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Amphetamine-type psychostimulants (ATS) are used worldwide by millions of patients for several psychiatric disorders. Amphetamine (AMPH) and "ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) are common drugs of abuse. The impact of chronic ATS exposure to neurons and brain aging is still undisclosed. Current neuronal culture paradigms are designed to access acute ATS toxicity. We report for the first time a model of chronic exposure to AMPH and MDMA using long-term rat cortical cultures. In two paradigms, ATS were applied to neurons at day 1 in vitro (DIV) (0, 1, 10 and 100 μM of each drug) up to 28 days (200 μM was applied to cultures up to 14 DIV). Our reincubation protocol assured no decrease in the neuronal media's drug concentration. Chronic exposure of neurons to concentrations equal to or above 100 μM of ATS up to 28 DIV promoted significant mitochondrial dysfunction and elicited neuronal death, which was not prevented by glutamate receptor antagonists at 14 DIV. ATS failed to promote accelerated senescence as no increase in β-galactosidase activity at 21 DIV was found. In younger cultures (4 or 8 DIV), AMPH promoted mitochondrial dysfunction and neuronal death earlier than MDMA. Overall, AMPH proved more toxic and was the only drug that decreased intraneuronal glutathione levels. Meanwhile, caspase 3 activity increased for either drug at 200 μM in younger cultures at 8 DIV, but not at 14 DIV. At 8 DIV, ATS promoted a significant change in the percentage of neurons and astroglia present in culture, promoting a global decrease in the number of both cells. Importantly, concentrations equal to or below 10 μM of either drug did not promote neuronal death or oxidative stress. Our paradigm of neuronal cultures long-term exposure to low micromolar concentrations of ATS closely reproduces the in vivo scenario, being valuable to study the chronic impact of ATS., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

3,4-Methylenedioxymethamphetamine (MDMA or "ecstasy"), is a widely abused, psychoactive recreational drug that is known to induce neurotoxic effects. Human and rat hepatic metabolism of MDMA involves N-demethylation to 3,4-methylenedioxyamphetamine (MDA), which is also a drug of abuse. MDMA and MDA are O-demethylenated to N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and alpha-methyldopamine (alpha-MeDA), respectively, which are both catechols that can undergo oxidation to the corresponding ortho-quinones. Ortho-quinones may be conjugated with glutathione (GSH) to form glutathionyl adducts, which can be transported into the brain and metabolized to the correspondent N-acetylcysteine (NAC) adducts. In this study we evaluated the neurotoxicity of nine MDMA metabolites, obtained by synthesis: N-Me-alpha-MeDA, alpha-MeDA and their correspondent GSH and NAC adducts. The studies were conducted in rat cortical neuronal cultures, for a 6 h of exposure period, under normal (36.5 degrees C) and hyperthermic (40 degrees C) conditions. Our findings show that thioether MDMA metabolites are strong neurotoxins, significantly more than their correspondent parent catechols. On the other hand, N-Me-alpha-MeDA and alpha-MeDA are more neurotoxic than MDMA. GSH and NAC conjugates of N-Me-alpha-MeDA and alpha-MeDA induced a concentration dependent delayed neuronal death, accompanied by activation of caspase 3, which occurred earlier in hyperthermic conditions. Furthermore, thioether MDMA metabolites time-dependently increased the production of reactive species, concentration-dependently depleted intracellular GSH and increased protein bound quinones. Finally, thioether MDMA metabolites induced neuronal death and oxidative stress was prevented by NAC, an antioxidant and GSH precursor. This study provides new insights into the neurotoxicity mechanisms of thioether MDMA metabolites and highlights their importance in "ecstasy" neurotoxicity.

Studies on 3,4-methylenedioxymethamphetamine ("ecstasy")-induced neurotoxicity mainly focus on damage of serotonergic terminals. Less attention has been given to neuronal cell death produced by 3,4-methylenedioxymethamphetamine and other amphetamines in areas including the cortex, striatum and thalamus. In the present study we investigated 3,4-methylenedioxymethamphetamine-induced neurotoxicity in neuronal serum free cultures from rat cortex. Since 3,4-methylenedioxymethamphetamine intake induces hyperthermia in both animals and humans, the experiments were performed under normal (36.5 degrees C) and hyperthermic conditions (40 degrees C). Our findings showed a dose-, time- and temperature-dependent apoptotic cell death induced by 3,4-methylenedioxymethamphetamine in cortical neurons. 3,4-Methylenedioxymethamphetamine-induced damage was potentiated under hyperthermia. The neurotoxicity was reduced by the serotonin 2A-receptor antagonists, ketanserin and (2R,4R)-5-[2-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]ethyl]-1-methyl-3-pyrrolidinol hydrochloride, in both normothermic and hyperthermic conditions. (+/-)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride, a model agonist for the serotonin 2A-receptor, also induced a dose- and time-dependent apoptotic cell death. Again, protection was provided by ketanserin and (2R,4R)-5-[2-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]ethyl]-1-methyl-3-pyrrolidinol hydrochloride against (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride-induced neurotoxicity, thereby indicating that the 3,4-methylenedioxymethamphetamine stimulation of the serotonin 2A-receptor leads to neurotoxicity. This study provides for the first time evidence that direct 3,4-methylenedioxymethamphetamine serotonin 2A-receptor stimulation leads to neuronal cortical death. alpha-Phenyl-N-tert-butyl nitrone a free radical scavenger and the nitric oxide synthase inhibitor Nomega-nitro-L-arginine as well as the NMDA-receptor antagonist MK-801 provided protection under normothermia and hyperthermia, thereby suggesting the participation of free radicals in 3,4-methylenedioxymethamphetamine-induced cell death. Since 3,4-methylenedioxymethamphetamine serotonin 2A-receptor agonistic properties lead to neuronal death, clinically available atypical antipsychotic drugs with serotonin 2A-antagonistic properties could be a valuable therapeutic tool against 3,4-methylenedioxymethamphetamine-induced neurodegeneration.