Multiple molecular and neuropharmacological effects of MDMA (Ecstasy)

3,4-Methylenedioxymethamphetamine (MDMA), commonly referred to as Ecstasy, is a widely abused, psychoactive recreational drug, which induces short- and long-term neuropsychiatric behaviors. This drug is neurotoxic to serotonergic neurons in vivo, and induces programmed cell death in cultured human serotonergic cells and rat neocortical neurons. Over the years it has been shown that MDMA alters the release of several neurotransmitters in the brain, it induces recompartmentation of intracellular serotonin and c-fos, and modifies the expression of a few genes. Recently, we observed changes in gene expression in mice treated with MDMA, and cloned and sequenced 11 cDNAs thus affected (4 correspond to known and 7 to unknown genes). The effect of MDMA on two of these genes, GABA transporter 1 and synaptotagmin IV was studied in detail. Characterization of the relationship between a given gene and certain physiological or behavioral effects of MDMA could shed light on the mechanism of the drug's action. However, establishing such a connection is difficult for several reasons, including that serotonergic neurons are not the only cells affected by MDMA. In this review, molecular and neurochemical events that occur in the brain following exposure to MDMA, and link between the observed molecular changes with known physiological effects of the drug are discussed. It is indicated that MDMA alters the expression of several proteins involved in GABA neurotransmission, thus having critical effect on thermoregulation and MDMA acute toxicity. This analysis should facilitate development of novel approaches to prevent deleterious effects, especially mortality induced by MDMA and other abused psychostimulants.     

Ketamine pretreatment exacerbated 3,4-methylenedioxymethamphetamine-induced central dopamine toxicity

Currently, joint use of ketamine and 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) represents a specific combination of polydrug abuse. Long-lasting and even aggravated central neuronal toxicity associated with mixing ketamine and MDMA use is of special concern. This study was undertaken to examine the modulating effects of ketamine treatment on later MDMA-induced dopamine and serotonin neurotoxicity. We found that repeated administration of ketamine (50 mg/kg x 7) at 1.5-h intervals did not render observable dopamine or serotonin depletion in catecholaminergic target regions examined. In contrast, three consecutive doses of MDMA (20 mg/kg each) at 2-h intervals produced long-lasting dopamine and serotonin depletions in striatum, nucleus accumbens and prefrontal cortex. More importantly, pretreatment with binge doses of ketamine (50 mg/kg x 7 at 1.5-h intervals) 12 h prior to the MDMA dosing regimen (20 mg/kg x 3 at 2-h intervals) aggravated the MDMA-induced dopaminergic toxicity. Nonetheless, such binge doses of ketamine treatment did not affect MDMA-induced serotonergic toxicity. These results, taken together, indicate that binge use of ketamine specifically enhances the MDMA-induced central dopaminergic neurotoxicity in adult mouse brain.     

Short- and long-term effects of MDMA ("ecstasy") on synaptosomal and vesicular uptake of neurotransmitters in vitro and ex vivo

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a commonly abused drug which has been shown to be neurotoxic to serotonergic neurons in many species. The exact mechanism responsible for the neurotoxicity of MDMA is, however, poorly understood. In this study, the effects of MDMA on the synaptosomal and vesicular uptake of neurotransmitters were investigated. Our results show that MDMA (0.5-20 microM) reduces both synaptosomal and vesicular uptake of serotonin and dopamine in a dose dependent manner in vitro, while the uptake of glutamate and gamma-aminobutyric acid (GABA) remains unaffected. Ex vivo experiments support the importance of the monoamines, with predominant dopaminergic inhibition at short-term exposure (3 x 15 mg/kg; 2-h intervals), and exclusively serotonergic inhibition at long-term exposure (2 x 10 mg/kg per day; 4 days). This study also compares MDMA and the structurally related antidepressant paroxetine, in an attempt to reveal possible cellular mechanisms for the serotonergic toxicity of MDMA. One important difference between paroxetine and MDMA is that only MDMA has the capability of inhibiting vesicular uptake of monoamines at doses used. We suggest that inhibition of the vesicular monoamine transporter-2, and a following increase in cytoplasmatic monoamine concentrations, might be crucial for the neurotoxic effect of MDMA.     

