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Toxicity of EcstasyBy Leon van Aerts, Ph.DIn the ecstasy consuming community the view that MDMA is a relatively safe drug prevails. However, knowledge of the toxicity of MDMA and of the risks involved with the use of ecstasy is scarce amongst those consuming it. In this chapter the existent scientific knowledge on the toxicity of MDMA is reviewed, and based on the available animal data, the dose-effects relationships of MDMA use in humans are estimated. Two areas of toxicity are of concern. These are the acute systemic toxicity and the neurotoxicity of MDMA. Acute systemic toxicity refers to the acute severe reactions that occur occasionally, and that may lead to hospitalization or even can be fatal. It is now well recognized that a greatly increased body temperature plays a pivotal role. Control of body temperature is therefore the most important means in preventing the severe reactions to MDMA. Recently, concerns have also been raised about a possible idiosyncratic liver toxicity. Most of the animal research has concentrated on the neurotoxicity of MDMA. Of main concern are the long term effects of MDMA on the serotonergic system. High doses or repeated administration of MDMA cause serotonin nerve terminal degeneration and serotonin axonal degeneration in animals. Lower doses result in changes, usually decreases, of serotonin neuronal markers, amongst which reduced serotonin brain tissue levels, reduced density of the serotonin reuptake transporter protein, and reduced activity of tryptophan hydroxylase. It is unclear if the changes that are observed after administration of low doses of MDMA are of a neuromodulatory nature, or if these are a reflection of neurodegenerative processes, or if it is a combination of these two possibilities. Although studies with a retrospective study design cannot establish a causal relationship, the observation that MDMA users had lower levels of a serotonin metabolite in their cerobrospinal fluid is an indication that changes in their serotonergic system may have occurred. Extrapolation of the available animal data to humans implies that neuromodulatory events in the serotonergic system of the brain will occur at recreational doses, and that nerve terminal degeneration is likely to occur at high doses. It is therefore advisable to refrain from the use of high doses, boosters or bingeing on ecstasy, as these will increase the likelihood of neurodegenerative processes. IntroductionIn this chapter information will be given on the toxicology of MDMA. It is a reflection of a study of the available scientific literature on this subject. It is not my purpose to add to the prevailing media scare around ecstasy, nor is my purpose to play down the potential dangers that are related with the use of ecstasy. The sole intention is to present the current knowledge on an intrinsic property of MDMA, namely its toxicity and, based on this knowledge, to make an estimation of health risks concerning the human use of ecstasy. The plea is made by the drug consuming community that the user himself should be free to decide which drugs he uses, so that, the user becomes his own risk manager in this matter. In order to make sound decisions on the risks that will or will not be taken, the risk manager must be provided with a solid risk assessment. A simplified presentation of such a risk assessment is given by presenting estimates of the effects that may be expected with the use of various doses of MDMA. Toxicity of MDMAToxicity is an intrinsic property of a substance, namely its ability to disturb the physiological balance of an organism to such an extent that the organism no longer can be considered healthy. In other words it becomes ill (Koeman, 1996). The Environmental Protection Agency (EPA) of the US considers the effects of substances as toxic or adverse when there is functional deterioration or pathological damage that affects the functioning of the whole organism or that reduces the ability of the organism to react to additional hazards. Concerning the toxicity of MDMA there are two main areas that deserve attention: acute systemic toxicity and neurotoxicity. The effect of the acute systemic toxicity on the health of the organism is obvious. In the case of neurotoxicity no immediate effect on the health of the animal or human is observed. Nevertheless, the deterioration of very specific parts of the nervous system, as observed in animals, must be considered to be potentially toxic, since such a deterioration may eventually affect the health of the animal or human, or reduce its capacity to cope with additional hazards. Acute systemic toxicity of MDMAThe first studies on the acute toxicity of MDMA were performed in 1953 and 1954 at Michigan University, supported by the US Army, declassified in 1969 and published by Hardman et al. (1973). They showed that the LD50 (dose at which 50% of the animals die) varied from 14 mg/kg, intravenously (i.v.) in the mongrel dog to 98 mg/kg, intraperitoneally (i.p.) in the guinea pig. In the monkey (Macaca mulatta) it was 22 mg/kg, i.v.. Compared to mescaline, MDMA was, depending on species, two to almost six times as toxic. Noteworthy is that 3,4-methylenedioxyamphetamine (MDA) was even more toxic with a LD50 in the monkey of 6 mg/kg, i.v., approximately four times the human recreational dose. At high doses MDMA caused a number of effects in the dog (5-50 mg/kg, i.v.) and monkey (10-75 mg/kg, i.v.) as a result of its action on the nervous