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Enzymatic–Nonenzymatic CellularAntioxidant Defense Systems Responseand Immunohistochemical Detection ofMDMA, VMAT2, HSP70, and Apoptosisas Biomarkers for MDMA (Ecstasy)Neurotoxicity

Irene Riezzo,1 Daniela Cerretani,2 Carmela Fiore,1 Stefania Bello,1 Fabio Centini,1

Stefano D’Errico,1 Anna Ida Fiaschi,2 Giorgio Giorgi,2 Margherita Neri,1

Cristoforo Pomara,1 Emanuela Turillazzi,1 and Vittorio Fineschi1*1Department of Forensic Pathology, Faculty of Medicine, University of Foggia, Foggia, Italy2Department of Pharmacology ‘‘G. Segre,’’ Faculty of Medicine, University of Siena, Siena, Italy

3,4-Methylenedioxymethamphetamine (MDMA)-inducedneurotoxicity leads to the formation of quinone metabo-lities and hydroxyl radicals and then to the productionof reactive oxygen species (ROS). We evaluated theeffect of a single dose of MDMA (20 mg/kg, i.p.) on theenzymatic and nonenzymatic cellular antioxidantdefense system in different areas of rat brain in theearly hours (<6 hr) of the administration itself, and weidentified the morphological expressions of neurotoxic-ity induced by MDMA on the vulnerable brain areas inthe first 24 hr. The acute administration of MDMA pro-duces a decrease of reduced and oxidized glutathioneratio, and antioxidant enzyme activities were signifi-cantly reduced after 3 hr and after 6 hr in frontal cortex.Ascorbic acid levels strongly increased in striatum, hip-pocampus, and frontal cortex after 3 and 6 hr. High lev-els of malonaldehyde with respect to control weremeasured in striatum after 3 and 6 hr and in hippocam-pus and frontal cortex after 6 hr. An immunohistochem-ical investigation on the frontal, thalamic, hypothalamic,and striatal areas was performed. A strong positivereaction to the antivesicular monoamine transporter 2was observed in the frontal section, in the basal gangliaand thalamus. Cortical positivity, located in the mostsuperficial layer was revealed only for heat shock pro-tein 70 after 24 hr. VVC 2009 Wiley-Liss, Inc.

Key words: MDMA; oxidative stress; HSP70; VMAT2;apoptosis

Previous experimental studies have concludedthat 3,4-methylenedioxymethamphetamine (MDMA)-in-duced neurotoxicity is characterized by the decrease ofserotonin and its metabolites, alterations most evident inneocortex of rats, striatum and hippocampus, becausedeficits in activities of tryptophan hydroxylase (TrypH),

the rate-limiting enzyme in the synthesis of the neuro-transmitter serotonin, occurred in the rat neostriatum,hippocampus, hypothalamus, and cortex (Schmidt andTaylor, 1987; Schmidt, 1987; Lyles and Cadet, 2003;Peng and Simantov, 2003). Decrease in activity of dopa-mine transporters and vesicular monoamine transporter 2(VMAT-2) was associated with increased axon caliber,large swollen varicosities, and dilated proximal axonstumps (Molliver et al., 1990). Evidence documents thatmicroglia activation is closely associated with neurotox-icity and not with other prominent pharmacologicaleffects of methamphetamine such as inhibition of DA or5HT transporters, stimulation of DA receptor, or hyper-thermia (Thomas et al., 2004). Microglia produces a va-riety of proinflammatory cytokines, prostaglandins, andreactive oxygen species in response to brain injury anddisease, which explains how they may contribute to orworsen neuronal damage. Again, studies in rats showedthat long-lasting MDMA induced a significant neurotox-icity, accompanied by an extensive fraction of shrunkencells with cytoplasm vacuolization and condensed nucleiindicating apoptotic rather than necrotic cell death(Atlante et al., 1999, 2001). The release of apoptogenicfactors into cytoplasm following neurotoxic insult maydestabilize the internal cytoskeleton, which may be amajor factor in the activation of cell apoptosis (Capelaet al., 2007a,b).

