 concentration vs. concentration p

etabolites induced neurotoxicity in cortical neuronal cul- M

ures is concentration-, temperature- and time-dependent;2) thioether MDMA metabolites are strong neurotoxinsomparatively to their parent catechols and MDMA;3) thioether MDMA metabolites induced delayed neu-onal death, accompanied by activation of caspase 3;4) thioether MDMA metabolites time-dependently inducedhe formation of reactive species in neurons; (5) thioetherDMA metabolites concentration-dependently depleted

ntracellular GSH levels and increased protein bound qui-one products in neurons; (6) NAC prevented the neuronaleath and oxidative stress induced by thioether MDMAetabolites.

Evidence indicates that further metabolism of N-Me-�-

ControlNAC 1mM

5-(GSH)-N-Me-α-MeDA5-(GSH)-N-Me-α-MeDA+NAC

#

SG

NHCH3OH

OH

5-(GSH)-N-Me-α-MeDA

ControlNAC 1mM

5-(NAC)-N-Me-α-MeDA5-(NAC)-N-Me-α-MeDA+NAC

SNAC

NHCH3OH

OH

5-(NAC)-N-Me-α-MeDA

ControlNAC 1mM

5-(NAC)-α-MeDA5-(NAC)-α-MeDA+NAC

SNAC

NH2OH

OH

5-(NAC)-α-MeDA

s in neuronal cortical cultures. Cortical neurons were incubated withA (B) and 5-(NAC)-�-MeDA (C), either with or without NAC 1 mMnitored during 6 h. Data are presented as percentage of fluorescencetwo to three independent experiments). Multiple comparisons by themeasurements test (* P�0.05, ** P�0.01, *** P�0.001 concentration1 mM).

5 6

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eDA and �-MeDA is required for metabolism-related

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–17571752

eurotoxicity. In fact, �-MeDA is not neurotoxic followingirect injection into the brain (Miller et al., 1996; Monks etl., 2004). Our results clearly show that catechol MDMAetabolites are not toxic to cortical neurons for a short timexposure of 6 h. However, in the same period, their corre-pondent GSH conjugates, 5-(GSH)-N-Me-�-MeDA and-(GSH)-�-MeDA were toxic. In particular the GSH conju-ate of N-Me-�-MeDA was an extremely potent neuro-

oxin. Longer incubation periods (24 h) of neuronal culturesith the catechol MDMA metabolites revealed a minor

oxicity, while the GSH conjugates increased dramaticallyheir neurotoxicity.

N-Me-�-MeDA and �-MeDA adducts with GSH areormed by hepatic MDMA metabolism (Hiramatsu et al.,

D) during 6 h under normothermic (36.5 °C) and hyperthermic (40 °C)onditions. In parallel experiments neuronal cultures were pretreatedith NAC (1 mM) followed by the thioether MDMA metabolites,-(GSH)-N-Me-�-MeDA (E) and 5-(NAC)-N-Me-�-MeDA (F), for 6 hnder normothermic conditions. Total intracellular GSH content areresented in nmol/mg protein (n�9 per condition out of three indepen-ent experiments). Multiple comparisons by the Student-Newman-euls post hoc test were performed after the Kruskal-Wallis test

* P�0.05, ** P�0.01 concentration vs. control; � P�0.05, �� P�0.01igher concentration vs. lower concentration; # P�0.05, ## P�0.01

able 1. Effect of MDMA and of the metabolites 5-(GSH)-N-Me-�-eDA and 5-(NAC)-N-Me-�-MeDA on cellular protein bound quinone

quinoprotein) levels

.

-(GSH)-N-Me-�-MeDA�M)

Quinoproteins(OD 530 nm/mg Prot)

4.57�0.1500 4.99�0.2100 5.15�0.16*00 5.94�0.19**

.

ondition Quinoproteins(OD 530 nm/mg Prot)

ontrol 4.69�0.13AC (1 mM) 4.66�0.12-(NAC)-�-MeDA (400 �M) 5.96�0.15**-(NAC)-N-Me-�-MeDA (400 �M) 6.39�0.23**-(NAC)-�-MeDA (400 �M)�NAC (1 mM) 6.03�0.22**-(NAC)-N-Me-�-MeDA (400 �M)�NAC(1 mM)

