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4-Methylthioamphetamine (MTA) is a phenylisopropylamine derivative whose use has been associated with severe intoxications. MTA is usually regarded as a selective serotonin-releasing agent. Nevertheless, previous data have suggested that its mechanism of action probably involves a catecholaminergic component. As little is known about dopaminergic effects of this drug, in this work the actions of MTA upon the dopamine (DA) transporter (DAT) were studied in vitro, in vivo and in silico. Also, the possible abuse liability of MTA was behaviourally assessed. MTA exhibited an in vitro affinity for the rat DAT in the low micromolar range (6.01 μM) and induced a significant, dose-dependent increase in striatal DA. MTA significantly increased c-Fos-positive cells in striatum and nucleus accumbens, induced conditioned place preference and increased locomotor activity. Docking experiments were performed in a homology model of the DAT. In conclusion, our results show that MTA is able to increase extracellular striatal DA levels and that its administration has rewarding properties. These effects were observed at concentrations or doses that can be relevant to its use in human beings.
4-Methylthioamphetamine (MTA) is a phenylisopropylamine derivative originally synthesized and evaluated as an anorectic drug more than 40 years ago . Subsequently, it was demonstrated that MTA is a potent, selective and non-neurotoxic serotonin (5-HT)-releasing agent in vitro [2, 3] and in vivo , an effect that is mediated via the 5-HT transporter (SERT) [5, 6]. MTA gained notoriety in the late 1990s as a street drug commonly known as ‘flatliner’, and its use has been associated with severe intoxications and several deaths [7-10]. Even though MTA is usually regarded as a selective serotonergic agent, it also potently inhibits monoamine oxidase-A (MAO-A) [4, 11], and both hyperthermia  and aortic contraction  induced by MTA in rodent models can be blocked by α-adrenergic antagonists. In addition, it has been shown that MTA induces dopamine (DA) release from rat striatal synaptosomes pre-loaded with [3H]-DA  at concentrations that might be relevant after recreational use in human beings [8, 14]. This background indicates that the overall mechanism of action of MTA may be more complex than originally thought and probably involves a catecholaminergic component. Information about neurochemical effects of MTA is relatively scarce as compared with other abused amphetamine derivatives such as methylenedioxymethamphetamine (MDMA, ‘ecstasy’). Also, despite its adverse effects, some potential clinical uses, for example related to its antidepressant and pro-apoptotic activities, have been suggested for this drug [4, 15, 16]. Consequently, we have now further characterized the dopaminergic effects of MTA. Thus, its actions upon the DA transporter (DAT) were studied in vitro and in silico. In addition, the consequences of its in vivo administration on striatal DA extracellular levels as well as its possible abuse liability were assessed.
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In the present study, we demonstrate that MTA is able to induce an increase in extracellular striatal DA levels and that the drug has rewarding effects. Furthermore, our results suggest that these effects are mediated, at least in part, by the interaction of MTA with the DAT.
Initial reports on the recreational use of MTA in human beings appeared in the late 1990s [7, 40, 41]. Its apparent slow onset of action and longer-lasting effects as compared for example with MDMA  might be a factor for the use of relatively high doses of the drug. This had been associated with several fatal and non-fatal intoxications, and in the cases where blood samples were measured, concentrations of MTA ranged from 0.7 to 28.9 μM [8, 9, 14, 43]. Even though blood levels of MTA found in clinical determinations cannot be straightforwardly extrapolated to actual concentrations in the brain, they give a reasonable range of possible MTA brain concentrations. In this context, it is of particular significance that dopaminergic effects of MTA were observed in rats at intra-striatal concentrations or systemic doses that could be relevant to its use in human beings.
In agreement with original reports on the MTA mechanism of action [2, 3], MTA exhibited an in vitro affinity for rDAT (defined here as the ability to displace a selective 3H-labelled transporter ligand) 5–6 times lower than that reported for rSERT  (table 1). Accordingly, the dopaminergic neurochemical effects we observed in vivo at doses of MTA of 5 mg/kg or lower were, although significant, much weaker than its effects on 5-HT. Thus, while a dose of 5 mg/kg induced an approximately 1.5 times striatal DA increase (this work), the same dose increases the extracellular concentration of hippocampal 5-HT more than 20 times, measured under similar conditions . Interestingly, when a higher dose of MTA (10 mg/kg) was administered, a much stronger effect (>20 times increase) upon striatal DA levels was observed. Beyond possible mechanistic implications (see below), this abrupt rise of extracellular DA concentrations further supports the notion that a dopaminergic component should be considered when assessing the overall effects of MTA, particularly at relatively high doses.