*Correspondence to: Vittorio Fineschi, MD, PhD, Department of Foren-

sic Pathology, University of Foggia, Ospedale Colonnello D’Avanzo, Via

degli Aviatori 1, 71100 Foggia, Italy. E-mail: [email protected]

Received 9 May 2009; Revised 13 July 2009; Accepted 17 July 2009

Published online 1 October 2009 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.22245

Journal of Neuroscience Research 88:905–916 (2010)

' 2009 Wiley-Liss, Inc.

The molecular mechanisms involved in the genesisof these toxic effects are not yet fully clarified, but theoxidative stress, exitotoxicity, and mitochondrial dys-function appear to be causal events that converge tomediate MDMA-induced neurotoxicity, as measured byloss of various markers of dopaminergic and serotonergicterminals. The increase in extracellular dopamine afterMDMA leads to the formation of quinone metabolities;the reactive intermediates produced during the oxidationof dopamine into reactive orto-quinones and/or amino-chromes can be conjugated with intracellular glutathione(GSH) to form the corresponding glutathionyl adducts.Quinone thioethers have ability to interfere with redoxcycle, to produce ROS, and to bind covalently to DNA(Miyazaki et al., 2006). In addition, the rise in extracel-lular glutamate produce by the substituted amphetaminesleads to an increase in intracellular calcium concentra-tions, which leads to the activation of nitric oxide syn-thase (NOS) and the consequent generation of reactivenitrogen species (RNS). The ROS and RNS can alterproteins, lipids, and DNA as well as inhibit mitochon-drial function to produce energy deficits in the nerveterminals. Recent studies support the idea that hyper-thermia is an important factor in MDMA-induced neu-ronal death (Capela et al., 2007a,b). Rats have beenreported in many studies to have an acute dose-depend-ent hyperthermic response following administration ofMDMA (Green et al., 2003). It is well established thatcytokines such as interleukin-1b, interleukin-6, and tu-mor necrosis factor-a increase body temperature bydirect or indirect mechanisms in the brain, and, in par-ticular, interleukin-1b is involved in the development ofthe hyperthermic response (Green et al., 2004).

The aim of our experimental study was to evaluatethe effect of a single dose of MDMA (20 mg/kg, i.p.) onenzymatic and nonenzymatic cellular antioxidant defensesystems in different areas of rat brain, in the early hours(<6 hr) of the administration itself, and to identify themorphological changes in expression of neurotoxicityinduced by MDMA and its metabolites on the vulnerablebrain areas in the first 24 hr. An immunohistochemicalinvestigation of the frontal, thalamic, hypothalamic andstriatal areas was performed with antibodies anti-MDMA,-glial fibrillary acidic protein (GFAP), -HSP 27, -HSP 70,-HSP 90 (heat shock proteins), -VMAT-2, -b-APP (bamyloid precursor protein), -TrypH, and -growth-associ-ated phosphoprotein 43 (GAP-43) and TUNEL assay forapoptosis to evaluate the specific morphological alterations.

MATERIALS AND METHODS

The experimental procedures followed the NIH Princi-ples of laboratory animal care (NIH publication no. 85-23 revised1996) and were approved by the University of Siena Commit-tee for Animal Experiments.

Animal Model and Experimental Protocol

For the evaluation of oxidative stress, 21 male albinorats (Wistar; Charles River) weighing 200–250 g were used to

analyze the effect of MDMA administration (20 mg/kg, i.p.)on rat brains. After treatment, animals were killed by decapita-tion at the following times: group 1 (9 rats) after 3 hr; group2 (9 rats) after 6 hr, and group 3 (3 rats) control group.

Both the histopathological examination of the brain andthe plasma concentration of MDMA and the metabolitemethylenedioxyamphetamine (MDAA) were carried out on25 rats, each weighing 200–250 g, divided into three groupsof seven animals each treated with MDMA 20 mg/kg, i.p.:group 1 killed 6 hr after treatment; group 2 killed 16 hr aftertreatment; and group 3 killed 24 hr after treatment of which 2died spontaneously within 4 hr after administration ofMDMA.

One control group of four animals was treated with sa-line i.p. and killed: 1 rat 6 hr after treatment, 1 rat 16 hr aftertreatment, and 2 rats 24 hr after treatment were used for his-tological examination and toxicological analysis. Plasma sam-ples obtained after the treatments were stored at –808C untilMDMA/MDAA gas chromatography/mass spectroscopy (GC-MS) analysis.