6.35�0.18**

Cortical neurons were exposed to growing concentrations (100, 200nd 400 �M) of the thioether MDMA metabolite, 5-(GSH)-N-Me-�-eDA (A) during 6 h under normothermic (36.5 °C) conditions (A). Inarallel experiments, neuronal cultures were pre-treated with NAC1 mM) followed by the thioether MDMA metabolites, 5-(NAC)-�-eDA and 5-(NAC)-N-Me-�-MeDA for 6 h under normothermic con-itions (B). Protein bound quinones values are presented in OD 530m/mg Prot (n�9 per condition out of 3 independent experiments).ultiple comparisons by the Student-Newman-Keuls post hoc testere performed after the Kruskal-Wallis test.P�0.05, concentration vs. control.* P�0.01, concentration vs. control.

ig. 8. Effect of MDMA and of the metabolites, N-Me-�-MeDA,-(GSH)-N-Me-�-MeDA and 5-(NAC)-N-Me-�-MeDA in the intracellu-

ar GSH levels. Cortical neurons were exposed to growing concentra-ions (100, 200 and 400 �M) of MDMA (A) and the metabolites, N-Me-

oncentration at 36.5 °C vs. concentration at 40 °C or concentration

s. concentration plus NAC 1 mM).

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–1757 1753

990). Conjugation of GSH with electrophiles such asuinones results in preservation or enhancement of theiriologic (re)activity. GSH-conjugated MDMA metabolitesan either be monoconjugated or undergo the addition of aecond molecule of GSH yielding a 2,5-bis-glutathionylonjugate (Jones et al., 2005). These systemically formedetabolites can be transported into the brain across thelood–brain barrier (Patel et al., 1993; Miller et al., 1996;

ig. 9. Effect of MDMA and of the metabolites, 5-(GSH)-N-Me-�-eDA and 5-(NAC)-N-Me-�-MeDA in the intracellular ATP levels.ortical neurons were exposed to growing concentrations (100, 200nd 400 �M) of MDMA (A) and the metabolites, 5-(GSH)-N-Me-�-eDA (B) and 5-(NAC)-N-Me-�-MeDA (C) during 6 h under normo-

hermic (36.5 °C) and hyperthermic (40 °C) conditions. In parallelxperiments neuronal cultures were pretreated with NAC (1 mM)ollowed by the metabolite 5-(GSH)-N-Me-�-MeDA (D), for 6 h underormothermia. Intracellular ATP content are presented in nmol/mgrotein (n�9 per condition out of three independent experiments).ultiple comparisons by the Student-Newman-Keuls post hoc testere performed after the Kruskal-Wallis test (** P�0.01 concentrations. control; �� P�0.01 higher concentration vs. lower concentration;# P�0.01 concentration vs. concentration plus NAC 1 mM).

annan et al., 1999). Once in the brain, these compounds M

an undergo further metabolism via the mercapturic acidathway by �-glutamyl transpeptidase (�-GT) and dipepti-ase to the corresponding cysteine and finally NAC con-

ugates (Monks and Lau, 1997). The half-life of the cys-eine conjugates is short and their uptake into the brain islso limited, and therefore the persistent metabolites arehe NAC conjugates, which appear to be slowly eliminatedrom the brain (Monks and Lau, 1997). In fact, recentxperiments using in vivo microdialysis have provided di-ect evidence for the presence of GSH and NAC conju-ates of MDMA metabolites in the brain after in vivo s.c.dministration of MDMA (Jones et al., 2005).

Our study demonstrated that 5-(NAC)-N-Me-�-MeDAnd 5-(NAC)-�-MeDA are potent toxins to cortical neuronsxposed for a short period of 6 h. Also, these NAC conju-ates were more toxic in the short and long term exposurehan their parent catechols and MDMA. NAC conjugatesetain the toxicity of their parent GSH conjugates. Conse-uently, the toxic potential of the NAC conjugates is highers they tend to accumulate in the brain. Additionally, it is

mportant to consider that all of the metabolites may ap-ear in the brain simultaneously after administration ofDMA. Therefore, a synergistic or additive toxic effect of

hese metabolites may occur at lower concentrations thanhat of each compound isolated.

