Exposure of human neuroblastoma SH-SY5Y cells to amphetamine-type stimulants leads to oxidative-antioxidative imbalance associated with DNA damage and acetylcholine antagonism

Abstract

Amphetamine-type stimulants (ATSs) are widely abused substances that impair central and peripheral nervous system functions. The mechanisms of their toxicity on human neuronal cells have not been fully clarified yet but include effects on oxidative-antioxidative balance and interaction with synaptic enzymes/receptors. The aims of this study were to determine oxidant/antioxidant status, DNA integrity and the activity of neurotransmitter system components (monoamine oxidase A, MAO-A and nicotinic acetylcholine receptors, nAChR) in human neuroblastoma SH-SY5Y cells after 24-h exposure to different ATSs. In this matter, we first evaluated cell viability by MTS assay. After determination of the concentrations (near IC₂₅) suitable for conduction of the alkaline comet assay, these were further studied for the extent of DNA damage, along with the levels of malondialdehyde, reactive oxygen species (ROS), glutathione (GSH) and activities of antioxidative enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). The activity of the MAO-A enzyme and nAChR activity were evaluated at concentration range 1–50 μM. Finally, the in silico pharmacokinetic parameters predictions of the tested ATSs were determined. When compared to untreated cells, the most notable result was obtained for amphetamine (AMP), where we observed a significant increase in ROS levels, SOD, GPx and CAT activity, and a decrease in GSH levels. Interestingly, all of the tested ATSs increased the activity of GPx, while the activities of the other enzymes were compound-dependent. The new psychoactive substance (NPS) mephedrone (4-MMC) had a similar effect as AMP, except that it did not affect CAT activity. At the tested concentrations, AMP, methamphetamine (METH) and 4-MMC also showed effects on DNA stability. None of the tested ATSs inhibited MAO-A in the tested concentration range, but AMP, METH, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) inhibited acetylcholine activation of human nAChR. Taken together, significant induction of oxidative stress parameters, increased level of DNA damage detected and inhibition of nAChR activity indicate potential mechanisms of ATS substances action, and represent the direction for further studies to clarify the toxicological risks associated with ATS and/or NPS consumption.