Biochemical Analysis: Oxidative Stress Evaluation

The hippocampus, striatum, and frontal cortex of thetreated and control animals were immediately resected andfrozen at –808C up to the time of the determination ofreduced and oxidized glutathione (GSH, GSSG), malondialde-hyde (MDA), and ascorbic acid (AA) levels and of superoxidedismutase (SOD), glutathione peroxidase (GPx), and glutathi-one reductase (GR) enzymatic activities.

GSH/GSSG and protein determination. Brain tis-sues were homogenized in EDTA K1- phosphate buffer, pH7.4 (1:3, w/v), at 08C. Total GSH was analyzed as describedby Tietze (1969) and GSSG was determined according toGriffith’s (1980) method. In the remaining aliquot, proteinswere assayed according to the method of Lowry (1951).

SOD, GPx, and GR assessment. To measure cyto-solic enzyme activity, the brain samples were homogenizedaccording to Whanger and Butler (1988). GPx activity wasmeasured according to Paglia and Valentine (1967). GR activ-ity was analyzed as described by Goldberg and Spooner(1983). Total superoxide dismutase (Cu/Zn superoxide dis-mutase and Mn-superoxide dismutase) was assayed by spectro-photometric method based on the inhibition of a superoxide-induced NADH oxidation according to Paoletti et al. (1986).The cytosolic protein concentration was determined by usingthe Lowry (1951) method with BSA as standard.

MDA assessment. The extent of lipid peroxidationin the rat brain was estimated by calculation of MDA levelswith an HPLC method and UV detection as described byShara et al. (1992).

AA assay. Brain tissues were homogenized in EDTA-K1- phosphate buffer, pH 7.4 (1:4, w/v), at 08C and analyzedas described by Ross (1994).

Morphological Examination

The brains of treated and control animals were fixed in10% buffered formalin for 48 hr. Paraffin-embedded tissuespecimens of brain were sectioned at 4 lm and stained with

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hematoxylin and eosin. In addition, immunohistochemicalinvestigation of the frontal section (A) and section C with thestructures of thalamus, hypothalamus, and striatum wasperformed with antibodies anti-MDMA, -GFAP, -HSP27,-HSP70, -HSP90, -VMAT2, -b-APP, -TrypH, and -GAP-43and TUNEL assay. We used 4-lm-thick paraffin sectionsmounted on slides covered with 3-amminopropyl-triethoxysi-lane (Fluka, Buchs, Switzerland). A pretreatment was necessaryto facilitate antigen retrieval and to increase membrane perme-ability to antibodies: for antibody anti-b-APP (Novocastra,Newcastle upon Tyne, United Kingdom), -TrypH (Novo-castra), and -GAP-43 (Santa Cruz, CA) boiling in 0.1 M citricacid buffer; for antibody anti-HSP27, -HSP70, and -HSP90,(Novocastra) boiling 0.25 M EDTA buffer; for antibody anti-VMAT2 (Chemicon, Temecula, CA) 5 min proteolyticenzyme at 208C (Dako, Copenhagen, Denmark); no pretreat-ment was necessary for antibody anti-GFAP (Dako, Copenha-gen, Denmark). For TdT enzyme the sections were immersedin proteinase K (20 lg/ml of TRIS) for 15 min at 208C. Theprimary antibody was applied in a 1:20 ratio for HSP27; in a1:50 ratio for VMAT2, HSP90, and TrypH; in a 1:100 ratiofor HSP70; in a 1:300 ratio for GFAP and b-APP; and in a1:2,000 ratio for GAP43. The incubation of primary antibodywas for 120 min at 208C for anti-VMAT2, -GFAP, -HSP27,-HSP90, and -GAP 43 and overnight for anti-b-APP,-HSP70, and -TrypH. For TUNEL assay (Chemicon, Teme-cula, CA), the sections were covered with the TdT enzyme,diluted in a ratio of 30% in reaction buffer (Apotag Plus Per-oxidase In Situ Apoptosis Detection Kit; Chemicon) andincubated for 60 min at 388C. Monoclonal antibodies thatspecifically recognize MDMA and MDAA were kindly sup-plied by Microgenics GmbH Products Europe (Passau, Ger-many): they were purified from mouse ascites and availablein two clones (clones 1A9 and 5C2; De Letter et al., 2003).Therefore, primary antibody anti-MDMA was tested onbrain rat samples treated with MDMA, without pretreatmentand with various pretreatments (boiling in 0.25 mM EDTAbuffer; boiling in 0.1 M citric acid buffer; ProteolyticEnzyme T 208C for 5 min; proteinase K for 15 min at208C) and at various concentrations (ratio 1:20, 1:50, 1:100,1:500, 1:1,000, 1:2,000). The samples tested were examinedunder a light microscope, which detected the best reaction.We used the primary antibody anti-MDMA before a pre-treatment with boiling in 0.25 mM EDTA buffer, in a ratio1:100, with an incubation for 120 min at 208C. The detec-tion system utilized was the LSAB1 Kit (Dako, Carpinteria,CA), a refined avidin-biotin technique in which a biotinyl-ated secondary antibody reacts with several peroxidase-con-jugated streptavidin molecules. The positive reaction wasvisualized by 3,3-diaminobenzidine (DAB) peroxidation,according to standard methods. Then, the sections werecounterstained with Mayer’s hematoxylin, dehydrated, cov-erslipped, and observed under a Leica DM4000B opticalmicroscope (Leica, Cambridge, United Kingdom). The sam-ples were also examined under a confocal microscope, and athree-dimensional reconstruction was performed (True Con-focal Scanner; Leica TCS SPE).

