Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Z ooo t',,,,,,.,;,,,1>,. ........ _ 4//,.,_,h_, ,,.,,.,,,.,; Ecstasy-induced _ _[iver toxicity in rat liver Beitia G, Cobreros A. Sainz L, Cenarruzabeitia ity in rat liver. Liver 2000: 20: 8-15. C: Munksgaard, 2(/00 E. Ecstasy-induced toxic- Abstract: BackgroundAims." The aim of the present study was to examine the effects of single and repeated administration of 3.4-methylenedioxymethamphetamine (MDMA, "ecstasy") on rat liver. Mefllods." Animals were given an acute (20 mg/kg) and repeated (20 mg/kg, b.i.d., for 4 consecutive day's) intraperitoneal dose of MDMA. and at various times after administration the hepatic and serum determinations were made. Resuhs. The effect of acute MDMA administration included increased triglyceride and cholesterol levels and an increase in all enzyme activities 6 h post administration. The toxic effect of MDMA was also observed in other hepatic processes. Gly'cogen content showed a marked decrease, which was accompanied by' a decrease in serum glucose levels. No significant changes in lipid peroxidation and hepatic GSH content were observed. In contrast, multiple MDMA administration produced some evidence of oxidative stress, namely, increased MDA content and decreased GSH content, a small decrease in liver glycogen at 3 h recovering 6 h post dose. no effect on blood glucose and increased AST and ALP activities but no effects ' on ALl" activity. Seven days after the last MDMA injection a tendency towards recovery was shown. Com'lusiom Our results show that the liver . toxicity' caused by' MDMA administration involves several mechanisms, 3,4-methylenedioxymethamphetamine (MDMA) commonly' known as "ecstasy" is a synthetic amphetamine derivative that rapidly became a widely abused drug (1). Patented in 1914 by the E. Merck Company as an appetite suppressant, it was never marketed, but in the 1970s and early 1980s the drug gained popularity as an adjunct to psychotherapy (2). Since MDMA's classification in 1985 as a Schedule I drug under the Controlled Substances Act (3), there has been an increase in clandestine synthesis and illicit use which has been accompanied by an equivalent increase in the number of MDMA toxicity reports. MDMA intoxication is predominantly associated with the ability of the drug to destroy serotonergic nerve terminals (4-7), but there are some reported cases of severe toxicity resulting from recreational misuse of small amounts of MDMA in which the pattern of toxicity included cardiac arrhythmias, fulminate hyperthermia, convulsions, disseminated intravascular coagulation, rhabdomyolysis, and acute renal failure (8-10). Hepatotoxicity effects have only recently been noted. However, there is increasing evidence that "ecstasy" is hepatotoxic, and liver changes have been reported following bi8 be ,'4--', o.- ! G. Beitia, A. Cobreros,L. Sainzand E. Cenarruzabeitia Department of Pharmacology, University of Navarra. 31008 Pamplona, Spain Key words: 3,4-methylenedioxymethamphetamine(MDMA)- liver - lipid per0xidation - reducedglutathi0ne(GSH)- glycogen Dr.E.Cenarruzabeitia. University of Navarra, SchoolofPharmacy, c/Irunlarrea 1, 31008Pamplona, Spain Received 23 October1998.acceptedfor publication5 August1999 opsy, which varied from foci of individual cell necrosis to centrilobular necrosis, hepatitis and extensive fibrosis (1 1-13). The principal evidence of hepatic lesions, resulting from MDMA in humans, is an unexplained jaundice or hepatomegaly (14, 15). Acute hepatitis has also been associated with repeated use of ecstasy (16, 17). The aim of the present study was to obtain evidence for the mechanism involved in MDMA-induced hepatotoxicity in rats. For this purpose, several parameters of rat liver function were determined. Materials and methods Drugs used The MDMA was obtained from the Audiencia Provincial de Navarra. All