system, including: lack of movement control, convulsions, muscle rigidity and tremors, vomiting and difficulties with breathing. Pathology was not performed. In a subchronic study (28 days) rats and beagle dogs were given MDMA orally at dose ranges of 0-100 mg/kg and 0-15 mg/kg, respectively.1 Besides clinical manifestations as mentioned above, the testicles of one out of three dogs in the 9 mg/kg group and one out of three in the 15 mg/kg group were reduced in size and two out of three dogs in the 15 mg/kg group had enlarged prostatic glands. Rats showed no gross lesions at necropsy. Some haematological parameters were slightly changed at the higher doses, but these changes were statistically not significant. Noteworthy is that no neuropathological effects were observed. However, the histological methods that were used were not properly described and may not have been appropriate to detect MDMA-induced lesions. Unfortunately, the acute toxicity of MDMA has also become apparent in humans. With the rise of rave culture in the UK reports of severe reactions, including fatalities after the use of 'ecstasy' appeared in the medical literature. They were reviewed by Henry et al. (1992). The predominant toxicity patterns that emerge from these reports are fulminant hyperthermia, convulsions, disseminated intravascular coagulation (DIC) (blood clotting in the blood vessels), rhabdomyolysis (dissolution of skeletal muscle), and acute renal (kidney) failure. DIC and rhabdomyolysis may be brought about by the hyperthermic condition, and rhabdomyolysis can also be caused by acute renal failure. Acute liver failure is another serious complication reported in association with the use of MDMA and can also precipitate from a hyperthermic condition.2 It is now well recognized that hyperthermia plays a central role in these events, and body temperature control is therefore an important means in preventing the serious conditions above. Providing the body with enough fluid is one way in achieving this, however it should be stressed that excessive drinking of water may lower the ionic strength (salt concentration) of the body fluids, cause tissues to swell p; a problem for the brains p; and can eventually lead to death. One fatality postmortemly examined by Milroy et al. (1996) was a case of water intoxication after the taking of ecstasy, and Matthai et al. (1996) described two cases, that were shown to have developed mild cerebral oedema (abnormal accumulation of fluid in brain tissue) due to unrestricted water intake. When very thirsty it is therefore wiser to drink isotonic fluids instead of solely water. Although raving for hours in a warm environment may aggravate the onset of a hyperthermic condition, it should be noted that MDMA by its pharmacologic action may lead to a rise in body temperature by itself. Severe reactions like hyperthermia, DIC and rhabdomyolysis were rare at the time it was used only in more relaxed settings in the 80s in the US. However, observations of this type were reported.3 Cardiac arrhythmia (irregularities in the heart rhythm) are often also noted in emergencies that are brought in and are probably another way by which death may precipitate, especially in those that are predisposed by having cardiac abnormalities. The increase in blood pressure and rise in heart rate caused by MDMA may be deleterious in people with heart problems.4 Besides acute liver failure as part of the above mentioned syndrome there is concern MDMA causes damage to the liver, resulting in acute hepatitis.5 The mechanism here is probably different and might be caused by the accumulation of a toxic metabolite from MDMA.6 There are only a few reports of acute hepatitis related to the use of ecstasy until now. Toxicological examinations were not performed, but the subjects admitted the use of ecstasy. Therefore, if there is a causal relationship, it is not clear whether the liver toxicity was caused by MDMA, or by another psychoactive compound that was contained in the "ecstasy" tablet, by a contaminant or by the consumption of another drug. If it is MDMA that causes the observed liver toxicity, than this phenomenon is clearly idiosyncratic (inherent to an individual's (genetic) condition), and the exact relation and mechanism are unresolved. Nevertheless it may be prudent to be cautious with the combination of ecstasy and substances which are known to put an extra burden on the liver, like alcohol and acetaminophen (Paracetamol). Furthermore, people having liver problems may be more sensitive to a possible toxic response of the liver. Additionally there are a few rare cases in which the use of ecstasy was mentioned. Amongst these were several in which there were vascular problems.7 However a clear causal relation with the use of ecstasy could not be established and the cause could be an undetected pre-morbid condition or the simultaneous use of amphetamine. In two reports the occurrence of pneumomediastinum (accumulation of air in the space between the lungs as a result of rupture of marginal alveoli in the lung, associated with the sudden onset of central chest pain) was reported.8 The first report mentioned vomiting as a possible cause and in the second the authors could only speculate that retching might have caused the problems in one case and presumed severe physical exercise in another. All cases of pneumomediastinum recovered in a few days. In one report two cases of aplastic anaemia (blood abnormality) were found after the use of MDMA.9 Since all of the cases