For semiquantitative analysis, slides were scored in ablinded manner by two observers (M.N., I.R.). Staining pat-

tern within each sample was categorized as absent (–), weak(1), moderate (11), or intense (111).

GC-MS Analysis of MDMA and MDAA

MDMA and MDAA were analyzed according a GC-MS method described by Peters et al. (2003). The analyteswere analyzed by gas chromatography/mass spectrometry inthe selected-ion monitoring mode after mixed-mode solid-phase extraction (HCX) and derivatization with heptafluoro-butyric anhydride. The method was fully validated accordingto international guidelines (De Letter et al., 2002a,b). It waslinear from 5 to 1,000 lg/l for all analytes. The limit of quan-tification was 5 lg/l for all analytes.

Statistical Analysis

Values are presented as means 6 SD. The unpairedtwo-way Student’s t-test was used to compare the resultsobtained for treated rats with the control group. P < 0.05 wasaccepted as indicative of significant difference among groups.

RESULTS

Enzymatic and Nonenzymatic AntioxidantCellular Defense System Evaluation

The acute administration of MDMA produces adecrease of GSH/GSSG ratio and oxidative stress in allbrain areas examined. SOD activity was significantly re-duced after 3 hr in hippocampus (–60.7%) and after 6 hrin striatum, hippocampus, and frontal cortex (–43.3%,–86.1%, and –23.4%, respectively). GR and GPxactivities were reduced after 3 hr (–22%) and after 6 hr(–33.3%) in frontal cortex (Fig. 1). AA levels stronglyincreased in striatum, hippocampus, and frontal cortexafter 3 hr (1159%, 184%, and 17.6%) and 6 hr(1162%, 1154%, and 123.4%), respectively. Highlevels of MDA with respect to control were measured instriatum after 3 hr (1276%) and 6 hr (1162%) and inhippocampus (171.8%) and frontal cortex (118.22%)after 6 hr (Fig. 2). The numerical values are summarizedin Table I.

Morphological Findings

The microscopic evaluation of the sections stainedwith H&E revealed the following: group III (24 hr): redneurons, nuclear shrinkage, and in the two dead rats(4 hr) perivascular hemorrhages; group I (16 hr): cyto-plasmic hypereosinophila; group I (6 hr): moderate peri-neuronal edema and initial neuronal cytoplasmic hyper-eosinophilia. The immunohistochemical study of thesamples, for each antibody revealed (Table II) the fool-owing.

Anti-MDMA. A markedly positive reaction wasnoted at the basal ganglia and thalamus in rats sacrificedat 6 hr (group I) after treatment. A positive reaction,gradually weaker, but more uniform, both in the frontalcortex and at the level of the striatum, hippocampus,and thalamus was reported in rats sacrificed after 16 hr(group II) and 24 hr (group III; Fig. 3).

