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The party drug 3,4-methylenedioxymethamphetamine -better known as MDMA or ecstasy- has numerous effects on the human body, characterized by a rush of energy, euphoria and empathy. However, also a multitude of toxic/neurotoxic effects have been ascribed to MDMA, based upon case reports and studies in animals. Given the intrinsic difficulties associated with controlled studies in human beings, most of our insights into the biology of MDMA have been gained through animal studies. The vast majority of these studies utilizes a pharmacological approach to elucidate the mechanisms by which MDMA exerts its effects. Advances in genetics during the last decade have led to the development of several mouse models (transgenic or knockout) that have greatly contributed to our understanding of MDMA biology. This review provides an overview of these genetically modified animal models, in the light of some characteristic effects of MDMA, e.g. hyperlocomotion, neurotoxicity, hyperthermia, behaviour or rewarding. Without a shadow of a doubt, the next decade will bring many more advanced animal models, such as mice with site-specific deletion or rescue of genes and more genetically modified rat models. These models will further improve our knowledge on the pharmacology and toxicity of MDMA and, possibly, may assist in developing therapies coping with potential damage in abusers of MDMA and other drugs, as well as in patients suffering from specific neuronal pathologies.

In this paper, the distribution and redistribution of the amphetamine derivative, 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is brought into focus. Animal experimental data were compared with internationally reported MDMA-related human fatalities: in general, these turned out to be parallel with each other. Due to its inherent properties (e.g. significant volume of distribution), MDMA is liable to postmortem redistribution. Indeed, very high concentrations have been found in cardiac blood and tissues located centrally in the body (blood-rich organs such as lungs and liver in particular). This confirms that post-mortem redistribution due to diffusion from higher to lower concentration can easily take place, mainly at longer post-mortem intervals and when putrefaction occurs. Therefore, we can conclude that for post-mortem quantitation of amphetamine and derivatives, and MDMA in particular, peripheral blood sampling (e.g. femoral vein) remains compulsory. However, if the latter is impossible, MDMA quantification in a few alternative matrices such as vitreous humour and iliopsoas muscle may provide additional information to come to a reliable conclusion. Furthermore, it should be stressed that--at present--it is impossible to estimate the individual susceptibility to the various possible adverse effects of MDMA, which implies that it is impossible to provide a "safe" or "therapeutic" blood MDMA level. Therefore, in current forensic practice, the post-mortem pathological and toxicological findings should form an entity in order to draw a well-grounded conclusion.

In this manuscript, a newly identified compound, 3,4-methylenedioxy-N,N-dimethylamphetamine (MDDM or also called MDDA), was quantified. The substance was identified in the biological specimens of a 31-year-old man who died following a massive 3,4-methylenedioxymethamphetamine (MDMA) overdose. In addition, the postmortem distribution of the identified substance in various body fluids and tissues was evaluated. For MDDM quantitation, a formerly reported and validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method was adapted. The following quantitative results of the MDDM quantitation were obtained: Femoral blood, aorta ascendens, and right atrial blood contained 2.5, 21.7, and 11.6 ng MDDM/ml, respectively. In left and right pleural fluid and pericardial fluid, concentrations of 47.0, 21.7, and 31.9 ng/ml, respectively, were found. MDDM levels in urine, bile, and stomach contents were 42.4, 1,101, and 1,113 ng/ml, respectively. MDDM concentrations in lungs, liver, kidney, and left cardiac muscle ranged from 12.8 to 39.8 ng/g, whereas these levels were below the limit of quantitation (< LOQ) in right cardiac and iliopsoas muscle. In conclusion, for the first time, MDDM was unambiguously identified in a fatal MDMA overdose. MDDM was probably present as a synthesis by-product or impurity in the MDMA tablets, which were taken in a huge amount by the victim, or MDDM was ingested separately and prior to the MDMA overdose. A third option, i.e., the eventual formation of MDDM as a result of postmortem methylation of MDMA by formaldehyde, produced by putrefaction processes or during storage under frozen conditions, is also discussed. The MDDM levels, substantiated in various body fluids and tissues, are in line with the distribution established for other amphetamine derivatives and confirm that peripheral blood sampling, such as that of femoral blood, remains the "golden standard".

