Keto Amphetamine Toxicity-Focus on the Redox Reactivity of the Cathinone Designer Drug Mephedrone

The beta-keto amphetamine (cathinone, beta-KA) designer drugs such as mephedrone (4-methylmethcathinone, 4-MMC) show a large degree of structural similarity to amphetamines like methamphetamine (METH). However, little is currently known about whether these substances also share the potential neurotoxic properties of their non-keto amphetamine counterparts, or what mechanisms could be involved. Here, we evaluate the cytotoxicity of beta-KAs in SH-SY5Y cells using lactate dehydrogenase (LDH) assays, assess the redox potential of a range of beta-KAs and non-keto amphetamines using the sensitive redox indicator 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), and explore the effect of 4-MMC on the formation of protein adducts using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) and on the mitochondrial respiratory chain using high-resolution respirometry. We show that treatment with beta-KAs increases LDH release. Further, we demonstrate that even under physiological pH, beta-KAs are effective and selective-as compared with their non-keto analogues-reductants in the presence of electron acceptors. Increased pH (range 7.6-8.0) greatly enhanced the reactivity up to sixfold. We found no evidence of protein adduct formation, suggesting the reactivity is due to direct electron transfer by the beta-KAs. Finally, we show that 4-MMC and METH produce dissimilar effects on the respiratory chain. Our results indicate that beta-KAs such as 4-MMC possess cytotoxic properties in vitro. Furthermore, in the presence of an electron-accepting redox partner, the ketone moiety of beta-KAs is vital for pH-dependent redox reactivity. Further work is needed to establish the importance of beta-KA redox properties and its potential toxicological importance in vivo.