mentioned in this paragraph are seldom found in association with the use of MDMA it is questionable whether there is a causal relationship. Mild unpleasant side effects are common with the use of MDMA. These include loss of appetite, trismus (jaw clenching), bruxism (teeth grinding), nausea, muscle aches, stiffness, ataxia (impairment of motor control), blurred vision, increased sweating, anxiety, tachycardia (increased heart rate), insomnia (sleeplessness) and fatigue.10 Most of these side effects subside within 24 hours, however complaints of muscle tension in the jaw continued for two days to six weeks, blurred vision up to three days and psychological effects like insomnia, depression and anxiety up to eight days.11 Psychiatric disorders like depression, anxiety disorder and psychosis associated with the use of MDMA are dealt with under Adverse Psychological Effects, page 112. Neurotoxicity of MDMANeurobiologyBrain cells communicate with each other by sending chemicals (neurotransmitters) to each other. The neurotransmitter is released from nerve terminals which are located at the end of an axon, a long fibre from the neuronal cell body to the effector region. After diffusing across the synapse, the gap which separates neurons, the neurotransmitter binds a receptor located on the surface of the receiving cell. Binding of the transmitter to some receptors induces, inhibits, or modulates currents of electrically charged particles (ions) across the cell membrane. Other receptors regulate the levels of "second messengers", small molecules which produce biochemical changes within the cell. There are also receptors which regulate gene expression, and still others which catalyse chemical modifications to themselves or other proteins (e.g. phosphorylation). Amongst the many neurotransmitters present in the brain, serotonin (5-hydroxytryptamine, 5-HT), and dopamine (both so-called monoamines), are the ones that are of utmost importance in both the pharmacologic and the toxicologic action of MDMA. Serotonin is synthesized within the nerve cell from the amino acid tryptophan, and tryptophan hydroxylase (TPH) is the rate-limiting enzyme in this synthesis. When serotonin is synthesized it can be stored within so-called presynaptic or membrane vesicles within the nerve terminal. 5-hydroxyindoleacetic acid (5-HIAA) is a metabolite of serotonin. The serotonin signal is limited in space and time through a process called reuptake. Most of the released neurotransmitter is reabsorbed by the nerve terminal for repackaging and reuse. The (re-)uptake site (carrier, transporter), a specialized proteinaceous structure located in the nerve terminal membrane, is responsible for this process. To investigate if the serotonergic system in the brain is affected by MDMA, the above mentioned serotonin neuronal markers can be measured by appropriate methods. Serotonin and 5-HIAA can be assayed by analytical chemistry, TPH can assessed with an enzyme assay, 5-HT uptake sites can be quantified by either measuring the rate of the uptake itself, or by binding of radioactively labelled ligands that bind specifically to the uptake carrier, and subsequently measuring the radioactivity or visualize the sites by autoradiography (exposing photographic material). A recently developed method uses 5-HT-uptake ligands that can be visualized in the living brain with positron emission tomography (PET). Furthermore, functionality can be tested by artificially stimulating the serotonin neurons electrically or challenging them chemically with a serotonin releasing agent. The ultimate proof of neuronal damage however is cutting the brain into very thin slices and observing them under the microscope (histology). Specific staining techniques have been developed, either to show neuronal damage (Fink-Heimer silver staining), or to mark serotonergic neurons with the use of specific antibodies (immunohistochemical staining). Alternatively, indirect evidence of neuronal damage can be assessed. For example, an increase in the volume of glial cells (a supporting and nourishing type of brain cell) (=gliosis) or an increase in glial fibrillary acidic protein (GFAP) are associated with neuronal damage and can be used as markers. Finally behavioural tests can be performed in animals or humans, however, as we know very little of how the various aspects of the serotonin system influence behaviour, such tests are difficult to interpret. Effects of MDMA on the serotonergic system in animalsMany of the above mentioned methods to investigate the effect of MDMA on the brain serotonergic system have been applied to a variety of mammals, including rats, guinea pigs, mice, and squirrel and rhesus monkeys. Here the evidence of MDMA-induced neurotoxicity in animals will be reviewed briefly. However, it should be noted, as will be discussed in the paragraph on up- and down-regulation, that the decreases in serotonin neuronal markers do not necessarily reflect neurodegenerative processes under all circumstances, as these markers are also under genetic control and may be affected by regulatory processes. Tissue 5-hydroxyindoles levelsThe most acute effect of MDMA is a rapid release of serotonin and dopamine from the presynaptic vesicles.12 Although in mice the effect is predominantly on the dopaminergic system,13 in most species the serotonergic system is in the long run more affected. The process of serotonin release is thought to be mediated by an interaction of MDMA with the serotonin uptake carrier leading to a