Volume 10 Supplement 1

16th Scientific Symposium of the Austrian Pharmacological Society (APHAR)

Open Access

4-Methylthioamphetamine (4-MTA) induces mitochondrial-dependent apoptosis in SH-SY5Y cells independently of dopamine and noradrenaline transporters

BMC Pharmacology201010(Suppl 1):A22

https://doi.org/10.1186/1471-2210-10-S1-A22

Published: 16 November 2010

Background

3,4-Methylenedioxymethamphetamine (MDMA or ‘ecstasy’) tablets are frequently contaminated by 4-MTA (‘flatliner’), an amphetamine derivative which is known to induce severe human intoxication and even death. Although an equipotent inducer of SERT-dependent 5-HT release in vivo, 4-MTA does not induce MDMA-like serotoninergic neurotoxicity in rats. Instead, 4-MTA users typically report unpleasant sympathomimetic effects such as tachycardia, tremors, stomach cramps, headache and sweating following ingestion. Here, for the first time we investigate the cytotoxic potency of 4-MTA in a catecholaminergic system.

Methods

SH-SY5Y cells express both dopamine and noradrenaline transporters (DAT, NET) in the presence of vesicular monoamine transporter 2 (VMAT2) and were therefore chosen as the ideal catecholaminergic model in which to examine the molecular mechanisms of 4-MTA and MDMA-induced cytotoxicity in vitro. Cell viability was determined using the MTT assay and validated using flow cytometry via PI exclusion. ROS production, mitochondrial membrane potential (MMP), apoptosis and the cell cycle were examined via flow cytometry using DCFH2DA, JC-1, annexin V/PI and PI respectively. The level of intracellular calcium was determined ratiometrically by confocal microscopy using two visible wavelength Ca2+-sensitive dyes, Fluo-3 and Fura Red.

Results

4-MTA was significantly more cytotoxic than MDMA at 24 h, demonstrating an EC50 of 0.60 mM in contrast to 2.01 mM for MDMA. In addition, the combination of MDMA and 4-MTA at low concentrations significantly increased cytotoxicity compared to that of each drug alone. 4-MTA-induced cell death was reduced by the anti-oxidant N-acetyl-L-cysteine (NAC) but not by the non-selective monoamine transport inhibitor indatraline, indicating that monoamine transport is not a requirement of 4-MTA-induced cytotoxicity. Drug-induced cell death was pre-empted by rapid intracellular Ca2+ influx, mitochondrial membrane depolarization (MMD), ROS production and caspase 9 activation. MDMA and 4-MTA also induced phosphatidlyserine exposure and caspase-dependent DNA fragmentation at 24 h indicative of cell death via apoptosis.

Conclusions

Although both MDMA and 4-MTA induced apoptosis via the mitochondrial death pathway, 4-MTA does so at more physiologically relevant concentrations and may therefore be a potent synergistic adjunct when mixed with MDMA.

Declarations

Acknowledgements

This work was supported by the Health Research Board (HRB), Ireland.

Authors’ Affiliations

(1)
Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna
(2)
Conway Institute of Biomolecular and Biomedical Research, University College Dublin

Copyright

© Montgomery et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd.

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