other reagents were of the highest grade commercially available and were obtained from E. Merck (Darmstadt, Germany) and Sigma Chemical Company (St. Louis, MO, U.S.A.). Catalase, glutathione reductase, glutathione peroxidase, glutathione-S-transferase and superoxide dismutase were purchased from Randox Laboratories Ltd. (Crumlin, UK). Ecstasy-induced injury Animalsand treatments Measurementof hepatic reducedglutathione(GSH)levels Male Wistar rats (200-220 g) were housed in plastic cages in a room with a controlled temperature (22°C) and maintained on a 12 h light-dark cycle with free access to food and water. Two different schedules of treatment with MDMA were used in this study. Acute treatment consisted of a single dose of MDMA (20 mg/kg, i.p.), the rats being sacrificed 3 h and 6 h later. Repeated treatment consisted of the same dose of MDMA (20 mg/kg, i.p.) which was given b.i.d, for 4 consecutive days, the rats being sacrificed 3 h, 6 h and 7 days later, The dose of MDMA (20 mg/kg, i.p.) was chosen because it is in a similar range of doses (10-20 rag/ kg) that have been shown to produce neurotoxicity in rats (17). MDMA dose refers to the hydrochloride. The time points (3 h, 6 h and 7 days) were selected because of the reported neurotoxic effects (18). Hepatic GSH content was estimated by a colorimetric method as previously described by Ellman (21). Samples of liver (0.3 g)were homogenised in 6 ml of 10% Trychloroacetic acid, containing 5 mM sodium ethylenediamine tetraacetic acid (EDTA), and later centrifuged at 3000×g for 15 min. The volume of the supernatants was fixed in 10 ml by addition of phosphate buffer (pH 7.4). Aliquots of 1.0 ml were taken and diluted to 10 ml with phosphate buffer. Then, 3.750 ml of these dilutions was mixed with a solution of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (0.08mg/ml in phosphate buffer) and incubated in a bath at 37°C for 30 rain. After cooling, the absorbance of each sample was determined at 412 nm, and the GSH concentration of these samples was calculated with a standard curve using a dissolution of GSH (0.12 mg/ml) as standard. Measurement of lipid peroxidation Measurement of antioxidant enzymatic activities: superoxide dismutase (SOD), glutathione peroxidase (GPx), The extent of lipid peroxidation, which can be measured by the formation of malondialdehyde (MDA) after the breakdown of polyunsaturated fatty acids, was measured by assay for thiobarbituric acid reactive substances (TBARS) at 535 rim, as described by Buegue and Aust (19). Briefly, liver samples were homogenised with 50 mM Tris buffer, pH 7.5. Then, 2 ml of TBA reagent (0.375% w/v thiobarbituric acid, 15°/,, w/v trichloroacetic acid in 0.25 N HC1) was added to a fraction from the homogenate samples. The mixture was heated for 10 min in a boiling water bath, allowed to cool and then centrifuged at 1000Xg for 10 rain. TBARS are presented as malondialdehyde equivalents, calculated using an extinction coefficient of 1.56×105 M-_/cm -_ Liver protein content was evaluated by the method of Bradford using bovine serum albumin (BSA) as standard (20). glutathione-S-transferase (GST), glutathi0ne reductase (GR)and catalase (CAT) Animals were sacrificed by decapitation and their livers quickly removed and placed in homogenisation medium (250 mM mannitol, 70 mM sucrose, 1 mM EDTA adjusted with Tris to pH 7.4). One part of the liver was immediately homogenised in a Potter-Elvehjem glass homogeniser (1 g of tissue plus 10 ml homogenisation medium). Unbroken cells, cell debris, and nuclei were sedimented at 800×g for 10 min, and the supernatant was used as homogenate. This was kept on ice while being sonicated at 300 w for 30 secondsin three 10 secondsintervals and centrifuged at 20000x g for 10 min. Aliquots ( 1.0 ml) of the resulting supernatant were pipetted into plastic tubes, stoppered, and stored at -70°C