The in vitro hepatotoxicity of the new recreational drug 3-methylmethcathinone (3-MMC) involves autophagy and apoptosis

3-Methylmethcathinone (3-MMC) has become one of the most popular recreational drugs worldwide after the ban of mephedrone, and it has been associated with several reports of intoxications and fatal cases. Current knowledge of the effects and toxicity of 3-MMC is mainly based on user reports and clinical intoxications, while non-clinical pharmacological and toxicological data are scarce. As the liver is a target organ due to its preponderant role in metabolism of toxicants, the aim of this study was to assess the potential hepatotoxicity of 3-MMC, by evaluating its in vitro toxicity to primary rat hepatocytes (PRH).

PRH were exposed for 24 h to 3-MMC (at concentrations that varied from 31 nM to 10 mM) to evaluate mitochondrial, lysosomal and membrane toxicity, apoptosis, and autophagy as well as the modulatory effects of cytochrome P450 inhibitors on the observed effects.

3-MMC induced toxicity at the lysosome at lower concentrations (NR NOEC 312.5 µM), when compared to mitochondria (MTT NOEC 379.5 µM) and to cellular lysis (LDH NOEC 1.04 mM). A significant concentration-dependent increase of intracellular reactive oxygen/nitrogen species was observed at concentrations higher than 10 µM. At this concentration, caspase -3, -8, and -9 activities were significantly elevated compared to control, but no differences were observed at higher concentrations, suggesting a shift in cell death mechanisms. 3-MMC also elicited concentration-dependent morphological changes consistent with autophagy, including formation of acidic vesicular organelles. No significant alterations were observed in the mitochondrial membrane potential (??m). Intracellular content of ATP slightly increased at 1 µM and significantly decreased at the highest concentrations (100 µM and 500 µM). A concentration-dependent decrease in antioxidant defences (GSH) with a concurrent increase of GSSG was also observed. CYP2D6 and CYP2E1 inhibition diminished 3-MMC-induced toxicity, but for CYP2E1 this effect was only observed for concentrations up to 1.3 mM.

Overall, our data point to a role of metabolism in the hepatotoxicity of 3-MMC, which appears to be associated to both autophagic and apoptotic mechanisms. The current work is a first attempt to better understand the toxicology of 3-MMC.