The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy" is a currently used or abused designer drug and fatalities are frequently encountered in forensic practice. However, the question remains open whether an MDMA blood level can be toxic or even potentially lethal. In order to provide insight in the interpretation of a detected MDMA concentration, the distribution of MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) in various body fluids and tissues was studied and discussed in two different fatalities. Apart from peripheral blood samples (such as femoral and subclavian blood), various blood samples obtained centrally in the human body and several body fluids (such as vitreous humour) were examined. In addition, various tissues such as cardiac muscle, lungs, liver, kidneys, and brain lobes were analysed. In contrast to the peripheral blood levels, high MDMA and MDA levels were found in cardiac blood and the majority of the organs, except for the abdominal adipose tissue. The high concentrations observed in all lung lobes, the liver and stomach contents indicate that post-mortem redistribution of MDMA and MDA into cardiac blood can occur and, as a result, blood sampled centrally in the body should be avoided. Therefore, our data confirm that peripheral blood sampling remains "the golden standard". In addition, a distinct difference in peripheral blood MDMA concentrations in our two overdose cases was established (namely 0.271 and 13.508 microg/ml, respectively). Furthermore, our results suggest that, if a peripheral blood sample is not available and when putrefaction is not too pronounced, vitreous humour and iliopsoas muscle can be valuable specimens for toxicological analysis. Finally, referring to the various mechanisms of death following amphetamine intake, which can result in different survival times (e.g. cardiopulmonary complications versus hyperthermia), the anatomo-pathological findings and the toxicological results should be considered as a whole in arriving at a conclusion.

We present a fatal case involving the combined ingestion of amphetamine, 3,4-methylenedioxymethylamphetamine, 3,4-methylenedioxyamphetamine, and paramethoxyamphetamine. Various postmortem specimens (e.g., several blood samples, urine, and tissue samples) were analyzed to study the distribution of the compounds and their metabolites in the human body. Quantitation took place using liquid chromatography-sonic spray ionization-mass spectrometry after pretreatment with a liquid-liquid extraction. The medico-legal findings were compatible with a disseminated intravascular coagulation induced by hyperthermia caused by the simultaneous intake of the amphetamine analogues.

Abuse of amphetamine derivatives such as 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) is an important issue in current forensic practice and fatalities are not infrequent. Therefore, we investigated an immunohistochemical method to detect the amphetamine analogues MDMA and MDA in human tissues. For the staining procedure, the Catalysed Signal Amplification (CSA) method using peroxidase (HRP) provided by Dako and specific monoclonal antibodies were used. Appropriate controls for validation of the technique were included. The distribution of these designer drugs was studied in various brain regions including the four lobes, the basal ganglia, hypothalamus, hippocampus, corpus callosum, medulla oblongata, pons, cerebellar vermis and, additionally, in the pituitary gland. A distinct positive reaction was observed in all cortical brain regions and the neurons of the basal ganglia, the hypothalamus, the hippocampus and the cerebellar vermis but in the brainstem, relatively weak staining of neurons was seen. The reaction presented as a mainly diffuse cytoplasmic staining of the perikaryon of the neurons, and often axons and dendrites were also visualised. In addition, the immunoreactivity was present in the white matter. In the pituitary gland, however, distinct immunopositive cells were observed, with a prominent heterogeneity. The immunohistochemical findings were supported by the toxicological data. This immunostaining technique can be used as evidence of intake or even poisoning with MDMA and/or MDA and can be an interesting tool in forensic practice when the usual samples for toxicological analysis are not available. Furthermore, this method can be used to investigate the distribution of these substances in the human body.

Drug concentrations in autopsy samples can also be influenced by post-mortem gastric diffusion when the stomach contains a substantial amount of the drug or by diffusion from the trachea when agonal aspiration or post-mortem regurgitation of vomit occurs. This was studied in a rabbit animal model in which MDMA solutions were infused post mortem either in the trachea or in the stomach. At 24, 48 or 72 h post mortem, samples including cardiac blood, vitreous humour, urine, bile, gastric content and several tissues were taken for toxicological analysis. After post-mortem tracheal infusion, MDMA can easily diffuse not only into the lungs but also in great quantities into the cardiac blood and, to a lesser extent, into the cardiac muscle. MDMA was also found in the closely adjacent diaphragm and in the upper abdominal organs, including the liver and the stomach. Following post-mortem infusion into the stomach, considerable MDMA levels were found in cardiac blood and muscle, both lungs, diaphragm and liver tissue when the solution was concentrated nearby the lower oesophageal sphincter. However, when the MDMA solution was present deeper in the stomach, MDMA levels were high in the spleen and the liver and relatively low in cardiac blood and muscle. In both experiments, MDA levels in most tissues were low or below the limit of quantitation, but were substantial in cardiac blood and muscle, lungs and diaphragm, indicating that MDMA can be metabolised to MDA after death. These results in the rabbit model indicate that the diffusion of MDMA out of the stomach content, or due to aspirated vomit and gastro-oesophageal reflux can lead to considerable post-mortem redistribution and thus should be taken into account in current forensic practice in order to draw the right conclusions when a peripheral blood sample is not available.