Comparative neurochemical profile of 3,4-methylenedioxymethamphetamine and its metabolite alpha-methyldopamine on key targets of MDMA neurotoxicity

The neurotoxicity of MDMA or "Ecstasy" in rats is selectively serotonergic, while in mice it is both dopaminergic and serotonergic. MDMA metabolism may play a key role in this neurotoxicity. The function of serotonin and dopamine transporter and the effect of MDMA and its metabolites on them are essential to understand MDMA neurotoxicity. The aim of the present study was to investigate and compare the effects of MDMA and its metabolite alpha-methyldopamine (MeDA) on several molecular targets, mainly the dopamine and serotonin transporter functionality, to provide evidence for the role of this metabolite in the neurotoxicity of MDMA in rodents. MeDA had no affinity for the serotonin transporter but competed with serotonin for its uptake. It had no persistent effects on the functionalism of the serotonin transporter, in contrast to the effect of MDMA. Moreover, MeDA inhibited the uptake of dopamine into the serotonergic terminal and also MAO(B) activity. MeDA inhibited dopamine uptake with a lower IC(50) value than MDMA. After drug washout, the inhibition by MeDA persisted while that of MDMA was significantly reduced. The effect of MDMA on the dopamine transporter is related with dopamine release from vesicular stores, as this inhibition disappeared in reserpine-treated animals. However, the effect of MeDA seems to be a persistent conformational change of this transporter. Moreover, in contrast with MDMA, MeDA did not show affinity for nicotinic receptors, so no effects of MeDA derived from these interactions can be expected. The metabolite reduced cell viability at lower concentrations than MDMA. Apoptosis plays a key role in MDMA induced cellular toxicity but necrosis is the major process involved in MeDA cytotoxicity. We conclude that MeDA could protect against the serotonergic lesion induced by MDMA but potentiate the dopaminergic lesion as a result of the persistent blockade of the dopamine transporter induced this metabolite.     

Impact of Cytochrome P450 2D6 Function on the Chiral Blood Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine (MDMA) and Its Phase I and II Metabolites in Humans

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) metabolism is known to be stereoselective, with preference for S-stereoisomers. Its major metabolic step involves CYP2D6-catalyzed demethylenation to 3,4-dihydroxymethamphetamine (DHMA), followed by methylation and conjugation. Alterations in CYP2D6 genotype and/or phenotype have been associated with higher toxicity. Therefore, the impact of CYP2D6 function on the plasma pharmacokinetics of MDMA and its phase I and II metabolites was tested by comparing extensive metabolizers (EMs), intermediate metabolizers (IMs), and EMs that were pretreated with bupropion as a metabolic inhibitor in a controlled MDMA administration study. Blood plasma samples were collected from 16 healthy participants (13 EMs and three IMs) up to 24 h after MDMA administration in a double-blind, placebo-controlled, four-period, cross-over design, with subjects receiving 1 week placebo or bupropion pretreatment followed by a single placebo or MDMA (125 mg) dose. Bupropion pretreatment increased the maximum plasma concentration (C_max) and area under the plasma concentration-time curve from 0 to 24 h (AUC₂₄) of R-MDMA (9% and 25%, respectively) and S-MDMA (16% and 38%, respectively). Bupropion reduced the C_max and AUC₂₄ of the CYP2D6-dependently formed metabolite stereoisomers of DHMA 3-sulfate, DHMA 4-sulfate, and 4-hydroxy-3-methoxymethamphetamine (HMMA sulfate and HMMA glucuronide) by approximately 40%. The changes that were observed in IMs were generally comparable to bupropion-pretreated EMs. Although changes in stereoselectivity based on CYP2D6 activity were observed, these likely have low clinical relevance. Bupropion and hydroxybupropion stereoisomer pharmacokinetics were unaltered by MDMA co-administration. The present data might aid further interpretations of toxicity based on CYP2D6-dependent MDMA metabolism.     