reversal of the serotonin flow.14 Within 24 hours after a single injection of a high dose in rats the greatly reduced levels of serotonin and dopamine have been restored to control level, however thereafter there is a progressive decline of tissue levels of serotonin and its metabolite 5-HIAA.15 These deficits can persist for weeks to more than a year after multiple doses of MDMA depending on which brain area and species are tested, with the (non-human) primate (squirrel and rhesus monkey) being the most sensitive with respect to the size of the dose that induces the protracted serotonin depletion as a well as to the extent and the duration of depletion.16 Tryptophan hydroxylase activity and serotonin uptake sitesTPH activity is also greatly reduced after repeated doses of MDMA in the rat.17 The acute inactivation of this enzyme involves the oxidation of sensitive sites of this enzyme, whereas the prolonged decreased activity after repeated injections with high doses of MDMA may reflect loss of serotonin nerve terminals.18 Loss of serotonin nerve terminals is also suggested by the reduction in the density of the serotonin uptake carriers as indicated by a reduced serotonin uptake capacity19 and decreased binding of specific serotonin uptake ligands.20 ImmunohistochemistryFurther evidence, suggestive of reduced serotonergic innervation, was provided by immunohistochemical staining. This method demonstrated that MDMA reduced the number of serotonin antibody positive nerve fibres, and that it was the fine fibre type axons ascending from the dorsal raphe nucleus (an area of serotonin neurons in the brain stem) that were specifically vulnerable to the action of MDMA.21 Morphological observationsEvidence of neuronal damage was revealed by the application of silver staining techniques, which showed degenerating axons and nerve terminals in the rat (lowest dose single oral 40 mg/kg).22 In the rhesus monkey injected subcutaneously with 5 mg/kg MDMA (twice daily for four days), abnormal inclusions within the cell bodies of dorsal raphe nucleus cells were found, which were not seen in the cell bodies of the median raphe nucleus or in non-serotonergic nuclei such as the substantia nigra or the locus ceruleus.23 The authors interpreted these inclusions as cytopathological changes. The nature and the meaning of these inclusions, however, remain unclear. Remaining fine fibre serotonergic axons visualized with immunostaining were often swollen and fragmented, providing further evidence of axonal degeneration (rat: eight doses of 20 mg/kg; monkey: eight doses of 5 mg/kg).24 Axon regenerationLoss of axons while the cell body stays intact leaves the possibility of axon regeneration. That this indeed happens has been shown in both the rat25 and the squirrel monkey.26 The recovery is region and time dependent. In the rat (exposed to repeated doses of 20 mg/kg) recovery of serotonin neuronal markers progresses in a rostral-caudal way (from the front to the back of the brain), with only partial recovery after 52 weeks in the occipital, temporal, and part of the frontal-parietal neocortex.27 In the monkey (exposed to repeated doses of 5 mg/kg) there is an initial but partial recovery of serotonin markers by 10 weeks, however after 18 months levels have dropped to the low of two weeks after treatment.28 In the squirrel monkey the pattern of reinnervation is different from what it was before treatment: Regions closer to the brain stem, where the damaged axons originate, appear to be hyperinnervated with serotonergic fibres, whereas the more distant parts remain poorly innervated after 18 months. Furthermore, the extent of regional redistribution of serotonin neuronal markers differed between animals, both in rats and in squirrel monkeys.29 Functional and behavioural effectsBesides neurochemical and morphological approaches, investigation of the functionality of the serotonergic system may help to assess the functional implications of the neurochemical and morphological changes observed after exposure to MDMA. Gartside et al. (1996) exposed rats to a neurotoxic dose-regimen of MDMA (eight doses 20 mg/kg). Two weeks later they observed that the firing activity of the dorsal raphe neurons had not diminished, nor were the basal extracellular serotonin levels in both the hippocampus and the frontal cortex (brain areas innervated by axons originating from the dorsal raphe nucleus) reduced, although the extracellular 5-HIAA levels were reduced by 50%. Moreover, no change was observed in the amount of serotonin released in the hippocampus in response to electrical stimulation (5 Hz) of either the dorsal or median raphe nucleus, but a marked reduction in the amount of serotonin released was observed in the frontal cortex after electrical stimulation of the dorsal raphe nucleus. It has also been reported that basal extracellular serotonin levels in the rat striatum could be maintained until tissue levels of serotonin are depleted by more than 95%.30 More evidence that basal cortical extracellular serotonin levels can be maintained after a MDMA-induced lesion, but that the maximal release is reduced was provided by Series et al. (1994), who challenged the serotonin system chemically with the serotonin releasing agent fenfluramine. Complex brain functions in the rhesus monkey that are first affected acutely by MDMA are related to behavioural tasks involving time estimation, motivation to work for food (which is not surprising considering the known appetite suppressing response to