The present study also intended to provide a structure–oxicity–activity relationship concerning the GSH conjuga-ion of the catechol MDMA metabolites, namely �-MeDA,n position 2 or 5 of the benzene ring and at both positionsFig. 10). The toxicity observed after 6 h of incubation forhe metabolites 2-(GSH)-�-MeDA and 5-(GSH)-�-MeDAas higher than that obtained for 2,5-(GSH)-�-MeDA, aSH bi-conjugated adduct. This correlated well with the

ower ability of the GSH bi-conjugated adduct to enter therocess of redox-cycle (Monks and Lau, 1997). The oxi-ation of the catechol in the presence of a nucleophile (likehiols) results in thioether catechols, which are more easilyxidized than the parent starting molecule (Macedo et al.,007). Recent studies have shown that the conjugation ofhe catechol with GSH or NAC at the 5 position reducedhe oxidation potential when compared with the non-con-ugated compound (Macedo et al., 2007). Noteworthy, theower oxidation potential observed for the 5-(GSH)-�-

eDA adducts of �-MeDA relative to the parent catecholorrelates well with the higher toxicity of this adduct in ratortical neurons.

Previous in vivo studies have proven that thioetherDMA metabolites promote neurotoxic effects when in-

ected directly into the brain. I.c.v. injections of 5-(NAC)-�-eDA and 5-(GSH)-�-MeDA into rats produced neurobe-avioral changes characteristic of peripheral administra-ion of MDMA/MDA as well as acute increases in brain-HT and DA concentrations (Miller et al., 1996). In addi-ion to the effects observed after i.c.v. administration of-(NAC)-�-MeDA and 5-(GSH)-�-MeDA, direct injection

nto the striatum, cortex, and hippocampus produced pro-onged depletions in 5-HT and neurobehavioral changesimilar to those obtained after in vivo administration of

DA and MDMA (Bai et al., 1999). 2,5-(GSH)-�-MeDA

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J. P. Capela et al. / Neuroscience 146 (2007) 1743–17571754

lso proved to decrease 5-HT levels in the striatum, hip-ocampus and cortex respectively, 7 days after injection inats (Miller et al., 1997). Recently, the metabolite 5-(NAC)--Me-�-MeDA was shown to produce acute changes in

ats’ behavior similar to that seen with MDMA and otherhioether conjugates of �-MeDA, and also significantly de-reased striatal and cortical concentrations of 5-HT and-HIAA in a dose-dependent manner (Jones et al., 2005).dditionally, we have previously shown that cultured cor-

ical neurons exposed to �-MeDA and 5-GSH-�-MeDAndergo apoptosis, as visualized by staining with the flu-

O

O

NHCH3

OH

OH

NHR

NHROH

OH

SG

NHROH

OH

SNAC

MDMA

N-Me-α-MeDA (R=CH3)

O-Demethylenation

5-(GSH)-α-MeDA (R=H)

5-(GSH)-N-Me-α-MeDA (R=CH3)

α-MeDA (R=H)

GSH Conjugation

Mercapturic acid pathway

5-(NAC)-α-MeDA (R=H)

5-(NAC)-N-Me-α-MeDA (R=CH3)

CatecholsCatechols ++++

GSHGSH conjugatesconjugates

55--GSH +++++GSH +++++

22--GSH +++GSH +++

2,52,5--GSH +++GSH +++

NACNAC conjugatesconjugates

55--NAC ++++++NAC ++++++

22--NAC ++++NAC ++++

2,52,5--NAC ++++NAC ++++

MDMAMDMA MetabolitesMetabolites NeurotoxicityNeurotoxicity ProfileProfile

MDMA +MDMA +

ig. 10. MDMA metabolites neurotoxicity profile and their relativeotency. The postulated MDMA metabolism pathway in humans andats is represented and the metabolites toxicity potential, according tour findings, is shown in the highlighted text boxes. The toxicity isiven comparatively to MDMA, where ������ stands for the maxi-um and � for the minimum toxicity. MDMA O-demethylation yields

atechols which are more toxic than MDMA. The catechols’ GSHdduction extraordinarily enhances their neurotoxicity. The first conju-ation at position 5 of the benzene ring, much more probable, yieldshe most toxic GSH conjugate. The GSH first conjugation is atosition 2, less probable, or the further adduction to the bis-adduct

eads to a minor ability of the metabolites to undergo redox-cycling andherefore they are less toxic than the 5-GSH conjugate. GSH metab-lites’ brain metabolism, via the mercapturic acid pathway, leads toersistent NAC conjugates. These NAC conjugates are slowly elimi-ated from the brain and retain the toxicity of their parent GSH me-abolites; consequently their potential neurotoxicity is higher.

rescent dyes ethidium bromide/acridine orange, used to m

istinguish apoptotic from necrotic death (Capela et al.,006a). Hence, data showing direct evidence of neuronalpoptosis, accompanied by activation of caspase 3, in aoncentration dependent manner exerted by thioetherDMA metabolites, confirm the important role of GSH- andAC-conjugated metabolites in MDMA-evoked neurotoxicity.