Uptake of 3,4-methylenedioxymethamphetamine and its related compounds by a proton-coupled transport system in Caco-2 cells

3,4-methylenedioxymethamphetamine (MDMA) is an illegal amphetamine-type stimulant (ATS) that is abused orally in the form of tablets for recreational purposes. The aim of this work is to investigate the absorption mechanism of MDMA and other related compounds that often occur together in ATS tablets, and to determine whether such tablet components interact with each other in intestinal absorption. The characteristics of MDMA uptake by the human intestinal epithelial Caco-2 cell line were investigated. The Michaelis constant and the maximal uptake velocity at pH 6.0 were 1.11 mM and 13.79 nmol/min/mg protein, respectively, and the transport was electroneutral. The initial uptake rate was regulated by both intra- and extracellular pH. MDMA permeation from the apical to the basolateral side was inferior to that in the reverse direction, and a decrease in apical pH enhanced MDMA permeation from the basolateral to the apical side. These facts indicate that this transport system may be an antiporter of H+. However, under physiological conditions, the proton gradient cannot drive the MDMA uptake because it is inwardly directed. Large concentration differences of MDMA itself drive this antiporter. Various compounds with similar amine moieties inhibited the uptake, but substrates of organic cation transporters (OCT1-3) and an H+-coupled efflux antiporter, MATE, were not recognized.     

Effects of MDMA Injections on the Behavior of Socially-Housed Long-Tailed Macaques (Macaca fascicularis)

3,4-methylenedioxy-N-methyl amphetamine (MDMA) is one of the few known molecules to increase human and rodent prosocial behaviors. However, this effect has never been assessed on the social behavior of non-human primates. In our study, we subcutaneously injected three different doses of MDMA (1.0, 1.5 or 2.0mg/kg) to a group of three, socially housed, young male long-tailed macaques. More than 200 hours of behavioral data were recorded, during 68 behavioral sessions, by an automatic color-based video device that tracked the 3D positions of each animal and of a toy. This data was then categorized into 5 exclusive behaviors (resting, locomotion, foraging, social contact and object play). In addition, received and given social grooming was manually scored. Results show several significant dose-dependent behavioral effects. At 1.5mg/kg only, MDMA induces a significant increase in social grooming behavior, thus confirming the prosocial effect of MDMA in macaques. Additionally, at 1.5 and 2.0 mg/kg MDMA injection substantially decreases foraging behavior, which is consistent with the known anorexigenic effect of this compound. Furthermore, at 2.0 mg/kg MDMA injection induces an increase in locomotor behavior, which is also in accordance with its known stimulant property. Interestingly, MDMA injected at 1.0mg/kg increases the rate of object play, which might be interpreted as a decrease of the inhibition to manipulate a unique object in presence of others, or, as an increase of the intrinsic motivation to manipulate this object. Together, our results support the effectiveness of MDMA to study the complex neurobiology of primates’ social behaviors.     

Enhancement of 3,4-methylenedioxymethamphetamine neurotoxicity by the energy inhibitor malonate

The acute and long-term effects of the local perfusion of 3,4-methylenedioxymethamphetamine (MDMA) and the interaction with the mitochondrial inhibitor malonate (MAL) were examined in the rat striatum. MDMA, MAL or the combination of MAL with MDMA was reverse dialyzed into the striatum for 8 h via a microdialysis probe while extracellular dopamine (DA) and serotonin (5-HT) were measured. One week later, tissue immediately surrounding the probe was assayed for DA and 5-HT tissue content. Local perfusion of MDMA increased DA and 5-HT release but did not produce long-term depletion of DA or 5-HT in tissue. Malonate also increased both DA and 5-HT release but, in contrast to MDMA, produced only long-term depletion of DA. The combined perfusion of MDMA/MAL synergistically increased the release of DA and 5-HT and produced long-term depletion of both DA and 5-HT in tissue. These results support the conclusion that DA, compared with 5-HT, neurons are more susceptible to mitochondrial inhibition. Moreover, MDMA, which does not normally produce DA depletion in the rat, exacerbated MAL-induced DA depletions. The effect of MDMA in combination with MAL to produce 5-HT depletion suggests a role for bio-energetic stress in MDMA-induced toxicity to 5-HT neurons. Overall, these results highlight the importance of energy balance to the function of DA and 5-HT neurons and to the toxic effects of MDMA.     