MDMA) and learning.31 It was shown that after chronic exposure of these monkeys to MDMA (daily for four months, doses escalating from 0.1 mg/kg to 20 mg/kg) baseline performance in the behavioural tests returned to the levels obtained before the exposure. However, the acute decrease in performance observed when the monkeys were given MDMA again five, twelve and nineteen months after the end of the chronic exposure period, was increasingly less pronounced, which disclosed the existence of a residual tolerance. It was suggested that in effect, the serotonergic system may be able to compensate for the loss of a majority of its central innervation during non-stressful or normal conditions. However, when challenged, the remaining components of the serotonergic system no longer have the capability of responding to the same degree as the fully intact system would.32 The reduction of maximal response has been demonstrated in the laboratory by electrical and chemical stimulation. Whether there are physiological conditions under which the serotonergic system is stressed to this extent is not known. Dose, route, regimen, and other variablesMany variables, including dose, route of administration, and regimen, determine the extent to which toxic or neuromodulatory effects will be observed. Many studies show that the effects of MDMA are dose-related.33 It has also become clear that repeated administration is more potent than single administration. Administering 10 mg/kg subcutaneously in the rat, Battaglia et al. (1988) showed that the reduction of serotonin neuronal markers was greatest when the number of doses was increased from two to eight (injections every twelve hours). Administering four times 10 mg/kg subcutaneously to the rat produced effects comparable to those seen after administering eight times 5 mg/kg.34 This suggests that dose and frequency of administration are additive, at least when within the near linear middle part of the dose-response curve. Scarce data on route dependency are available. In the squirrel monkey subcutaneous administration was, depending on the brain region examined, two to three times more effective than intragastric administration.35 However, in the rhesus monkey intragastric administration was twice as potent compared to the subcutaneous route, as assessed by the maximal serotonin uptake capacity of hippocampal synaptosomes and serotonin concentrations in cortical and hippocampal brain regions.36 Finnegan et al. (1988) compared intragastric with subcutaneous administration in the rat and found similar dose-related reductions in serotonin levels in the hippocampus two weeks after administration. A number of variables may affect the toxicity of MDMA. Besides the already mentioned species differences and dose, route, and regimen dependency, differences in sensitivity have been observed between different strains within one species.37 Gender differences have also been observed.38 This is a common phenomenon in the rat, but rarely seen in larger animals.39 The stage of development may also influence the long-term effects, as Broening et al. (1994, 1995) showed that newly born rats (ten days old) quickly replenished their cortical serotonin tissue levels and showed no reductions in serotonin uptake sites one week after an oral administration of MDMA (40 mg/kg), whereas young adult rats (ten weeks) did not recover the loss in serotonin levels and showed a progressive decline in serotonin uptake sites during the following week. In both rats and mice it has been shown that increased body temperature facilitates the neurotoxic action of MDMA, and that neurotoxicity can be diminished by reducing the ambient temperature, by co-administering drugs that decrease the body temperature, or by restraining the animals.40 Up- and down-regulation of serotonin neuronal markersIf serotonergic neurons would stop producing serotonin, but otherwise would be intact, this could result in a lowering of serotonin and 5-HIAA levels, reduction of TPH activity and disappearance of serotonin antibody reactive staining of serotonergic nerve fibres. It has therefore been argued that reduction of these neuronal markers after exposure to MDMA does not necessarily mean that the axons and nerve terminals have degenerated, but that a long lasting pharmacological action of MDMA may account for the observed effects.41 O'Callaghan and Miller (1993) observed in the Long-Evans rat no increase in GFAP at doses (10-30 mg/kg twice daily for seven days) where there was a marked decrease in serotonin levels. Intraventricular injection of the well-known serotonergic neurotoxicant 5,7-dihydroxytryptamine produced similar reductions in tissue serotonin levels in the presence of an increase of GFAP, which shows that the GFAP marker can be used to demonstrate serotonergic neuronal damage. These findings contrast with those of Slikker et al. (1988). The latter showed that a single oral administration of 40 mg/kg MDMA in the Sprague-Dawley rat increased the number of silver stained nerve terminals by 90%, indicating nerve terminal degeneration. Also Jensen et al. (1993), using a cupric silver stain, observed increased silver staining in the neocortex of the rat after administration of 25 mg/kg MDMA twice daily for two days. However, it should be noted that the affected neurons were not serotonergic. Commins et al. (1987), who also observed silver stained non-serotonergic neocortical neurones after the administration of MDMA, speculated that these neurons could be affected by 