Rat Brain Responses to Ecstasy 907


TrypH. TrypH did not provide significantresults, except for a weak positive reaction in the exter-nal cortical layers of rats sacrificed after 24 hr (groupIII); the other samples showed a constant negativity.

VMAT2. We found a strong positive reaction tothe VMAT2. In detail, we reveal that 1) group I (6 hr)had a positive reaction, particularly in thalamus areas; 2)group II (16 hr) had a weak and uniform positive reac-tion in the frontal section, in the basal ganglia, and inthalamic areas; c) group III (24 hr) had a marked positivereaction in the deep layers (Fig. 4).

HSP 27, HSP 70, HSP 90. A cortical positivitylocated in the most superficial layer was revealed onlyfor HSP70 in rats of group I (6 hr). Positivity, graduallyincreasing, was observed, in both sections, in the groupII (16 hr) and rats sacrificed after 24 hr (group III); thereaction was localized exclusively in the cortex but witha more extended involvement of the cortical layers (Fig.

5). A constant negativity was noted in response toHSP90 and -27.

Apoptosis (TUNEL). A weak positive reactionwas shown in the deep layers of rats sacrificed at 6 hr(group I) and 16 hr (group II) after treatment. A markedpositivity was revealed in the superficial layers (or corti-cal) of rats sacrificed after 24 hr (group III; Fig. 6). Allgroups showed a negative reaction to GAP43, b-APP,and GFAP.

MDMA plasma levels. The MDMA plasmalevels decreased dramatically with respect to the valuesdetected during the first 6 hr after i.p. administration; inrats that died after 4 hr, concentrations were similar tothose observed at hour 6 (Table III).

DISCUSSION

This study demonstrates that the administration ofa single dose of MDMA (20 mg/kg ip) was able to alter

Fig. 1. GR and GPx activities were reduced after 3 hr (–22%) and after 6 hr (–33.3%) in frontal cortex.SOD activity was significantly reduced after 3 hr in hippocampus (–60.7%) and after 6 hr in striatum,hippocampus, and frontal cortex (–43.3%, –86.1%, and –23.4% respectively).

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significantly the antioxidant defense system, producingan oxidative stress that may result in lipid peroxidation,with consequent deterioration of the homeostasis ofCa21 and neuronal damage. Numerous mechanismshave been suggested to be responsible for MDMA toxic-ity, such as oxidative stress, metabolic compromise, andinflammation (Cerretani et al., 2008). The role of oxida-tive stress in mediating MDMA toxicity is further illus-trated by a decrease in the activity of the endogenousantioxidants gluthatione peroxidase, catalase, and super-oxide dismutase observed after MDMA administration(Yamamoto and Raudensky, 2008). Although decreasesin endogenous antioxidant enzyme may mediateincreased free radical production, acute increases in DAhave been shown to play a predominant role in theincreases in oxidative stress associated with MDMAadministration. The generation of free radical speciessubsequent to increases in DA is derived mainly from

autooxidation of DA or enzymatic oxidation by monoa-mine oxidase (MAO) to result in the production ofsuperoxide and hydrogen peroxide (Yamamoto andRaudensky, 2008). Furthermore, MDMA depleted intra-cellular GSH levels in a concentration-dependent man-ner, an effect that was attenuated by N-acetylcysteine,an antioxidant and GSH precursor that also reducedMDMA-induced toxicity in neuronal cultures from ratcortex (Capela et al., 2007b). It has been described(Capela et al., 2007a) that thioether MDMA metabolitestime dependently increased the production of reactivespecies, concentration dependently depleted intracellularGSH and increased protein bound quinones, and finallyinduced oxidative stress and neuronal death. It is postu-lated that hepatic metabolism is a key factor in the pro-duction of MDMA-induced neurotoxicity to 5-HT-containing neurons. The mechanisms by which MDMAcauses its deleterious effects have yet to be completely

Fig. 2. AA levels increased in striatum, hippocampus, and frontal cortex after 3 hr (1159%, 184%, and117.6%) and 6 hr (1162%, 1154%, and 123.4%, respectively). High levels of MDA were measuredin striatum after 3 hr (1276%) and 6 hr (1162%); in hippocampus (171.8%) and in frontal cortex(118.22%) after 6 hr. Acute administration of MDMA produces a decrease of GSH/GSSG ratio andoxidative stress in all brain areas examined.



