‘Ecstasy’ and amphetamine neurotoxicity to cultured rat cortical neurons in a continuous exposure model

Silvana da Costa Araújo1, Vera Marisa Costa1, Frederico C. Pereira2, Eduarda Fernandes3, Maria de Lourdes Bastos1, Félix Dias Carvalho1, João Paulo Capela1,4. 1REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal, 2Farmacologia e Terapêutica Experimental, Instituto Biomédico de Investigação da Luz e Imagem (IBILI), Faculdade de Medicina, Universidade de Coimbra, 3000-548 Coimbra, Portugal, 3REQUIMTE, Laboratório de Química-Física, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal, 4Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, 4200-150 Porto, Portugal

Background: Amphetamine-like psychostimulants are used worldwide by millions of patients in the clinical treatment of narcolepsy, obesity, and even attention deficit hyperactivity disorder in children and adolescents despite concerns on their high abuse potential and neurotoxicity. Amphetamine itself (AMPH) or ‘Ecstasy’ (3,4-methylenedioxymethamphetamine or MDMA) are also major drugs of abuse. Consequences to the brain of chronic exposure to these drugs are presently undisclosed.

Aims: Examine the potential toxic effects of continuous exposure to MDMA or AMPH on cultured neurons from rat cortex.

Methods: Primary neuronal cultures of cortex were obtained from Wistar rat embryos (E-17 to E-19). One day after seeding [1 day in vitro (DIV)], cultures were exposed to MDMA or AMPH in the concentrations of 0, 1, 10, 100 or 200 μM. Cultures were fed at 4 and 8 DIV, by addition of new medium that, in treated cells, contained the appropriate concentration of the drug. At 4, 8, and 14 DIV several assays were performed: mitochondrial dysfunction by the tetrazolium-based (MTT) assay, life-death cellular assay based on the lactate dehydrogenase (LDH) release, and the activity of caspase 3. In the neuronal medium, the concentrations of AMPH and MDMA were quantified by HPLC at the latter time-points, 8 and 14DIV, and in the higher concentrations tested. Statistical analysis was conducted using one-way ANOVA to compare means from different treatment groups, and two-way ANOVA was used to compare means from different groups coming from different time-points. Analysis was followed by a post-hoc test, if a significant p value had been obtained (p<0.05).

Results: The concentration of 100μM of AMPH and MDMA was maintained within the range up to 14DIV. From 8DIV to 14DIV, there was a slight, but statistically significant, concentration increase (10 to 15%) in the medium of neurons exposed to 200μM of both drugs, most likely due to evaporation of culture medium. MDMA promoted mitochondrial dysfunction at all time points tested for the 200μM concentration, while only at the 8DIV for 100μM. Moreover, cell death was also significant for the highest concentration (200μM) at all time-points, but only significant for the 100μM concentration at 8DIV. Concerning AMPH, mitochondrial dysfunction was significant at all time points tested for both 100 and 200μM concentrations. Moreover, at 8DIV mitochondrial dysfunction was significant for 1 and 10μM. AMPH promoted evident cell death for the highest concentration at all time-points, and also for 100μM at 4DIV and 8DIV. Only for the highest concentration tested of both drugs, 200 μM, at 8 DIV there was an increase in caspase 3 activity. Overall, both drugs promoted a concentration dependent neurotoxicity, which was more pronounced at the 8DIV time-point in the parameters measured. On the other hand, AMPH proved to be more toxic than MDMA.

Conclusions: A cultured neuronal model of continuous exposure to amphetamines can be very valuable to determine their chronic brain consequences using lower concentrations than the ones usually applied in other in vitro studies. Further studies are required to reveal other effects of exposure to low concentrations of amphetamines in neurons and their consequences in the aging process.

Acknowledgments: This work was supported by “Fundação para a Ciência e Tecnologia” (FCT) [PTDC/SAU-FCF/102958/2008]-QREN initiative with EU/FEDER financing through COMPETE. VMC and JPC acknowledge FCT for their Post-doc grants (SFRH/BPD/63746/2009 and SFRH/BPD/30776/2006).