until assayed. The enzyme activities of SOD, GR, GPx, GST and CAT were measured using a Cobas MIRA clinical analyser (Roche Diagnostic Systems, Inc., Basel, Switzerland). Analyses, and calculations were performed automatically accord- Measurement of hepatic triglyceridesand cholesterol levels ing to the programmed instructions. Animals were sacrificed by decapitation and their livers immediately removed and placed in homogenisation medium (250 mM mannitol, 70 mM sucrose, 1 mM EDTA adjusted with Tris to pH 7.4). One part of the liver was immediately homogenised in a Potter-Elvehjem glass homogeniser (1 g of tissue plus 10 ml homogenisation medium). Cholesterol and triglycerides from this homogenate were measured by automated assays using Hitachi kits for each determination(HITACHl 704analyser). Assay of serum enzymatic activities and glucose serum levels In order to determine serum transaminase and alkaline phosphatase activities and glucose serum levels, animals were anaesthetised using sodium pentobarbital, 50-60 mg/kg body weight, and blood samples were removed from the retro-orbital sinus. The enzymatic activities and glucose levels were measured by automated enzymatic assays 9 deitia et ai. ElControl r_MDMA3 h NMDMA6 h o .'-',× < ._ 7" DControl I_MDMA3h t:1MDMA6h NMDMA7 d 2] 1.5' L "r Acute treatment (20 mg/kg, - .J.* i,.ll._ 111.4 _.... Results Effectof MDMA onlipidperoxidation ///,, II1"1 /,1./,' h'll tffJ _5_ 555.I I//l "1"" _//'/_ Repeatedtreatment 1 ,_ < _ 0.5 _" _ _ Fie, 1. Effect of single test (ANOVA), followed b\ the Scheffc test. The minimuna number of animals used to determine mean values and statistical differences was eight for each treatment. i.p.) and repeated A single dose of MDMA (20 m_/kg, i.p.) did not cause any significant change in lipid peroxidation. However, MDMA (20 mg/kg, i.p., b.i.d., for 4 consecutive days) produced an increase in lipid peroxidation, the eflect being significant 3 h (18%).and 6 h (26%) after last injection. Seven days later, MDA levels returned to control values (Fig. 1). (20 mg/kg. i.p., b.i.d.. 4 consecutive days) administration of MDMA on lipid peroxidation in rat liver. Animals were sacrificed 3 h, 6 h and 7 days after the last MDMA administration. Data are the Effect of MDMA on hepatic triglycerides and cholesterol mean__SEM A series of experiments was performed to evaluate hepatic levels of triglycerides and cholesterol after group of 8-10 (one-way rats. analysis *p<0,05, **p<0.01 of the variance vs. to control followed by Scheffe test), using Technicon-Ba,xer kits for each enzyme (Technicon R.A.1000 autoanalyser), Measurement of hepatic glycogen content Hepatic glycogen content was determined according to the method of McGarry and Kawajima (22). Hepatic samples (0.3 g) were digested with 5 M KOH and were precipitated with ethanol. Later, samples were centrifuged at 1000Xg for 15 min. An aliquot of the precipitate was mixed with 5 m] of anthrone reagent (0.05% w/v anthrone, 1% w/v thiourea in sulfuric acid __7"_'"'_,, v/v). The colour was developed by' placing the tubes in a boiling bath for 10 min. The absorbance of each sample was determined at 620 nm and the glycogenconcentration of these samples was calculated with a standard curve using a glycogen kit (E. Merck, Darmstadt, Germany). levels acuteand repeatedMDMAadministration.Acute MDMA treatment caused a significant increase in hepatic triglycerides and cholesterol contenL the effect being significant 6 h after drug injection. Repealed administration of MDMA produced a significant increase in cholesterol levels, both 3 h and 6 h after drug administration, which returned to control values seven days later. However, no significant change in hepatic triglycerides content was observed after repeated MDMA treatment at any' time of sacrifice (Table 1). Effect of MDMA on reduced glutathione (GSH) hepatic content A single