Post-mortem redistribution is known to influence blood and tissue levels of various drugs. An animal model was used in an attempt to elucidate this problem for the amphetamine analogue, 3,4-methylenedioxymethamphetamine (MDMA). Rabbits received 1 mg/kg MDMA intravenously (iv) and were killed 2 h later in order to simulate the state of complete distribution in the body. MDMA and 3,4-methylenedioxyamphetamine (MDA) concentrations were determined in blood, urine, bile, vitreous humour, and various tissues (eye globe walls, brain, cardiac muscle, lungs, liver, kidneys, iliopsoas muscle and adipose tissue) using a high pressure liquid chromatographic (HPLC) procedure with fluorescence detection. In the first group (control group, sampling immediately post mortem) considerable MDMA concentrations were found in the brain and both lungs. In addition, our data indicate the elimination of MDMA by hepatic biotransformation and excretion via the bile. When the animals were preserved either 24 or 72 h post mortem (second group), an increase of MDMA and MDA levels in the liver and the eye globe walls was noticed. In the lungs, on the other hand, they tended to decline as a function of increasing post-mortem interval. MDMA levels in cardiac and iliopsoas muscle were fairly comparable and remained stable up to 72 h after death. In the third group, ligation of the large vessels around the heart took place immediately post mortem, but significant differences in blood and tissue MDMA concentrations between rabbits of group 2 and 3 could not be demonstrated. We therefore conclude that post-mortem redistribution of MDMA at the cellular level (viz. by pure diffusion gradient from higher to lower concentrations) is more important than its redistribution via the vascular pathway. Finally, MDA levels were relatively low in all samples, thus indicating that this is not a major metabolite in the rabbit, at least within the first 2 h after administration.

In this study, regional tissue distributions of the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") and its metabolite 3,4-methylenedioxyamphetamine (MDA) in a fatal overdose are presented. Quantitation of MDMA and MDA levels occurred in blood samples taken centrally (right and left heart and main adjacent great vessels) and peripherally (subclavian and femoral blood). In addition, MDMA and MDA concentrations were determined in cardiac and iliopsoas muscle, both lungs, liver, both kidneys, spleen, the four brain lobes, cerebellum and brainstem, and adipose tissue. Finally, MDMA and MDA levels were determined in serum, vitreous humor, urine, and bile. For all samples, a fully validated high-pressure liquid chromatography procedure with fluorescence detection was used. The found substances were also identified with liquid chromatography-tandem mass spectrometry. Our data confirm that blood sampling from an isolated peripheral vein is recommended for MDMA and MDA. In addition, the vitreous humor MDMA level indicates that this fluid can be an interesting alternative when a suitable blood sample is missing. Considering the substantial differences in concentrations in blood samples taken from various sites in the body and the high levels in some tissues (e.g., in liver), we concluded that the influence of postmortem redistribution should be taken into account in the interpretation of toxicological data when an appropriate peripheral sample cannot be obtained or when blood samples are not available because of putrefaction.

As drug instability and redistribution are factors known to affect the interpretation of post-mortem blood levels, we questioned whether post-mortem vitreous humour concentrations could be useful as predictors for the MDMA load at the time of death. In a first series of in vivo experiments using rabbits, 3,4-methylenedioxymethamphetamine (MDMA) concentrations in plasma, blood and vitreous humour were studied as a function of time after intravenous (i.v.) administration of MDMA. Equilibration between the vascular compartment and vitreous humour was attained about 1 h after i.v. MDMA administration. In a second series of experiments, the post-mortem stability of MDMA in vitreous humour in relation to ambient temperature was investigated. Post-mortem MDMA concentrations in vitreous humour were closer to the ante-mortem blood levels when compared to cardiac blood samples. These preliminary investigations in the rabbit model indicate that measurements of vitreous humour concentrations could also be of interest for predicting the blood concentration at the time of death in humans.