5,6-dihydroxytryptamine, a neurotoxicant produced by serotonergic afferents. These conflicting results suggest that the reduction in serotonin levels after administration of repeated doses of 10-30 mg/kg MDMA is largely caused by a lower concentration of serotonin in the remaining nerve terminals, while the extent of nerve terminal degeneration is too little to be detected by the GFAP marker. An alternative explanation for the absence of an increase in GFAP after administration of 10-30 mg/kg MDMA twice daily for seven days could be that MDMA directly or indirectly affects the glial cells in such a way that the GFAP response is inhibited with these doses. It has been shown that serotonin inhibits the expression of GFAP in cultured astrocytes (Le Prince et al., 1990). Furthermore, MDMA-induced prolonged activity of glial glycogen phosphorylase may compromise the energy state of the astrocytes (Poblete and Azmitia, 1995). Nevertheless, when the dose was increased to 75 mg/kg (twice daily for two days) an increase in GFAP could be observed.42 Reduction of another serotonin marker, the density of serotonin uptake sites, also does not necessarily reflect a reduction of serotonin nerve terminals, as it has been shown that acute serotonin depletion by administration of the tryptophan hydroxylase inhibitor para-chlorophenylalanine down-regulates the messenger RNA (mRNA) template for the transporter protein shortly after the treatment.43 However the density of the transporter protein was not reduced at early time-points, but was reduced 14 days after treatment,44 while the level of mRNA for the transporter was increased seven days after treatment. These results show that changes in serotonin transporter mRNA are not temporally related to changes in serotonin transporter protein levels.45 Chronic administration of the serotonin re-uptake inhibiting antidepressants imipramine and fluoxetine also reduced the expression of serotonin transporter mRNA in the rat brain.46 An experiment by Ali et al. (1993) showed that at an exposure level of 1.25 mg/kg (administered orally twice daily for four days), rhesus monkeys had an increased serotonin level in the caudate 30 days later. This result suggests that the first long-lasting effect of MDMA exposure above the NOAEL is an up-regulation of the serotonin system in some brain areas. A related discussion affects the putative neurotoxicity of the psychostimulant methamphetamine. It had been reported that high doses of this substance caused long-lasting reductions in dopamine concentrations and other dopamine nerve terminal markers in the striatum of non-human primates.47 Postmortem examination of 12 chronic human methamphetamine users revealed that three dopamine nerve terminal markers (dopamine, tyrosine hydroxylase, and the dopamine transporter protein) were reduced. However, two other markers (DOPA decarboxylase and the vesicular transporter protein) were not reduced.48 Since the latter two markers are also reduced in Parkinson's disease, the authors concluded that at the doses taken by the subjects no permanent loss of striatal dopamine nerve terminals occurred. However, in Parkinson's disease the dopaminergic neurons are destroyed, whereas the alleged neurotoxicity of methamphetamine involves only the nerve terminals of the dopaminergic neurons. Unfortunately, only concentrations of these markers were measured and no immunohistochemistry was applied. This leaves open the possibility that the unchanged markers were located at a higher concentration in fewer nerve terminals. Considering the morphological evidence cited in previous paragraphs, the view that the loss of neuronal markers after repeated 20 mg/kg doses in the rat or repeated 5 mg/kg doses in the squirrel monkey is solely caused by a pharmacological action of MDMA becomes difficult to interpret. At lower doses it may be possible that regulatory mechanisms influence the levels of serotonin neuronal markers observed, or that both degenerative and regulatory processes result in a reduction of serotonin neuronal markers. The fenfluramine issueDexfenfluramine is a substituted amphetamine used as an appetite suppressant. Although it, like MDMA, is a serotonin releasing agent, its psychoactive effect is different. A number of studies in animals, both rats and primates, have shown that fenfluramine administration, like MDMA, produces acute and long-lasting reductions in serotonin neuronal markers,49 decreases serotonin neuronal function50 and affects the regulation of the serotonin transporter.51 However, MDMA and fenfluramine seem to affect the dopaminergic system in different ways. MDMA depleted striatal dopamine and increased GFAP in the mouse, whereas fenfluramine had no effect.52 Moreover, chlormethiazole, a GABA agonist that reduces dopaminergic function, reduces MDMA-induced serotonergic toxicity in rats, whereas fenfluramine-induced neurotoxicity was not affected by chlormethiazole co-administration.53 Therefore, the precise mechanisms by which MDMA and fenfluramine reduce the serotonin neuronal markers may be different, although functional consequences may be the same. The decision of the FDA to approve the use of dexfenfluramine as an anorectic drug is often mentioned in the discussion of the neurotoxicity of MDMA, since indications of neurotoxicity are very similar. However, it should be noted that doses of dexfenfluramine taken by humans (approximately 0.3 mg/kg) are much lower than those used in the animal experiments and are lower than the doses of