Uptake of 3,4-methylenedioxymethamphetamine and its related compounds by a proton-coupled transport system in Caco-2 cells

3,4-Methylenedioxymethamphetamine (MDMA) is an illegal amphetamine-type stimulant (ATS) that is abused orally in the form of tablets for recreational purposes. The aim of this work is to investigate the absorption mechanism of MDMA and other related compounds that often occur together in ATS tablets, and to determine whether such tablet components interact with each other in intestinal absorption. The characteristics of MDMA uptake by the human intestinal epithelial Caco-2 cell line were investigated. The Michaelis constant and the maximal uptake velocity at pH 6.0 were 1.11 mM and 13.79 nmol/min/mg protein, respectively, and the transport was electroneutral. The initial uptake rate was regulated by both intra- and extracellular pH. MDMA permeation from the apical to the basolateral side was inferior to that in the reverse direction, and a decrease in apical pH enhanced MDMA permeation from the basolateral to the apical side. These facts indicate that this transport system may be an antiporter of H⁺. However, under physiological conditions, the proton gradient cannot drive the MDMA uptake because it is inwardly directed. Large concentration differences of MDMA itself drive this antiporter. Various compounds with similar amine moieties inhibited the uptake, but substrates of organic cation transporters (OCT1-3) and an H⁺-coupled efflux antiporter, MATE, were not recognized.     

Effects of combined treatment with mephedrone and methamphetamine or 3,4-methylenedioxymethamphetamine on serotonin nerve endings of the hippocampus

Aims

Mephedrone is a stimulant drug of abuse with close structural and mechanistic similarities to methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Although mephedrone does not damage dopamine nerve endings it increases the neurotoxicity of amphetamine, methamphetamine and MDMA. The effects of mephedrone on serotonin (5HT) nerve endings are not fully understood, with some investigators reporting damage while others conclude it does not. Presently, we investigate if mephedrone given alone or with methamphetamine or MDMA damages 5HT nerve endings of the hippocampus.

Main methods

The status of 5HT nerve endings in the hippocampus of female C57BL mice was assessed through measures of 5HT by HPLC and by immunoblot analysis of serotonin transporter (SERT) and tryptophan hydroxylase 2 (TPH2), selective markers of 5HT nerve endings. Astrocytosis was assessed through measures of glial fibrillary acidic protein (GFAP) (immunoblotting) and microglial activation was determined by histochemical staining with Isolectin B4.

Key findings

Mephedrone alone did not cause persistent reductions in the levels of 5HT, SERT or TPH2. Methamphetamine and MDMA alone caused mild reductions in 5HT but did not change SERT and TPH2 levels. Combined treatment with mephedrone and methamphetamine or MDMA did not change the status of 5HT nerve endings to an extent that was different from either drug alone.

Significance

Mephedrone does not cause toxicity to 5HT nerve endings of the hippocampus. When co-administered with methamphetamine or MDMA, drugs that are often co-abused with mephedrone by humans, toxicity is not increased as is the case for dopamine nerve endings when these drugs are taken together.     

Changes in leptin, ghrelin, growth hormone and neuropeptide-Y after an acute model of MDMA and methamphetamine exposure in rats.

