João Paulo Capela

Molecular and cellular mechanisms of ecstasy-induced neurotoxicity: an overview.

Abstract“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.

Toxicity of amphetamines: an update.

Amphetamines represent a class of psychotropic compounds, widely abused for their stimulant, euphoric, anorectic, and, in some cases, emphathogenic, entactogenic, and hallucinogenic properties. These compounds derive from the β-phenylethylamine core structure and are kinetically and dynamically characterized by easily crossing the blood–brain barrier, to resist brain biotransformation and to release monoamine neurotransmitters from nerve endings. Although amphetamines are widely acknowledged as synthetic drugs, of which amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are well-known examples, humans have used natural amphetamines for several millenniums, through the consumption of amphetamines produced in plants, namely cathinone (khat), obtained from the plant Catha edulis and ephedrine, obtained from various plants in the genus Ephedra. More recently, a wave of new amphetamines has emerged in the market, mainly constituted of cathinone derivatives, including mephedrone, methylone, methedrone, and buthylone, among others. Although intoxications by amphetamines continue to be common causes of emergency department and hospital admissions, it is frequent to find the sophism that amphetamine derivatives, namely those appearing more recently, are relatively safe. However, human intoxications by these drugs are increasingly being reported, with similar patterns compared to those previously seen with classical amphetamines. That is not surprising, considering the similar structures and mechanisms of action among the different amphetamines, conferring similar toxicokinetic and toxicological profiles to these compounds. The aim of the present review is to give an insight into the pharmacokinetics, general mechanisms of biological and toxicological actions, and the main target organs for the toxicity of amphetamines. Although there is still scarce knowledge from novel amphetamines to draw mechanistic insights, the long-studied classical amphetamines—amphetamine itself, as well as methamphetamine and MDMA, provide plenty of data that may be useful to predict toxicological outcome to improvident abusers and are for that reason the main focus of this review.

Pro-oxidant effects of Ecstasy and its metabolites in mouse brain synaptosomes.

3,4‐Methylenedioxymethamphetamine (MDMA or ‘Ecstasy’) is a worldwide major drug of abuse known to elicit neurotoxic effects. The mechanisms underlying the neurotoxic effects of MDMA are not clear at present, but the metabolism of dopamine and 5‐HT by monoamine oxidase (MAO), as well as the hepatic biotransformation of MDMA into pro‐oxidant reactive metabolites is thought to contribute to its adverse effects.

The neurotoxicity of amphetamines during the adolescent period.

Amphetamine‐type psychostimulants (ATS), such as amphetamine (AMPH), 3,4‐methylenedioxymethamphetamine (MDMA), and methamphetamine (METH) are psychoactive substances widely abused, due to their powerful central nervous system (CNS) stimulation ability. Young people particularly use ATS as recreational drugs. Moreover, AMPH is used clinically, particularly for attention deficit hyperactivity disorder, and has the ability to cause structural and functional brain alterations. ATS are known to interact with monoamine transporter sites and easily diffuse across cellular membranes, attaining high levels in several tissues, particularly the brain. Strong evidence suggests that ATS induce neurotoxic effects, raising concerns about the consequences of drug abuse.

Differential Effects of Methyl-4-Phenylpyridinium Ion, Rotenone, and Paraquat on Differentiated SH-SY5Y Cells