dose of MDMA did not cause any significant change in hepatic GSH content. The effect ! Table 1. Effect of single and repeated administration of MDMAon choiester0i and triglycerides content inratliver Survival Treatment Histology Statistical analysis Data were reported as mean_ + SD and were ann- lysed using the one-way analysis of the variance 10 Cholesterol Triglycerides (mg/g liver) (mg/g liver) Control 0.93±009 8.81-_-0,48 MDMA MDMA Control MDMA MDMA MDMA 1.06+_0.10 9.91=0.41 1.4±0.10" 11,12:0,50" 0.78_*0.09 943+-0.29 1.11+_0.05"*10.24±0.64 1.40_.0.05"**8.78=0.88 0.85*_0.04 7.19+_0.26 time Single Immediately after the sacrifice of the animals, liver sections were excised and immersed in 10% buffered neutral formalin for 24 h. Fixed portions were then processed, embedded in paraffin blocks, sectioned at about 5 #m, mounted on glass slides, and stained with hematoxylin-eosin and Masson stains. 3h 6h Repeated 3h 7 6h days _, Animalsreceivedsaline (control group), MDMA(20 mg/kg i.p.) or MDMA(20 mg/kg i.p., b.i.d for 4 days) for single or repeated treatment respectively. Rats were sacrificed 3 h, 6 h and 7 days after the Last MDMA injection. Valuesare mean±SEM from 8-10 rats. *p<0.05. **p<0.01 ***p<0.001 vs control group using one-way analysis of the variance lollowed by Schefie test. ' Ecstasy-induced injury FlControl [3Control EIMDMA mMDMA 3h WMDMA6h A 1.6. 1-. 'g" 1.2 "-_ g tathione reductase in liver were determined in rats alter single or repeated MDMA administration. Liver enzymes behaved differently. With the exceptionof GPx, the catalyticactivitiesof the antioxidant enzymes in tile liver did not show any significant change between different groups (data not shown). However, MDMA (20 mg/kg, i.p.) caused a slightdecreasein glutathioneperoxidase(GPx) activity,the effectbeingsignificant7 daysafter repeated MDMA treatment (Fig. 2B). 3 h t':IMDMA 6h WMDMA 7d T i 0.8 ,//,,r .... 0.4- Effect of MDMAon serum transaminase and alkaline phosphatase activities Acute treatment B Repeated treatment Y 1.5. r-J :/:r i:i , :::_: : :::::. _ :::: ::::: :::: :ii 0 _:, ..... Acutetreatment ! 0.5' Fig. 2. Effect " " "• •: of single (20 m_kg, on serum ALT, AST and ALP activitiesis shownin Table2. AST activity showedan increasingtendencyafter acuteMDMA treatment, but the significant increase became evi- 2- ,:, The effect of MDMA _/,-_ , dent 6 h after drug administration. Repeated MDMA treatment, causeda significantincreasein (/Z, ,/// "" ,,-,-, _, [] Control EtMDMA 3 h EMDMA 6 h ¢//. Ill* A rnControl E_MDMA3 h t':1MDMA6 h IIMDMA 7 d 5 Repeatedtreatment i.p.) and repeated "" 4 (20 m_kg, i.p., b.i.d., 4 consecutive days) administration of MDMA on GSH content (A) and on glutathione peroxidase activiD (B) in rat liver. Animals were sacrificed 3 h, 6 h and 7 days - after the Z r'n N Last MDMA administration. Data are the mean+_SEM rats. *p<0.05. **p<0.01 xs. to control group lone-way of the _ariance follo_ed b._ Scheffe test). _ _ _ "_ of 8 10 analysis 3 _0 2 * 1 0 of repeated MDMA treatment was more marked. A significant reduction of approximately 22% and 27% in GSH hepatic content was found 3 h and 6 h respectively after the last injection. Seven days later, hepatic glutathione levels returned to control values (Fig.2A). i Acute treatment B Repeatedtreatment 240 180 - _a r,_ ,..-, Effectof MDMAon antioxidanthepaticenzymeactivities To establish the mechanism of toxicity as oxygen radical-mediated, there are a number of direct and indirect methods that can be employed. Direct methods include the measurementof superoxide hydrogen peroxide, or hydroxyl radical. These species are very reactive and their quantitation can be difficult. Therefore, indirect methods of study are often used. One such method is the measurement of changes ill endogenous antioxidant enzyme activitv. , In this sense, the five principal antioxidant enzymes superoxide dismutase, catatase, glutathione peroxidase, glutathione-S-transferase and glu- { "I" 8 "1- . "l- "_ 120 _ 60 : 0 Acute treatment (20 m_kg. Fig. 3. Effect of single i.p., b.i.d., hepatic 4 consecutive gl?,cogen content days) Repeatedtreatment i.p.)and repeated administration (A) and on glucose (20 mjkg. of MDMA serum on lcxels IB) in rat. Animals were sacrificed 3 h.the 6 hmean_-SEMof and 7 days after8 the last MDMAadmmistration. Data are 10rats. */,<0.05. **p<0.01 the _ariance follov,,ed vs. to control b? Scheffe group (one-way analssis of test). 