As mentioned, the potent 5-HT-releasing action of MTA is related to its being a SERT substrate . As some dopaminergic effects of compounds with similar properties (e.g. MDMA) have been shown to be mediated by the activation of 5-HT receptors [45, 46], a possible serotonergic influence on the effects of MTA on striatal DA was evaluated by concomitantly perfusing the 5-HT uptake blocker citalopram. In addition, as some of the effects of MTA upon DA extracellular levels might be mediated by its actions upon noradrenergic transmission, drug effects were evaluated in presence of the noradrenalin uptake blocker desipramine. The lack of effect of either citalopram or desipramine on the MTA-induced increase in DA indicates that this MTA effect is largely independent of its actions on 5-HT and/or noradrenalin transport. As MAO-A inhibitory properties have been reported for MTA , further studies are necessary to evaluate the possibility that part of the effects of MTA are related to MAO-A inhibition.
Our immunohistochemical results demonstrated that MTA administration enhances neuronal activity in both the striatum and nucleus accumbens. This suggests that MTA also increases DA levels in the latter region and that therefore it might have rewarding properties. This hypothesis was behaviourally tested, and similarly to other abused amphetamine derivatives, MTA increased locomotor activity and induced CPP. Altogether, our results document reward-like effects of this recreational drug. This finding adds a further concern regarding MTA misuse in human beings and underlines the need for additional work on monoamine-releasing agents .
Even though, by definition, theoretical simulations yield speculative information, the two-pocket arrangement found for the rDAT is in accordance with the existence of two substrate binding sites experimentally found in LeuTAa . In addition, both cavities were positioned in regions similar to those recently proposed as possible binding sites for DA (and other substrates and blockers) in the human DAT . In general terms, the binding interactions proposed here for MTA in the rDAT are in good agreement with those suggested for structurally similar DAT substrates such as DA or amphetamine at S1 in the rDAT  and S2 in the human DAT [48, 49]. Interactions between the ligand amino group and Asp79 and Asp475 (Asp476 in the human DAT) seem to be of particular importance for MTA binding at S1 and S2, respectively. This observation agrees with the role of both acidic residues in the recognition of DAT substrates and blockers, as demonstrated by mutagenesis studies .
Regarding the possible mechanism of action of MTA, the IC50 values for radioligand displacement and for [3H]DA uptake inhibition are very similar, yielding a binding/uptake ratio ≈ 1.2 (table 1). On the other hand, it has been shown that MTA is about 10 times more potent inhibiting [3H]5-HT uptake than displacing [3H]citalopram binding, as assessed using a rat hippocampal synaptosome preparation . As discussed by Schmitt et al.  (and references therein), these differential potencies on binding/uptake inhibition are an indication of whether a ligand is a transporter substrate or a blocker. In this context, our results agree with the evidence showing that MTA is a substrate of SERT, while suggesting that it might be a DAT blocker. However, using superfused striatal synaptosomes (an experimental model which dissociates releasing effects from uptake inhibition), Gobbi et al.  have unequivocally shown that MTA behaves as a DA-releasing agent at concentrations of 10 μM or higher.
To theoretically evaluate this issue, we performed flexible docking experiments of MTA and DA at S1. The distance between Asp79 and Tyr156 after the docking of a given ligand has been proposed as indicative of the nature of its interaction with the DAT [48, 51]. Our simulation study revealed that after docking of DA or MTA, the distances between these residues (as measured between the respective side-chain oxygen atoms) were 1.9 Å and 3.2 Å, respectively, which are indicative of a preserved hydrogen bond (fig. S3). On the other hand, docking of WIN 35,428, which was included to compare our docking results with those of previous studies [48, 51], resulted in a distance of 4.2 Å, which is greater than the maximum distance (3.5 Å) proposed for a hydrogen bond (fig. S3). Therefore, these data also support the view that MTA could be a DAT substrate.
These experimental (binding/superfusion) and theoretical (flexible docking) observations agree with the profile we observed in microdialysis studies, where the effect of MTA on DA levels at 5 mg/kg resembles that of uptake blockers, while the effect at 10 mg/kg is more similar to that produced by DA-releasing agents . In this context, it is tempting to speculate that the mechanism of action of MTA at the DAT might switch from an uptake blockade at low concentrations to a substrate-like behaviour at higher concentrations. Recent experimental and computational studies [47, 49] have shown that transport by the DAT (and LeuTAa) requires not only the presence of the substrate in S1, but also the binding of a second substrate molecule at the more external S2. This latter event would trigger a series of conformational transitions of the protein, leading to the intracellular release of the substrate initially bound at S1 . Based on these findings, we propose that MTA could block DAT by binding with higher affinity at S1 but, as the drug concentration increases, the binding of a second MTA molecule at S2 might trigger both the transport of the compound and the efflux of DA. Further experiments are necessary to test this hypothesis.
In conclusion, neurochemical effects on DA and their functional consequences have been demonstrated in vivo for MTA. Even though MTA can still be considered as a predominantly serotonergic drug, it also can modify dopaminergic neurotransmission, and this relative promiscuity should be considered while assessing the global effects of the drug, particularly when used by human beings.