MDMA that are taken by humans, whereas the neurotoxic potential of both substances are very similar, dexfenfluramine being slightly more potent with respect to the serotonin neuronal markers. Still, when extrapolating the animal data to the human, the dose range of dexfenfluramine at which reduced, but reversible, brain serotonin concentrations can be expected in the human is close to or overlaps the dose range taken by humans. However, the doses at which neuronal damage may be expected that can be detected by experimental techniques independent of serotonin content, or where irreversible reductions of serotonin neuronal markers are to be expected are higher than the doses taken by humans. Effects of MDMA on the serotonergic system in humansMany experiments are done in animals which may be impossible to do in humans. Therefore indirect approaches must be made to assess the effects of MDMA on the serotonergic system in humans. However, such indirect ways of assessing the integrity and function of the serotonin system in humans are often difficult to interpret. Furthermore, due to the complexity of neural systems, knowledge of all elements, how they work and how they interact is incomplete. This too makes it difficult to understand what the meaning is of a change in a parameter if such changes are observed. This must be born in mind when looking at the effects of MDMA on the serotonin system in humans. In a small scale study by Peroutka et al. (1987) cerobrospinal fluid (CSF) 5-HIAA levels of five MDMA users were compared to literature controls. In this study no differences were observed. In a larger and internally controlled study, levels of 5-HIAA in CSF of 30 MDMA users was measured and appeared to be reduced by 32% as compared to levels in 30 controls.54 In a previous study55 squirrel monkeys were administered 5 mg/kg MDMA subcutaneously twice daily for four days. In this study the monkeys showed, two weeks later, 73-94% depletions of serotonin and 5-HIAA in brain, 42-45% depletions of serotonin and 5-HIAA in spinal cord and 60% reductions in CSF 5-HIAA. McCann et al. (1994a) therefore concluded that the observed reduction in CSF 5-HIAA in human MDMA users may have reflected a partial depletion of the subjects brain serotonin levels, and therefore could be interpreted as a sign of MDMA-induced neurotoxicity. A common problem with retrospective studies of this kind is that they cannot establish a causal relationship, as the difference in CSF-5-HIAA levels may have been pre-existing. For example, differences in personality related to serotonin levels and pre-disposing to MDMA use could be a confounding factor that cannot be excluded. To see if the implicated reduction in brain serotonin levels caused any change in functionality of the serotonin system in the same subjects, they were submitted to a tryptophan challenge. This is a test in which tryptophan is given intravenously and prolactin levels in blood are measured. Tryptophan, which is a serotonin precursor, will increase serotonin release, which then elicits an increase in prolactin levels (Charney et al., 1982). If serotonin system functionality is reduced this increase in prolactin is expected to be smaller. However, in the study of McCann et al. (1994a) MDMA users showed an increase in blood prolactin levels similar to those in control subjects. Previously, Price et al. (1989) had found a blunted response to the tryptophan challenge on prolactin release in nine MDMA users, that were pre-selected from the McCann study (1994a) for their low CSF 5-HIAA level, as compared to nine healthy controls. However, this decrease was statistically not significant. If the lower average CSF 5-HIAA level in MDMA users observed by McCann et al. (1994a) was not pre-existing, then it indicates that a change in the serotonin system had occurred, but the nature of this change is unclear. It could be a reflection of neurotoxicity, but regulatory events leading to different metabolism and disposition of serotonin and its metabolite 5-HIAA may just as well have been the cause. The lack of a change in the tryptophan challenge suggests several possibilities: 1) that the serotonin system, at least as far as this neuroendocrine response is concerned, is still intact, 2) the system has only deteriorated to a degree where there is no loss in neuroendocrine functionality, or 3) that the prolactin release system itself has become sensitized and is maintaining release at lower than normal serotonin levels. In the study by McCann et al. (1994a) personality assessments were made as well. It was found that MDMA users had greater harm avoidance behaviour, showed less hostility and were less impulsive than controls. No significant changes in pain sensitivity, pain endurance and pain tolerance between MDMA users and controls were observed. In another study on sleep performance it was found that MDMA users had less stage 2 sleep compared to controls.56 In a study of nine individuals Krystal et al. (1992) did not find any indication of depressive mood or affective disorder, however MDMA users scored slightly lower in memory tests in the absence of any clinical signs of cognitive impairment. However, the subjects were compared with literature controls, not matched controls. Furthermore, 7 grams of tryptophan was administered minimally three hours before the testing. Although neuroendocrine response was observed to be returned to baseline, a residual effect on neuropsychological performance cannot be excluded. The role of the