Paraquat (PQ), a cationic nonselective bipyridyl herbicide, has been used as neurotoxicant to modulate Parkinsons disease in laboratory settings. Other compounds like rotenone (ROT), a pesticide, and 1-methyl-4-phenylpyridinium ion (MPP+) have been widely used as neurotoxicants. We compared the toxicity of these three neurotoxicants using differentiated dopaminergic SH-SY5Y human cells, aiming to elucidate their differential effects. PQ-induced neurotoxicity was shown to be concentration and time dependent, being mitochondrial dysfunction followed by neuronal death. On the other hand, cells exposure to MPP+ induced mitochondrial dysfunction, but not cellular lyses. Meanwhile, ROT promoted both mitochondrial dysfunction and neuronal death, revealing a biphasic pattern. To further elucidate PQ neurotoxic mechanism, several protective agents were used. SH-SY5Y cells pretreatment with tiron (TIR) and 2-hydroxybenzoic acid sodium salt (NaSAL), both antioxidants, and N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase inhibitor, partially protected against PQ-induced cell injury. Additionally, 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenyl-propyl)piperazine (GBR 12909), a dopamine transporter inhibitor, and cycloheximide (CHX), a protein synthesis inhibitor, also partially protected against PQ-induced cell injury. In conclusion, we demonstrated that PQ, MPP+, and ROT exerted differential toxic effects on dopaminergic cells. PQ neurotoxicity occurred through exacerbated oxidative stress, with involvement of uptake through the dopamine transporter and protein synthesis.

Mitochondria: key players in the neurotoxic effects of amphetamines

Abstract Amphetamines are a class of psychotropic drugs with high abuse potential, as a result of their stimulant, euphoric, emphathogenic, entactogenic, and hallucinogenic properties. Although most amphetamines are synthetic drugs, of which methamphetamine, amphetamine, and 3,4-methylenedioxymethamphetamine (“ecstasy”) represent well-recognized examples, the use of natural related compounds, namely cathinone and ephedrine, has been part of the history of humankind for thousands of years. Resulting from their amphiphilic nature, these drugs can easily cross the blood–brain barrier and elicit their well-known psychotropic effects. In the field of amphetamines’ research, there is a general consensus that mitochondrial-dependent pathways can provide a major understanding concerning pathological processes underlying the neurotoxicity of these drugs. These events include alterations on tricarboxylic acid cycle’s enzymes functioning, inhibition of mitochondrial electron transport chain’s complexes, perturbations of mitochondrial clearance mechanisms, interference with mitochondrial dynamics, as well as oxidative modifications in mitochondrial macromolecules. Additionally, other studies indicate that amphetamines-induced neuronal toxicity is closely regulated by B cell lymphoma 2 superfamily of proteins with consequent activation of caspase-mediated downstream cell death pathway. Understanding the molecular mechanisms at mitochondrial level involved in amphetamines’ neurotoxicity can help in defining target pathways or molecules mediating these effects, as well as in developing putative therapeutic approaches to prevent or treat the acute- or long-lasting neuropsychiatric complications seen in human abusers.

The mixture of "ecstasy" and its metabolites is toxic to human SH-SY5Y differentiated cells at in vivo relevant concentrations.

The neurotoxicity of “ecstasy” (3,4-methylenedioxymethamphetamine, MDMA) is thought to involve hepatic metabolism, though its real contribution is not completely understood. Most in vitro neurotoxicity studies concern isolated exposures of MDMA or its metabolites, at high concentrations, not considering their mixture, as expected in vivo. Therefore, our postulate is that combined deleterious effects of MDMA and its metabolites, at low micromolar concentrations that may be attained into the brain, may elicit neurotoxicity. Using human SH-SY5Y differentiated cells as dopaminergic neuronal model, we studied the neurotoxicity of MDMA and its MDMA metabolites α-methyldopamine and N-methyl-α-methyldopamine and their correspondent glutathione and N-acetylcysteine monoconjugates, under isolated exposure and as a mixture, at normothermic or hyperthermic conditions. The results showed that the mixture of MDMA and its metabolites was toxic to SH-SY5Y differentiated cells, an effect potentiated by hyperthermia and prevented by N-acetylcysteine. As a mixture, MDMA and its metabolites presented a different toxicity profile, compared to each compound alone, even at equimolar concentrations. Caspase 3 activation, increased reactive oxygen species production, and intracellular Ca2+ raises were implicated in the toxic effect. The mixture increased intracellular glutathione levels by increasing its de novo synthesis. In conclusion, this study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at low micromolar concentrations, which represents a more realistic approach of the in vivo scenario, elicited toxicity to human SH-SY5Y differentiated cells, thus constituting a new insight into the context of MDMA-related neurotoxicity.

