Bacterial Plate Assays and Electrochemical Methods: An Efficient Tandem for Evaluating the Ability of Catechol−Thioether Metabolites of MDMA (“Ecstasy”) to Induce Toxic Effects through Redox-Cycling

Several catechol-thioether metabolites of MDMA (ecstasy), three monoadducts, 5-(glutathion-S-yl)-N-methyl-alpha-methyldopamine (1), 5-(N-acetylcystein-S-yl)-N-methyl-alpha-methyldopamine (2), and 5-(cystein-S-yl)-N-methyl-alpha-methyldopamine (3), and two bi-adducts, 2,5-bis(glutathion-S-yl)-N-methyl-alpha-methyldopamine (4) and 2,5-bis(N-acetylcystein-S-yl)-N-methyl-alpha-methyldopamine (5), have been synthesized through an environmentally friendly one-pot electrochemical procedure. Their cytotoxicity profiles were further characterized using simple Escherichia coli plate assays and compared with those of N-methyl-alpha-methyldopamine (HHMA), dopamine (DA), and its corresponding catechol-thioether conjugates (monoadducts 6-8 and bi-adducts 9 and 10). Toxicity mediated by reactive oxygen species (ROS-TOX) was detected in the OxyR- assay, using cells sensitive to oxidative stress due to a deficiency in the OxyR protein. Toxicity arising from the high susceptibility of quinone toward endogenous nucleophiles (Q-TOX) was detected using OxyR+ cells, in the presence of tyrosinase, to promote catechol oxidation to the corresponding o-quinone. At the exclusion of 5-(cystein-S-yl) mono-conjugate 3, which was devoid of any toxicity, all compounds produced ROS-TOX, which was enhanced in the presence of tyrosinase, suggesting that the generated o-quinone (or o-quinone-thioether) species can enter redox cycles through its semiquinone radical, leading to the formation of ROS. The sequence order of toxicity was HHMA approximately = 1 approximately = 2 approximately =5 >> 7 > DA approximately = 4 > 10 > 6 > 8. In contrast, no Q-TOX arising from the binding of quinones with cellular nucleophiles was evidenced, even in the presence of tyrosinase. Finally, taking into account that several different pathways could contribute to the overall MDMA toxicity and that HHMA and catechol-thioether conjugates 1-5 have not been undoubtedly established as in vivo toxic metabolites of MDMA, it can be suggested that these compounds could participate in the toxic effects of this drug through the efficiency of redox active quinonoid centers generating ROS.