11 I',eitia el al. Table 2 Effect of single ar)d repeated adminisl)at)orl Treatment ol MDMA on serum ALT AST and ALP activilies Time S)ngle ALTiU/L) in rat AST (U/L) ALP {ULI 3h 6h Control MDMA MDMA 2868 - 2.20 29.33 " 151 4434-537" 8392 : 5.89 86.70:443 133 971478"" 251 67 : 3 67 36117 : 11.76' 384.50-35.59"" 3h 6h Control MDMA MDMA 3200 _125 34.80:204 3100- 321 79.83- 1.45 10200-291"** 98.00-2.55*" 25043 - 10.05 33738-76.99* 336.86' 291" 7days MDMA 33831.28 101 43-3.67"** 38529-32.14"* Repeated Animals received saline ieontrol group). MDMA (20 mg/kg ip ) or MDMA (20 mg/kg i.p bi.d for 4 days) for single or repeatedtreatment respectively.Blood samples were taken 3 h. 6 h and 7 days after the last MDMAinjection Valuesare mean-SEM from 8-I0 rats. "p. 0.05 **p<0.01 "**p- 0.001 vs control group using one-way analysis o1 the variance followed by Scheffe test AST activity. Seven days later AST activit\ remained significantly increased. A single injection of MDMA significantly increased ALl- activity 6 h after drug administration. Repeated MDMA treatment did not elicit an\ significant change in ALT activity at any time of sacrifice, MDMA caused a significant increase in ALP acti\it\ at both. 3 h and 6 h after single or repeated drug administration. Furthermore. seven days acute treatment. Seven days after repeated MDMA treatment, glscogen hepatic levels returned to control values (Fig. 3A). Serum glucose levels after single or multiple administration of MDMA are shown in Fig. 3B. MDMA caused a substantial reduction in the glucose levels6 h after a singleinjection.However, no significant effect on glucose serum levels after repeated MDMA treatment was observed. later. creased. ALP Histological examination activit\ remained significantl) in- Figure 4A shows the liver section of a control rat Effectof MDMAon glycogenhepaticlevelsand glucose serumlevels MDMA caused a significant decrease in glycogen hepatic content, the effect being more marked after showing, in the centre off the photograph, the portal tract. Striking changes were identified in the liver.Acute MDMA treatmentcausedcell necrosis particularly in portal areas with inflammatory infiltrate consisting in lymphocytes and macro- /-7,,,.4 L.i\cr histology sho\_ ing the portnl lracl in the centre of the photograph. Hepatic necrosis with the portal area being infiltrated by I__mphoc._tcs and macrophagcs following administration off NII)M a, &nimal received saline (control group) (A) or MDMA (20 mg/kg, i.p.) (B) being sacriiiccd 6 hours laler. 12 Ecstasy-induced injury Fi%. 5. Liver histology showing the hepatic xein in the centre of the photograph. Hepatic necrosis with inflammatory infihrate around hepatic vein following administration or MDMA. Animal receixed saline (control group) IA) or MDMA {20 mg/kg, i.p.. b.i.d., l\)r 4 days) (B) being sacrificed 6 hours htter. phages denser in portal tracts, 6 h post administration (Fig. 4B). Figure 5A shows the liver section of a control rat showing, in the centre of the photograph, the hepatic vein. Repeated rejection produced cell necrosis and inflammatory infiltrate around the hepatic _ein. These effects were more marked 6 h after the last administration (Fig. 5B) bi,tt seven days later no changes were observed, Discussion Hepatotoxicity is one of the medical consequences of MDMA consumption (14). Jaundice, hepatomegaly, centrilobular necrosis and hepatitis are some of the MDMA-induced liver conditions (11. 