Our study, clearly demonstrated that thioether MDMAetabolites increase reactive species, including ROS andNS production in neurons. The generation of reactivepecies in neurons increased with the time of exposurend was significant as early as 2 and 3 h after treatment. Inact, GSH and NAC conjugates remain redox active andan originate quinones that are highly redox active mole-ules. These can undergo redox-cycling, which originatesemiquinone radicals and leads to the generation of ROSnd RNS (Capela et al., 2006a). In accordance, 5-(GSH)--MeDA and 2,5-(GSH)-�-MeDA were shown to produceOS in SK-N-MC cells transiently transfected with theuman 5-HT transporter (Jones et al., 2004). MDMA ad-inistration has proven to increase free radical formation

n the rat brain (Colado et al., 1997). ROS production seenfter MDMA administration has been attributed to theDMA-induced inhibition of the 5-HT transporter (Shanka-

an et al., 1999, 2001; Jones et al., 2004) and also to theA oxidation process (Aguirre et al., 1998; Sprague et al.,998; Jones et al., 2004). Additionally, it seems likely thathe oxidation of thioether MDMA metabolites contributes,t least in part, to the ROS/RNS generated in the brain ofDMA-administered animals.

Hyperthermia was shown to potentiate the neurotoxic-ty of thioether MDMA metabolites. Taking into accounthat MDMA metabolites produce ROS/RNS and decreaseSH intracellular levels, the hyperthermia-induced oxida-

ive stress will potentiate their toxicity. MDMA was showno cause acute dose-dependent hyperthermia in laboratoryxperimental animals, and in humans MDMA-induced hy-erthermia can be fatal (Henry, 1992; Green et al., 2003).hile the long term MDMA-induced neurotoxic effects

ave been shown to be related to the acute hyperthermiaroduced by the drug (Broening et al., 1995; Malberg andeiden, 1998), neurodegeneration has also been reported

o occur in normothermic animals (Broening et al., 1995;arfel and Seiden, 1995). Furthermore prevention ofDMA-induced hyperthermia decreases the neurotoxicitynd many drugs that protect against MDMA-induced neu-otoxicity also decrease the animals body temperatureFarfel and Seiden, 1995; Malberg et al., 1996). In animaltudies, researchers are faced with several factors affect-

ng body temperature, during the experimental procedure.n this study, the high body temperature was simulated bylacing the cortical neurons in an incubator at an environ-ent temperature of 40 °C, after addition of MDMA andetabolites (Capela et al., 2006b). Nevertheless, whileyperthermia can potentiate the neurotoxic events, thenderlying bases of MDMA-evoked neurotoxicity are notependent on the hyperthermia induced by MDMA (Jonest al., 2004).

Incubation of cortical neurons with thioether MDMA

etabolites for a short time of exposure, either at normo-

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hermia or hyperthermia depleted the intracellular GSHevels in a concentration-dependent manner. This deple-ion may be considered a marker of oxidative stress andeads to an increase of the neuronal vulnerability to furtherxidative injury mediated by the metabolites. The antioxi-ant and GSH synthesis precursor, NAC, prevented neu-onal death. NAC pre-treatment prevented the formation ofeactive species and increased the intracellular GSH levelsn neurons, thereby providing protection against thehioether MDMA metabolites neurotoxicity. By acting as aysteine donor, NAC maintains intracellular GSH levelsnd is neuroprotective for a range of neuronal cell typesgainst a variety of stimuli in vitro and in animal modelsHussain et al., 1996; Hart et al., 2004). The importance ofhe neuronal redox status for the neurotoxic events wasnderlined in the present study, since an important protec-ive effect was observed with NAC.