MDMA impairs mitochondrial neuronal trafficking in a Tau‑ and Mitofusin2/Drp1‑dependent manner

Abstract Identification of the mechanisms by which drugs of abuse cause neuronal dysfunction is essential for understanding the biological bases of their acute and long-lasting effects in the brain. Here, we performed real-time functional experiments of axonal transport of mitochondria to explore the role of in situ mitochondrial dysfunction in 3,4-methylenedioxymethamphetamine (MDMA; “ecstasy”)-related brain actions. We showed that MDMA dramatically reduced mitochondrial trafficking in hippocampal neurons in a Tau-dependent manner, in which glycogen synthase kinase 3β activity was implicated. Furthermore, we found that these trafficking abnormalities were rescued by over-expression of Mitofusin2 and dynamin-related protein 1, but not of Miro1. Given the relevance of mitochondrial targeting for neuronal function and neurotransmission, our data underscore a novel mechanism of action of MDMA that may contribute to our understanding of how this drug of abuse alters neuronal functioning.

In vitro models for neurotoxicology research

The nervous system has a highly complex organization, including many cell types with multiple functions, with an intricate anatomy and unique structural and functional characteristics. The study of its (dys)functionality following exposure to xenobiotics, neurotoxicology, constitutes an important issue in neurosciences. Despite the extensive use of in vivo models to reveal the neurotoxicological phenomena, the existence of difficulties related to the increasing cost and time required for neurotoxicity studies with experimental animals, as well as the animal ethical concerns, have limited their use. Consequently, in vitro alternatives, providing an understanding of the mechanistic basis, at the molecular and cellular level, have earned a notable consideration in the field of neurotoxicological research. In this field, the selection of the most appropriate in vitro neuronal system relies on specific endpoints that are of particular relevance for the neurotoxicological phenomena that will be studied. Furthermore, application of specific endpoints to various neuronal cellular models should be done in a careful way to build reliable and feasible testing strategies. This review addresses the use of in vitro models for neurotoxicity research, aiming to contribute to a better understanding and guidance of in vitro neurotoxicological studies. As such, subcellular systems, namely isolated mitochondria and synaptosomes, and cellular models, including immortalized cell lines, primary cultures, co-cultures, organotypic cultures, neural stem cells and blood–brain barrier models, as well as their inherent advantages and limitations, are discussed.

Effect of 3,4-methylenedioxyamphetamine on dendritic spine dynamics in rat neocortical neurons--involvement of heat shock protein 27.

Along with chronic neurotoxic effects, the long-term consumption of amphetamines has been associated to psychiatric symptoms and memory disturbances. Dendritic spine dynamics have been discussed as a possible morphological correlate. However, the underlying mechanisms are still elusive. 3,4-Methylenedioxyamphetamine (MDA), a major drug of abuse and a main metabolite after 3,4-methylenedioxymethamphetamine (MDMA) intake, provokes a loss of dendritic spine-like protrusions in primary cultures of rat cortical neurons. 3,4-Methylenedioxyamphetamine also induced a rapid activation of the p38 mitogen activated protein kinase (p38 MAPK) pathway and phosphorylation of heat shock protein 27 (hsp27) indicative for its decreased chaperone activity. Concurrent pharmacological inhibition of the p38 MAPK by SB203580 abolished hsp27 phosphorylation and diminished the loss of dendritic spine-like protrusions. Moreover, upon MDA treatment dendritic spine-like protrusions were stabilized in neurons constitutively expressing hsp27. In parallel experiments we observed a robust activation of the heat shock transcription factor 1 (HSF-1) and a subsequent increase of hsp27 and hsp70. The regulation of small heat shock proteins corroborates the existence of a neuronal stress response after MDA treatment. Pharmacological targeting of small heat shock protein phosphorylation may provide a new strategy to preserve spine integrity after amphetamine exposure.