14). The most reported form of liver injury is hepatitis (15-17, 23). The mechanism of MDMA-induced hepatic injury is unclear but a spectrum of severity seems to exist as assessed by histological changes varying from mild to moderate lobular hepatitis and to features of massive hepatic parenchymal collapse with areas of nodular regeneration. The severity of liver damage does not seem to correlate with either the amount or frequency of MDMA ingested suggesting an idiosyncratic type of reaction (13). In the present study, the major biochemical events indicative of liver injur,<'after acute and repeated MDMA administration to rats. wereinxestigated. The attack of reactive oxygen species on polyunsaturated fatt\ acids, essential constituents of biological membranes, has been sho_vn to result in peroxidative damage of these lipids (24). Malondialdehyde, the reaction product of lipid peroxidation usually determined in experiments, exerts several biological effects including cross-linking of proteins and nucleic acids, and is presumably mutagenic and carcinogenic. Lipid peroxidation and subsequent cellular damage are regarded as an inaportant mechanism underlying the toxicity of several xenobiotics (25t. Living organisms contain various free radical scavenging systems for the clarification of the pathologic role of free radicals, it is therefore essential to estimate the changes in both the generation and the scavenging of free radicals. The reduced form of glutathione (GSH), the major intracellular thiol, neutralises many kinds of radicals, either directly or in association witl'lglutathione peroxidase (26). Depletion of GSH, especially in the liver, can cause irreversible damage and cell death. GSH plays an important role in protecting cells against reactive 02 intermediates and free radicals. Glutathione deficiency worsens free radical-induced toxicity, while free radical production depletes glutathione. Decreased ghitathione is an early marker of free radical insult and is evident before overt cell death occtlrs (27). A single dose of MDMA did not change glutathione levels, and free radical production from MDMA was not quantitati,,ely significant enough to deplete glutathione and allo,<vuncontrolled reaction with ,,ital cclh.llar components. In contrast, re- 13 Beitia et al, peated MDMA injection produccd a sufficicnl quantity of free radicals to deplete glutathione, Furthermore, glutathione peroxidase activity showed a tendency to decrease after MDMA injection. In addition, not only GSH itself but also tile GSH redox cycle, catalvsed by the enzyme glutathione peroxidase, could contribute to radical scavenging, Numerous studies have documented that decreases in glutathione precede lethal cell injury from free radical generators (28). The leakage of intracellular enzymes, suggests irreversible damage. In this sense, ALl- and AST serum activities as indi- suggested that MDMA could be regarded as a chemical stressor as M DMA-induced neurochcn> teal, behavioural and endocrine aherations closely resemble those elicited by exposure to acute stress (,__). McGuinness et al. established that during chronic stress hormone infusion the rise in epinephrine exerts potent stimulatorv effects on glucose production principally by' enhancing hepatic glycogenolysis (33). Thus, these results SUgGest that MDMA-induced stimulation of glycogenolysis could be associated with the sympathomimetic elfects observed after acute MDMA administration cators of liver injury were determined. Three hours after a single dose of MDMA, no significant changes were observed. In contrast, a significant increase in ALT and AST activities, 6 h after drug administration were obtained. Repeated MDMA treatment produced a significant increase ill AST activity but no effect on ALT activity was observed, In all cases, MDMA produced a disproportionate increase in AST activity compared with ALT activitv. This effect is frequently all index of important cell necrosis. Furthermore, ALP activity as indicatire of cholestasis was determined. MDMA caused an increase in ALP activity, the effect being more marked after a single dose. Seven days after repeated MDMA treatment. ALP activity remained increased. These effects resemble those seen in humans in which increases in AST, ALT and ALP activities have been reported (13. 