Previous studies have proven in vitro that the concen-ration of N-Me-�-MeDA and �-MeDA decreases over timen biological media, due to their oxidation to the correspon-ent aminochromes (Carvalho et al., 2004). The reactive

ntermediates produced during the oxidation of these cat-cholamines into reactive ortho-quinones and/or amin-chromes, can be conjugated with GSH to form the corre-ponding glutathionyl adducts. It was previously shownhat GSH depletion is one of the toxic events observedn rat cardiomyocytes exposed to N-Me-�-MeDA and-MeDA (Carvalho et al., 2004). Aminochromes can alsondergo further oxidation, leading to the formation of aelanin type polymer (Shen and Dryhurst, 1996). In fact, aark brown/black turbidity, characteristic of these poly-ers, appeared in the neuronal medium, not only after

ncubation with thioether MDMA metabolites (early eventt 3 h), but also after incubation with N-Me-�-MeDA and-MeDA, as a late stage event. NAC pre-treatment pre-ented the brown/black turbidity formation in the neuronaledium, therefore preventing the formation of melaninolymers. It might be postulated that the decrease in the

ntracellular GSH levels is largely dependent on the GSHonjugation of the reactive intermediates of MDMAetabolites.

Quinone thioethers have the ability to interfere withedox-cycle, produce ROS, to arylate tissue macromole-ules and also to covalently bind to DNA (Kleiner et al.,998; Miyazaki et al., 2006). In accordance, we found aoncentration dependent increase in quinoproteins, in theultivated neurons after exposure to the thioether MDMAetabolites. Ortho-quinones, aminochromes, and GSH

onjugates are known to cause irreversible inhibition ofnzymes that possess either a GSH binding site and/orysteine residues critical for enzyme function (Monks andau, 1997). Likewise, inhibition of GSH reductase andSH-S-transferase by quinones, as well as inhibition ofSH reductase, selenium-dependent GSH peroxidase,nd GSH-S-transferase by aminochromes, was previouslyeported (Remião et al., 2002). Interestingly, pre-treatmentith NAC did not prevent the increase in the formation ofuinoproteins. NAC may not prevent the redox-cycle of the

uinone thioethers and their possible adduction to macro-

olecules, like proteins. Instead, NAC possibly acts byapturing ROS/RNS, generated by the MDMA thioetheretabolites and promoting their oxidizing products conju-ation with GSH, thereby slowing the formation of otheroxic oxidation products such as semi-quinones and do-aminochromes. This would, also, explain why the neuro-al medium of the NAC plus thioether metabolites treat-ent does not display the typical color that results from the

ormation of aminochromes and melanin type polymer,hich occurs in the thioether MDMA metabolite-treatedeurons.

The neurotoxicity of 5-(GSH)-N-Me-�-MeDA and 5-NAC)-N-Me-�-MeDA is of relevance to the extrapolationf the results to humans. N-Me-�-MeDA is a main humanlasma metabolite of MDMA, while in rats it is �-MeDA (de

a Torre and Farre, 2004; Easton and Marsden, 2006).arious studies conducted in humans have evaluatedDMA metabolism and have identified and quantified theDMA catechol metabolites �-MeDA and N-Me-�-MeDA

n the plasma (Segura et al., 2001, 2005; Pizarro et al.,004). The metabolic reactions in human MDMA metabo-

ism are similar to the ones present in rats, only differing inerms of metabolic kinetic rate (de la Torre and Farre,004). Thus, it might be expected that NAC- and GSH-onjugated MDMA metabolites are also present in humanrains and that they may achieve neurotoxic concentra-ions, as occurs in rats. In addition, the fraction of N-Me--MeDA converted to neurotoxic metabolites may varyreatly in human individuals exposed to similar doses ofDMA, because enzymes that participate in the activationf MDMA (CYP2D6) and inactivation of MDMA metabolitescatechol-O-methyltransferase) are highly polymorphic inhe human population (de la Torre et al., 2005; Carmo etl., 2006). Finally, species differences in the rate of O-emethylenation of MDMA and MDA may underlie speciesifferences in the neurotoxic response to these amphet-mine analogs (de la Torre and Farre, 2004; Easton andarsden, 2006).

CONCLUSION

n conclusion, MDMA metabolism leading to reactivehioether MDMA metabolites, which produce ROS/RNSnd toxic oxidation products, has a key role in the neuro-oxic events produced by “ecstasy.”

cknowledgments—This work was supported by the “Fundaçãoalouste Gulbenkian” (Ref. FCG 10/04). J.P.C. was the recipientf a PhD grant from “Fundação para a Ciência e Tecnologia” (Ref.FRD/BD/10908/2002).

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(Accepted 22 March 2007)(Available online 30 April 2007)