clarified. However, there is good evidence to suggestthat the mechanism of neurotoxicity involve the forma-tion of MDMA metabolic products such as N-methyl-a-methyl dopamine (MeDA), orthoquinones, and quinonethioethers (Thiriet et al., 2002); MeDA is unstable and ismetabolized in the presence of NADPH into a quinonethat forms an adduct with GSH. The cathecol thioetermetabolites are capable of redox cycling and generatingROS, which is a key feature of MDMA-induced neuro-toxicity (de la Torre and Farre, 2004). GSH and relatedenzymes participate in the protection of neurons from avariety of stresses, and changes in GSH metabolism havebeen associated with neurodegenerative processes of thebrain (Erives et al., 2008).

In our experiment, after a single MDMA injectionin rats, we observed presence of oxidative stress in stria-tum, hippocampus, and frontal cortex, with alteration of

antioxidant enzymes, decrease of GSH/GSSG ratio, andincrease in MDA levels. The alteration of the antioxi-dant defense system starts from the early hours (acutephase or reversible) with a subsequent gradual increase,thus giving relief not only to the formation of free radi-cals but also with regard to the MDMA metabolites indetermining neuronal toxicity. Although there is amplescientific evidence confirming MDMA-induced seroto-nergic depletion, few studies have so far examined theeffects of MDMA on markers of neurotoxic damage oron the possible morphological markers indicative ofMDMA-induced encephalic damage and their expressionhistory.

The experimental use of anti-MDMA has allowedus to reveal the encephalic distribution of the substance.In this regard, there has been a unique study on ence-phalic human tissue, taken from two MDMA-related

TABLE I. Enzymatic and Nonenzymatic Antioxidant Cellular Defence System Evaluation Expresses in Numeric Values

Control 3 Hours 6 Hours

MDA frontal cortex (nmoli/g) 13.23 6 2.151 13.31 6 2.709 15.64 6 0.384a

MDA hyppocampus 9.18 6 2.227 12.05 6 4.341 15.77 6 5.015a

MDA striatum 7.06 6 1.420 20.60 6 8.069a 18.53 6 7.428a

AA frontal cortex (lmol/g) 443.37 6 48.305 521.46 6 25.803a 547.06 6 47.456a

AA hyppocampus 233.00 6 124.112 427.91 6 61.748a 592.98 6 87.833c

AA striatum 143.583 6 14.055 353.125 6 66.282c 420.130 6 92.395c

GSH/GSSG ratio frontal cortex 17.588 6 7.421 6.801 6 2.084b 5.864 6 0.928c

GSH/GSSG ratio hyppocampus 10.747 6 4.665 4.173 6 1.280b 4.679 6 4.782a

GSH/GSSG ratio striatum 7.391 6 2.261 4.038 6 0.669a 4.249 6 0.771a

GPx frontal cortex (U/mg protein) 17.353 6 2.075 15.070 6 3.979 11.744 6 2.303c

GPx hyppocampus 17.601 6 3.864 19.440 6 2.029 18.908 6 7.304

GPx striatum 15.122 6 4.633 19.191 6 1.771 19.439 6 6.354

GR frontal cortex (U/mg protein) 24.878 6 4.694 19.409 6 2.402a 21.345 6 1.679

GR hyppocampus 25.208 6 5.389 29.659 6 6.013 27.366 6 1.037

GR striatum 21.733 6 6.285 25.668 6 3.485 29.446 6 4.759

SOD frontal cortex (U/mg protein) 1.746 6 0.479 2.103 6 0.301 1.335 6 0.109d

SOD hyppocampus 2.090 6 0.611 0.820 6 0.409c 0.287 6 0.220c–e

SOD striatum 1.568 6 0.491 1.448 6 0.416 0.893 6 0.434

aP < 0.05 compared with control group.bP < 0.02 compared with control group.cP < 0.01 compared with control group.dP < 0.001 compared with the 3-hr group.eP < 0.05 compared with the 3-hr group.