15.29, 301. These findings were corroborated with histological examination which, according to the post-mortem changes was reported in deaths associated with MDMA intake (12-17). Hepatic necrosis and illflammatory infiltrate were evident and overall findings were consistent with a drug related hepatitis. There appears to be a relationship between glutathione deficiency and glycogen metabolism. Recently: it has been reported that glutathione depletion caused by various drugs stimulates glycoten breakdown (tl). Our results show that (34). Taken together with the present results, the liver toxicity caused by acute MDMA treatment involves several mechanisms which cause increased lipid levels, increased liver enzymes and impaired gluconeogenesis leading to a fall ill blood glucose and depletion of liver glycogen. In contrast repeated treatment produced some evidence of oxidative stress, namely, increased MDA content and decreased GSH content, but no evidence of hepatocyte damage. As far as we know. these results could be the first major step for research in liver injury'. Furthermore. according to the sympathomimetic effects reported after acute MDMA mjeclion (34), these results could be explained by the acute effect of the last dose rather than any longterrn chronic effects. - repeated administration of MDMA (20 mg/kg, i.p.) causes a significant decrease in hepatic glycogen content which is not accompanied by a modification in serum glucose levels. In this sense, the effect on hepatic glycogen content could be explained by the significant GSH depletion observed after repeated MDMA treatment. On tile other hand, 3 h after a single dose off MDMA a 60" ,, reduction of liver glycogen content, witllout additional was tent crease effect on seruna glucose levels observed. A larger reduction of glycogen COlaI)/ (approximately 83,,t and a signiticant deof plasma glucose levels were found 6 h after MDMA. B\ contrast glutathione levels did not change after acute MDMA treatment. It has been recently 14 Acknowledgements We thank Caju Pamplona i\_r a fello_ship 1o one of us (G.B.B.j. We also _ish to thank Mrs. Mariu Luz Muro for her excellent contributions to this work. References 1. BRONSO>, M E. BARRIOS-Z_MI_F,A?VO k. JIANG_i eta]. Behavioural and de\elopmemal effects of t,_o 3.4-moth\len- edioxymethamphetamine (MDMA) derixatixes. Drug AIcoholDepend1994: 36:161 6. 2 GREER G. TOLBERI R. Subjeclixe reporls of Ihe effects of MDMA in a clinical setting..1 Psychoactixe Drugs 1986: is: 319-27. 3. Law>, J (2. Federal Register 1988:53: 5156. 4. SPRA(iL'[ J E. EVIiR\IAN S L, NICHOLS D E. _n integrated hypothesis for the serotonergic axonal loss induced by 3.4methvlenedioxymethamphe(amine. Neurotoxicology 1998: 19:427 42. 5. A(;tlRm N. B.,_RlOXt'l\()M. L-XSH_R..XS B. Dtl Rio J. The role of dopaminergic systems in the pcrinatal sensiti\it 3 to 3.4-mcthylencdiox)methamphclaminc-induccd neurotoxicit\ in rats..1 Pharmacol ExpThcr 1998: 2,";6:1159 65. 6. S..xl_()L K E. kl<\v R. Rl(u.,xm)s .1 B. et al. Meth._lencdiox._methamphetaminc-ihduced serotonin deficits urc follo_ed b, partial reco\er\ oxer u 52-x_eek period. Part I: synaptosomal uptake and tissue concentrations. J Pharmacol Exp Ther 199(_: 27(x 846 54. 