Club drug abuse is a growing problem in the United States. Beyond addiction and toxicity are endocrine effects which are not well characterized. Specifically, the changes in appetite following exposure to drugs of abuse are an interesting but poorly understood phenomenon. Serum hormones such as leptin, ghrelin, growth hormone (GH), and neuropeptide-Y (NP-Y) are known to affect appetite, but have not been studied extensively with drugs of abuse. In this work, we examine the effects of club drugs 3,4-methylenedioxymethamphetamine (MDMA) (ecstasy) and methamphetamine (METH) (doses of 5, 20 and 40 mg/kg) on serum concentrations of these hormones in adult male Sprague-Dawley rats 6, 12, 24 and 48 hours after drug administration. In a dose-dependent manner, MDMA was shown to cause transient significant decreases in serum leptin and GH followed by a base line recovery after 24 hours. Conversely, serum ghrelin increased and normalized after 24 hours. Interestingly, serum NP-Y showed a steady decrease in both treatment of MDMA and METH at different time points and dosages. In humans, abuse of these drugs reduces eating. As evident from these data, acute administration of METH and MDMA had significant effects on different serum hormone levels involved in appetite regulation. Future studies should be performed to see how chronic, low dose drug administration would affect hormone levels and try to answer questions about the physiological mechanisms involved in the anorexic paradigm observed in drug use.     

Serotonin mediates rapid changes of striatal glucose and lactate metabolism after systemic 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") administration in awake rats

The pathway for selective serotonergic toxicity of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") is poorly understood, but has been linked to hyperthermia and disturbed energy metabolism. We investigated the dose-dependency and time-course of MDMA-induced perturbations of cerebral glucose metabolism in freely moving rats using rapid sampling microdialysis (every minute) coupled to flow-injection analysis (FIA) with biosensors for glucose and lactate. Blood samples for analysis of glucose and lactate were taken at 30-45 min intervals before and after drug dosing and body temperature was monitored by telemetry. A single dose of MDMA (2-10-20 mg/kg i.v.) evoked a transient increase of interstitial glucose concentrations in striatum (139-223%) with rapid onset and of less than 2h duration, a concomitant but more prolonged lactate increase (>187%) at the highest MDMA dose and no significant depletions of striatal serotonin. Blood glucose and lactate levels were also transiently elevated (163 and 135%) at the highest MDMA doses. The blood glucose rises were significantly related to brain glucose and brain lactate changes. The metabolic perturbations in striatum and the hyperthermic response (+1.1 degrees C) following systemic MDMA treatment were entirely blocked in p-chlorophenylalanine pre-treated rats, indicating that these effects are mediated by endogenous serotonin.     

Protection against 3,4-methylenedioxymethamphetamine-induced neurodegeneration produced by glutathione depletion in rats is mediated by attenuation of hyperthermia

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

Duloxetine inhibits effects of MDMA ("ecstasy") in vitro and in humans in a randomized placebo-controlled laboratory study

This study assessed the effects of the serotonin (5-HT) and norepinephrine (NE) transporter inhibitor duloxetine on the effects of 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) in vitro and in 16 healthy subjects. The clinical study used a double-blind, randomized, placebo-controlled, four-session, crossover design. In vitro, duloxetine blocked the release of both 5-HT and NE by MDMA or by its metabolite 3,4-methylenedioxyamphetamine from transmitter-loaded human cells expressing the 5-HT or NE transporter. In humans, duloxetine inhibited the effects of MDMA including elevations in circulating NE, increases in blood pressure and heart rate, and the subjective drug effects. Duloxetine inhibited the pharmacodynamic response to MDMA despite an increase in duloxetine-associated elevations in plasma MDMA levels. The findings confirm the important role of MDMA-induced 5-HT and NE release in the psychotropic effects of MDMA. Duloxetine may be useful in the treatment of psychostimulant dependence. TRIAL REGISTRATION: Clinicaltrials.gov NCT00990067.     

Studies,using in vivo microdialysis,on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine ('ecstasy')-induced dopamine release and free radical formation in the mouse striatum

The present study examined the mechanisms by which 3,4-methylenedioxymethamphetamine (MDMA) produces long-term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3-h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 +/- 64% of baseline). This increase was enhanced to 576 +/- 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca(2+) medium inhibited the MDMA-induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca(2+) -dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg x 3) increased the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre-treatment attenuated the MDMA-induced increase in 2,3-DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre-treatment. GBR 12909 did not modify the MDMA-induced changes in body temperature. These data suggest that MDMA-induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA-induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.     