TABLE II. Semiquantitative Analysis: Staining Pattern Within Each Sample Was Categorized as Absent (–), Weak (1), Moderate

(11), Intense (111)

Group III (24 hr) Group II (16 hr) Group I (6 hr) Control group (IV)

Section A Section C Section A Section C Section A Section C Section A Section C

Anti-MDMA 1 1 11 11 111 111 – –

TrypH 1 1 – – – – – –

VMAT2 111 111 11 11 11 11 – –

HSP 70 111 111 11 11 1 1 – –

Apoptosis (TUNEL) 111 111 11 11 11 11 – –

GAP-43 – – – – – – – –

b-APP – – – – – – – –

GFAP – – – – – – – –

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fatal cases, in which the use of the same antibodyallowed to show a marked immunoreactivity corre-sponding to neurons in all cortical regions, basal ganglia,hypothalamus, hippocampus, cerebellar vermis, andwhite matter (De Letter et al., 2003). Our study,although confirming the topographic distribution ofMDMA, revealed a different location of the substancewith time, with a markedly positive reaction in the basalganglia and thalamus in rats sacrificed at 6 hr (group I)after treatment, whereas a weaker uniform positivity wasseen for the frontal cortex, striatum, hippocampus, andthalamus in the second (16 hr) and third (24 hr) groups.

It is well known that MDMA reduced the activityof TrypH, the rate-limiting enzyme in the synthesis ofthe neurotransmitter serotonin, converting 5-hydroxy-tryptophan to serotonin. In the literature, the deficit inactivities of TrypH occurred in the rat neostriatum, hip-pocampus, hypothalamus, and cortex. The negative

expression of anti-TrypH in our study was explained bythe MDMA-induced serotonergic deficit probably beingdue to adaptive changes of gene expression and functionof proteins, expression of a metabolic state of quiescenceor exhaustion, rather than a neurotoxic damage. More-over, the scientific work refers only to radioimmuno-logic dosage, without providing any data on tissueexpression of antibody itself, in reference to the adminis-tration of MDMA.

A strong positive reaction to the anti-VMAT2was observed. Although there are no reports on the tis-sue expression (by immunohistochemical reaction) ofVMAT-2 after administration of MDMA, the findingsfrom our study are in agreement with those reported inthe literature. After administration of methamphetamine,there is a fast (1–48 hr) decrease in cytoplasmic functionof the transporter VMAT2 corresponding to the stria-tum, associated with a reduced cytoplasmic reactivity

Fig. 3. A: Low-magnification views of sections of brain that wereincubated with anti-MDMA: markedly positive reaction was noted atthe basal ganglia and thalamus in rats sacrificed at 6 hr. B: Weakerbut uniform positivity was found in the frontal cortex, striatum, hip-

pocampus, and thalamus of the second (16 hr) and third (24 hr)groups (C). D: Confocal laser scanning microscopy showed markedlypositive cytoplasmic reaction (in green) on the basal ganglia and thal-amus in rats sacrificed at 6 hr (group I) after treatment.



(Chu et al., 2008). In our study, this finding is welldefined in relation to the times at which the rats weresacrificed.

High doses of MDMA lead to an excessive sympa-thetic activation, and hyperthermia can induce a physio-logical dysregulation sufficient to produce nonspecificneuronal damage, involving not only the serotonergiccells (Baumann et al., 2007), as evaluated from theresponse obtained by immunohistochemistry using anti-bodies to HSP27, HSP70, and HSP90. The literatureincludes only one study conducted on rats treated withMDMA in different doses and sacrificed after 3 days,which revealed a positive reaction to HSP27, located atall cortical areas and hippocampus (Adori et al., 2006).We observed a cortical positivity for HSP70, located inthe most superficial layers, after 6 hr and graduallyincreasing after 16 and 24 hr. A constant negative reac-tivity was noted in response to HSP90 and -27.

We did not find a positive reaction for GAP43, amarker of synaptic plasticity and a key regulator in nor-mal pathfinding and arborization of serotonergic axonsduring early brain development. GAP43 is frequentlyused as a marker for sprouting, because it is located ingrowth cones, is maximally present during nervous sys-tem development, and is reinduced in injured andregenerating neural tissues. The role of GAP43, still tobe precisely defined, may be connected to intracellularregulation events, which allows the growing axon toadapt to the changing environmental framework. Thus,negative expression could be due to the neurodegenera-tive timing (Casoli et al., 2004). We found no b-APPexpressivity (a transmembrane glycoprotein type 1expressed preferentially in the brain in various isoformsand used by many authors as an immunohistochemicalmarker of the axonal damage in different etiopathogene-sis), indicative of the absence of early axonal damage.