7. Gt:_)_!l.s_ G A. t.a_l.._,_u)r() B K. NAsI_ .l E Pmentiation of 3.4-methylenedioxymethamphetaminc-induced dop- Ecstasy-induced injury 8. amine release ceptor agonists. CtlADWICK and [ S, serotonin neurotoxicity Eur J Pharmacol LINSLtY A, I994: FREI{\It)NT by 5-HT(2) 264:325 A J. DORAN re- 21. ELLMAN G L. Tissue 30. ophys B. Ec- 22. sulph,,dryl groups. Arch Biochcnl Bi- 1959: 82:70-7. _,I('GARRY I D, N.,XW-_.JIM.\ M. From dietary glucose to li,_er stasy. 3.4-methylenedioxynlethan_phetamme (MDMA). a tatality associated ,aith coagulopath,, and hyperthcrmia..1 R Soc Med 1991 84: 371. FxltAI. 1 H. S.XLLO,_n D E Y..',(,_oof_M. BtII. G M. Acute renal failure after ecstas}. BriI Med J 992 305: 29. R.XNDALL T. Ecstas>-fuelcd "'ruNe'" parties become dunces of death for English }ouths. JA.MA 1992: 268:1505 6. MILROY C M. CI.ARK J C. F()RREST A i \M Patholog} of deaths associated with "'ecstas\" and 'e;e" misuse. J Clin Pathol 1996: 49:149 53. KHAKOO S I. COLES C J, ARMsrRoN(; J S. BARRY R E. gl}cogen, the full c_cle round. Annu Rex Nutr 1'-)87: 7:51 73. 23. FU)Lli_: H. DHIIA.(}N A. GtRTNIR D. Bt r_{}t _;HS A. Chronic ecstasy 3.4-nleth_lencdioxxmethanlp letl n _c} abuse: it recurrent and unpredictable c:m_,e o1 sc,,cre acute hepatitis. J Hcpatol 1996: 25:563 6. 24. H.',I.LIWELL B, CtlIRI('() S. Lipid peroxidation: its mechanism. measurement and significance. \m J (lin Nutr 1_)93: 57: 715S 25. 25. Kt.l,,OSl! l. HIGtT(HI H. K.\lo S. et _.11.Ethanol-reduced oxidative stress in the liner. Alcohol Clin Exp Res 1996: 20: Hepatotoxicity and acceleTated fibrosis following 3.4meth>lenedioxymethamphetamine tecstasy") usage. J Clin Gastroenterol [995: 20: 244-7. 13. Et.LIS A J. WENDON J A, PORTM.X.NNB. VV'ILLIAMSR. Acute 77A 85. 26. KoB..xY.XSul H. NOXAMI T. KUROK.',W_ T. et al. Changes in the glutathione redox s,_stcm during ischemia and reperfusion in rat liver. Scand J Gastroenterol 1992: 27: 71l 6. liver damage and ecstas_ ingestioq. Gut !996: 38:454 8. I4. HI!NRY J A, JEFFRtCfS K J. DAWt.IN(; S. Toxicity and deaths 27. MITCHELL J B. Russo A. The role _1 glutathione ation and drug induced cEtotoxicit.,,. Br J Cancer from 3.4-methylenedioxymethumphetamine. Lancet 1992: 340: 384_7. 15. DYKHUIZEN R S, BRUNT P W. :_TKINSON P. et al. Ecstas,_ (suppl 8): 105- 12. 28. P{}wls O. Free radical formation Free Rudic Biol Med 1989: 6:63 induced hepatitis mimicking viral hepatitis. Gut 1995: 36: 939_,I. t6. SHEARMAN J D. CHAPMAN R W G. SATSANGI J, RYI]_2¢ N 29. BROWN C. OSTERLOH J. Multiple sexere complications from recreational ingestion of MDMA (ecstasx)[Letter]. JAMA 1987: 258:780 1. G. Misuse of ecstas,, [Letter]. Brit Med J 1992: 305: 309. 17. GORARI) D A. DAVIES S E. CLARK N'I k. Misuse of ecstas._ 30. IBRAHIM H E SALLOMI D E YAOOOB M. BELL G M. Acute renal failure after ecstas}. Brit Med J 1002: 305: 29. [Letter]. Brit Med J 1992: 305: 309. 18. AGUIRRE N. GAt.BFTE J L, LASHERASB, DEL Rio J. Methyl- 31. BRAUN L. CSAL.X M. Poussw A. ctal. Glutathione depletion induces glycogenolysis dependent ascorbate s.,,nthesis in cnedioxymethamphetamine induces opposite changes in central pre-and postsynaptic 5-HTt \ receptors in rats. Eur J Pharmacol 1995: 281: 10l 5. 19. BUEGWEJ A. AUNT S D. Microsomal lipid peroxidation, isolated murine hepatocytes. FEBS Lett 1996: 388:173 6. 32. (/ONNOR T J, N|CNAMARA N/[ G. FINN D, et al. ,-',cute 3.4methvlenediox.vmethamphetamine (MDMA) administration produces a rapid and sustained sappression of immune Methods Enz>mol 1978: 52: 20. BRADFORD M M. A rapid q uantitation of microgram the principle of protein-dye 72:..48 _4. function in the rat. Immunopharmacology 1998: 38: "__ 60 33. MCGUINNESS O P, SHAW V. Bt.:NSON E M. et al. Am J Physiol 1997: _ _ 8674 81. 34. GREER G. SIRASSMAN R J. Information on "'Ecstasy". Am J Psychiatry1985:142:1391. 9. 10. 1l. I2. _0_-,. and sensitive method for the quantities of protein utilizing binding. Anal Biochem 1976: b\ antitumor 101. m radi1987:55 quinones. 15