Minoxidil prevents 3,4-methylenedioxymethamphetamine-induced serotonin depletions: role of mitochondrial ATP-sensitive potassium channels, Akt and ERK

Preconditioning has emerged as a valid strategy against different neurotoxic insults. Although the mechanisms underlying preconditioning are not fully understood, the activation of ATP-sensitive potassium (KATP) channels has been proposed to play a pivotal role in neuronal preconditioning. In the present work we examine whether minoxidil a KATP channel activator protects against the long-term toxicity caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in rats. Our data show that intrastriatal administration of minoxidil prevents MDMA-induced long-term indole depletions in the rat striatum. This effect was not related to an effect on core temperature, as pre-treatment with minoxidil did not significantly alter MDMA-induced hyperthermia. Taking into account that minoxidil opens both sarcolemmal and mitochondrial KATP channels, we examined the role of each type of channels in the protective effects of minoxidil using specific inhibitors. The administration of HMR-1098, a blocker of the sarcolemmal KATP channels, along with minoxidil did not affect the protection afforded by the latter. On the contrary the selective mitochondrial KATP channel blocker 5-hydroxydecanoic acid completely reversed the protection afforded by minoxidil, thereby implicating the involvement of mitochondrial (but not sarcolemmal) KATP channels. Furthermore our data show the participation of Akt and extracellular signal-regulated kinases in minoxidil-afforded protection. Intrastriatal administration of wortmannin or PD98059 (inhibitors of phosphatidylinositol-3-kinase and mitogen-activated protein kinase/extracellular regulated protein kinase, respectively), along with minoxidil abolished the protective effect of minoxidil against the serotonergic toxicity caused by MDMA. These results demonstrate that minoxidil by opening mitochondrial KATP channels completely prevents MDMA toxicity and that Akt and MAP kinases are involved in minoxidil-afforded neuroprotection.     

MEIC—A new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests

A new international project to evaluate the relevance for human systemic and local toxicity of in vitro tests of general toxicity of chemicals has been organized by the Scandinavian Society of Cell Toxicology under the title Multicenter Evaluation of In Vitro Cytotoxicity (MEIC). The basic assumptions underlying the project, as well as the practical goals and the design of the program are outlined. The list of the first 50 reference chemicals is presented. The chemicals are an otherwise unbiased selection of compounds with known human acutely lethal dosage and blood concentrations, including LD50-values in the rat or mouse. Most agents also have other data on human toxicity and toxicokinetics, including more extensive animal toxicity data. International laboratories already using or developing in vitro tests of various partial aspects of general toxicity are invited to test the substances, the results of which will be evaluated by us. The predictivity of the in vitro results for both partial and gross human toxicity data will be determined with combined use of univariate regression analysis and soft multivariate modeling. The predictivity of the in vitro results will be compared with the predictivity of conventional animal tests for the same chemicals. Finally, batteries of tests with optimal prediction power for various types of human toxicity will be selected. The need for and possible uses of such batteries are discussed.     

3,4-Methylenedioxymethamphetamine ("Ecstasy") induces apoptosis of cultured rat liver cells

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) has been shown to be hepatotoxic for human users, but molecular mechanisms involved in this effect remained poorly understood. MDMA-induced cell damage is related to programmed cell death in serotonergic and dopaminergic neurons. However, until now there has been no evidence of apoptosis induced by MDMA in liver cells. Here we demonstrate that exposure to MDMA caused apoptosis of freshly isolated rat hepatocytes and of a cell line of hepatic stellate cells (HSC), as shown by chromatin condensation of the nuclei and accumulation of oligonucleosomal fragments in the cytoplasm. In both cell types, apoptosis correlated with decreased levels of bcl-x(L), release of cytochrome c from the mitochondria and activation of caspase 3. In HSC, but not in hepatocytes, MDMA induced poly(ADP-ribose)polymerase (PARP) proteolysis. These results suggest that apoptosis of liver cells could be involved in the hepatotoxicity of MDMA.     