serotonin system in human behaviour is complex and it has been implicated in the regulation of sleep, mood, anxiety, pain, aggression, memory and appetite.57 The findings mentioned above therefore suggest that some change in the serotonin system may have occurred. However, these data cannot answer whether or not structural changes of the serotonin system have ensued from the MDMA exposure. It is clear from the neurochemical, neuroendocrine and neuropsychological data discussed above that the evidence for MDMA-induced neurotoxicity in humans is far from conclusive. In ongoing studies at John's Hopkins University newly developed positron emission tomography (PET) techniques are employed to further investigate the effects of MDMA use in humans on the brain serotonin system.58 This method is based on ligand binding to the serotonin uptake transporter. However, even if reductions in ligand binding are found, it will be difficult to interpret such reductions, as they may be caused by either reduction in nerve terminals as a result of degeneration, or by down-regulation of the uptake transporter. In the end it will be necessary to do postmortem neuropathological examinations in order to produce more conclusive evidence. Such examinations should include immunostaining of brain sections, making use of antibodies to serotonin, different parts of the serotonin uptake carrier59 and the vesicular monoamine transporter protein. Measuring the concentrations of these components alone may produce inconclusive evidence, as was the case with postmortem evaluation of chronic methamphetamine users.60 Protection against neurotoxicity and dangerous interactions with other drugsIt has been shown that selective serotonin re-uptake inhibitors (SSRIs) reduce the neurotoxicity of MDMA in animals,61 SSRIs like fluoxetine (Prozac®) are used as antidepressants. As the uptake site facilitates the MDMA-induced release of serotonin,62 and the serotonin release is thought to be important for the psychoactive response to MDMA, it may be anticipated that fluoxetine reduces this response to MDMA. However, a report from McCann and Ricaurte (1993) describes the experience of four subjects that have experimented with the combination of MDMA and fluoxetine. The subjects claim that some side effects, notably nausea and vomiting increased with some of them, but also that fewer after effects like fatigue occurred. The entactogenic properties of MDMA remained, however doses of MDMA taken (totals 450, 450, 300 and 125 mg) were rather high. In ongoing research by Katherine Bonson at the National Institute of Health in the US (personal communication) additional reports on the combination of fluoxetine (either acute or chronically) and MDMA have been collected. Although some subjects reported no change in response to MDMA after fluoxetine, other case reports suggest an increase as well as a decrease in response to MDMA following fluoxetine use. However, when sertraline, another SSRI, was taken chronically, it reduced the psychoactive responses to MDMA in seven of eight individuals. It should also be emphasized that the combination of MDMA with an MAO inhibitor has been known to induce a hypertensive crisis and could lead to death. Thus, ayahuasca and other cocktails that contain MAO inhibitors (e.g. harmaline) should therefore also not be combined with MDMA. To report experiences with combinations of MDMA or other psychoactive drugs and prescription or other drugs, contact Dr. Bonson at kbonson@codon.nih.gov. Gudelsky (1996) recently showed that large quantities of reducing agents (vitamin C, cysteine) had a protective effect with respect to the neurotoxicity of MDMA in rats. These experiments add to the accumulating evidence that at some point in the toxic mechanism of MDMA oxidative processes are involved. High concentrations of antioxidants may reduce the oxidative stress. However, the concentrations that were applied in the experiment of Gudelsky (1996) were extremely high and a human would have to take about 15 grams of vitamin C applying the same weight-to-body weight ratio. It is questionable if such large quantities are still to be considered healthy. However, taking into account the increased clearance and other pharmacokinetic parameters that likewise change as a result of the lower body mass of the rat, it is conceivable that a quantity of 1-2 gram of vitamin C may afford some protection in humans. However, if such a protective effect can be generalized to the primate, should be investigated by primate studies. Also some more dose finding experiments should be done. Because oxidative mechanisms may play a role in the neurotoxicity of MDMA, and perhaps also in the suspected liver toxicity, it may be wise to refrain from combining MDMA with drugs that also result in oxidative stress, for example, the widely used analgesic acetaminophen (Paracetamol). From animal experiments it has become clear that high body temperature enhances the neurotoxicity of MDMA (see above). Therefore control of body temperature is not only of importance in preventing acute severe toxicity of MDMA, but in minimizing the risk of neurotoxicity as well. Besides reducing the ambient temperature, facilitating cooling of the body by light clothing, and reducing physical activity, the availability of drinking water is of importance.63 However, to avoid water intoxication one should not drink water alone, but should also maintain electrolyte and carbohydrate intake.
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