Fig. 4. Immunohistochemical visualization of anti-VMAT2. A: Strong positive reaction in the thalamusareas at 6 hr (B). C: Group II (16 hr) showed a weak but uniform positive cytoplasmic reaction in thebasal ganglia. D: Confocal laser scanning microscopy: Group III (24 hr) showed a marked positive nu-clear reaction (in green) in the deep layers.

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Massive doses of MDMA are associated with highlevels of GFAP in different brain areas, but this increaseis not related to the degree of serotonergic depletion.Positivity to GFAP, expression of reactive gliosis, andastrocytic response resulting from neuronal damage aresometimes located only in areas corresponding to thehippocampus (Adori et al., 2006), but after repeated dos-ing with methamphetamine, not MDMA, and after lon-ger times from sacrifice. In other studies, no glial reac-tion was reported after administration of MDMA, imply-ing an important distinction between the effects ofmethamphetamine and those of MDMA (Baumannet al., 2007). Our results are indicative of the absence ofreactive astrocytes (Sharma and Ali, 2008).

Finally, in agreement with the literature, althoughreferring only to an immunohistochemical reaction forcaspases 3 and 1 (Capela et al., 2007b), we found a grad-ually increase in apoptosis (TUNEL) from 6 hr to 24 hrafter MDMA administration (Neri et al., 2007). A dos-

age effect is suggested by the fact that at lower doses theeffects of MDMA may be repaired by factors such asHSP70 (Fornai et al., 2004). At higher doses, repairmechanisms may be overcome, and irreversible damageresults (Granado et al. 2008).

In conclusion, although oxidative stress and theproduction of ROS are already established in the earlyhours after administration of a single dose of MDMA (3and 6 hr) or in the so-called acute or reversible phase,there are very few morphologic changes in the brain,except for sporadic and limited hemorrhage, a gradualand progressive neuronal cytoplasmic hypereosinophilia,and an increase in apoptotic phenomenon in the samelocations where it has been shown that there is moreaction of MDMA. Our study has demonstrated that oxi-dative stress is responsible for MDMA-selective neuro-toxicity from the early stages, with gradually increasingneuronal alterations demonstrated with the most com-mon immunohistochemical methods.

Fig. 5. A: Low-magnification view of HSP70 immunolabeling located in the most superficial layer wasrevealed after 6 hr (B). C: In group II (16 hr) and in rats sacrificed after 24 hr (D), positivity was local-ized exclusively in the cortex but with a more extended involvement of the cortical layers.



Fig. 6. A: A marked positivity was revealed in the superficial layers of rats sacrificed after 24 hr. Weakapoptotic reaction in the deep layers of rats sacrificed after 16 hr (C). B: Marked positivity was revealedin the superficial layers of rats sacrificed after 24 hr. D: Confocal laser scanning microscopy shows amarked positive nuclear reaction (in red apoptotic bodies) after 24 hr.

TABLE III. MDMA and MDAA Plasma Levels

Groups Total rats Number MDMA (lg/ml) MDAA (lg/ml)

6 Hours 1 0.368 0.032

6 Hours 2 0.297 0.029

6 Hours 3 0.310 Not detected

6 Hours 7 4 0.153 0.014

6 Hours 5 0.814 0.060

6 Hours 6 0.448 0.038

6 Hours 7 0.523 0.041

16 Hours 8 0.174 0.019

16 Hours 9 0.101 0.013

16 Hours 10 0.099 0.009

16 Hours 7 11 0.141 0.013

16 Hours 12 0.122 0.012

16 Hours 13 0.130 0.011

16 Hours 14 0.206 0.022

24 Hours 15 0.032 0.008



24 Hours 7 18 0.018 Not detected


24 Hours, died at 4 hr 20 0.330 0.030

24 Hours, died at 4 hr 21 0.390 0.038

Control 4 22-25 Not detected Not detected

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