Systematic reviews of animal experiments demonstrate poor human clinical and toxicological utility

The assumption that animal models are reasonably predictive of human outcomes provides the basis for their widespread use in toxicity testing and in biomedical research aimed at developing cures for human diseases. To investigate the validity of this assumption, the comprehensive Scopus biomedical bibliographic databases were searched for published systematic reviews of the human clinical or toxicological utility of animal experiments. In 20 reviews in which clinical utility was examined, the authors concluded that animal models were either significantly useful in contributing to the development of clinical interventions, or were substantially consistent with clinical outcomes, in only two cases, one of which was contentious. These included reviews of the clinical utility of experiments expected by ethics committees to lead to medical advances, of highly-cited experiments published in major journals, and of chimpanzee experiments--those involving the species considered most likely to be predictive of human outcomes. Seven additional reviews failed to clearly demonstrate utility in predicting human toxicological outcomes, such as carcinogenicity and teratogenicity. Consequently, animal data may not generally be assumed to be substantially useful for these purposes. Possible causes include interspecies differences, the distortion of outcomes arising from experimental environments and protocols, and the poor methodological quality of many animal experiments, which was evident in at least 11 reviews. No reviews existed in which the majority of animal experiments were of good methodological quality. Whilst the effects of some of these problems might be minimised with concerted effort (given their widespread prevalence), the limitations resulting from interspecies differences are likely to be technically and theoretically impossible to overcome. Non-animal models are generally required to pass formal scientific validation prior to their regulatory acceptance. In contrast, animal models are simply assumed to be predictive of human outcomes. These results demonstrate the invalidity of such assumptions. The consistent application of formal validation studies to all test models is clearly warranted, regardless of their animal, non-animal, historical, contemporary or possible future status. Likely benefits would include, the greater selection of models truly predictive of human outcomes, increased safety of people exposed to chemicals that have passed toxicity tests, increased efficiency during the development of human pharmaceuticals and other therapeutic interventions, and decreased wastage of animal, personnel and financial resources. The poor human clinical and toxicological utility of most animal models for which data exists, in conjunction with their generally substantial animal welfare and economic costs, justify a ban on animal models lacking scientific data clearly establishing their human predictivity or utility.     

Molecular and Cellular Mechanisms of Ecstasy-Induced Neurotoxicity: An Overview

“Ecstasy” [(±)-3,4-methylenedioxymethamphetamine, MDMA, XTC, X, E] is a psychoactive recreational hallucinogenic substance and a major worldwide drug of abuse. Several reports raised the concern that MDMA has the ability to induce neurotoxic effects both in laboratory animals and humans. Despite more than two decades of research, the mechanisms by which MDMA is neurotoxic are still to be fully elucidated. MDMA induces serotonergic terminal loss in rats and also in some mice strains, but also a broader neuronal degeneration throughout several brain areas such as the cortex, hippocampus, and striatum. Meanwhile, in human “ecstasy” abusers, there are evidences for deficits in seronergic biochemical markers, which correlate with long-term impairments in memory and learning. There are several factors that contribute to MDMA-induced neurotoxicity, namely, hyperthermia, monoamine oxidase metabolism of dopamine and serotonin, dopamine oxidation, the serotonin transporter action, nitric oxide, and the formation of peroxinitrite, glutamate excitotoxicity, serotonin 2A receptor agonism, and, importantly, the formation of MDMA neurotoxic metabolites. The present review covered the following topics: history and epidemiology, pharmacological mechanisms, metabolic pathways and the influence of isoenzyme genetic polymorphisms, as well as the acute effects of MDMA in laboratory animals and humans, with a special focus on MDMA-induced neurotoxic effects at the cellular and molecular level. The main aim of this review was to contribute to the understanding of the cellular and molecular mechanisms involved in MDMA neurotoxicity, which can help in the development of therapeutic approaches to prevent or treat the long-term neuropsychiatric complications of MDMA abuse in humans.