Molecular toxicological mechanisms of new psychoactive substances "in vitro"

In recent years, many “new psychoactive substances” (NPS), such as amphetamine and synthetic cathinone derivatives, have dramatically appeared on the illegal market, and the abuse of these drugs is now a global crisis. The mechanisms of cytotoxicity associated with NPS are still unclarified. The aims of this thesis were to comprehensively evaluate the mechanisms of NPS-induced myotoxicity, hepatotoxicity and neurotoxicity in vitro, and as well as to assess the role of hyperthermia on methcathinone-induced neurotoxicity. Therefore, we treated human hepatoma HepG2 cells, mouse muscle C2C12 cells, and human neuroblastoma SH-SY5Y cells with amphetamines and synthetic cathinones at concentrations from 50-2000 μM. In the first paper, we focused on the toxicological effects in C2C12 cells of the following synthetic cathinones: 3,4-methylenedioxymethcathinone (methylone), 4-methylmethcathinone (4-MMC, mephedrone), 3-methylmethcathinone (3-MMC), methylenedioxypyrovalerone (MDPV), α-pyrrolidinopentiophenone (α-PVP), and naphthylpyrovalerone (naphyrone). All the investigated synthetic cathinones showed a concentration-dependent impairment of the cell membrane integrity, a drop in intracellular adenosine triphosphate (ATP) content, and an increase of mitochondrial superoxide concentrations. α-PVP and naphyrone impaired basal and maximal cellular respiration and inhibited the activities of complex I and II of the electron transport chain (ETC). These results indicated mitochondrial dysfunction associated with these drugs. In conclusion, α-PVP and naphyrone showed mitochondrial toxicity after 24 h exposure. In comparison, the cytotoxic effects of methylone, 4-MMC (mephedrone), 3-MMC and MDPV were related to an impairment of glycolysis rather than inhibition of mitochondrial pathways. The goal of the second paper was to investigate the pharmacological profile and the potential hepatotoxicity of para-halogenated amphetamines and cathinones in vitro. We determined the pharmacological profile in transporter-transfected human embryonic kidney 293 cells (HEK 293). Amphetamine, 4-fluoroamphetamine (4-FA), 4-chloroamphetamine (PCA), methcathinone (MC), 4-fluoromethcathinone (4-FMC) and 4-chloromethcathinone (4-CMC) inhibited the norepinephrine transporter (NET) and the dopamine transporter (DAT). Moreover, the inhibition of these compounds on the dopamine versus the serotonin transporter, showed selectivity in their activity, which decreased together with the increasing size of the para-substituents, resulting in an inhibition of the serotonin uptake. Concerning the assessment of hepatocellular toxicity, we found that all substances induced membrane toxicity, depletion of the intracellular ATP content and formation of reactive oxygen species (ROS) in HepG2 cells. The decrease in the ATP content was at a lower concentration than the damage of the cell membrane integrity, which suggests mitochondrial toxicity. Furthermore, amphetamines and 4-CMC impaired the mitochondrial respiratory chain, confirming their nature as mitochondrial toxicants. Finally, both 4-FA and 4-CMC induced apoptosis and necrosis in HepG2 cells. Taken together, para-halogenation of amphetamines and cathinones increase the risk for serotonergic neurotoxicity, which may induce hyperthermia in vivo. The toxicity rank of the substitutes was the following: chloride > fluoride > hydrogen. The purpose of the third study was to characterize the mechanisms of neurotoxicity of amphetamine, 4-FA, PCA, methcathinone (MC), 4-FMC and 4-CMC. 4-FA, PCA and 4-CMC strongly impaired membrane integrity, depleted ATP intracellular content and decreased the mitochondrial membrane potential of undifferentiated and differentiated neuronal SH-SY5Y cells, indicating mitochondrial toxicity. Moreover, PCA and 4-CMC inhibited the function of the ETC, increased ROS and induced apoptosis for both cell types. Besides that, caspase 3 and 9 were activated after 4-CMC exposure. In conclusion, PCA and 4-CMC impaired the function of mitochondria and induced apoptosis in undifferentiated and differentiated SH-SY5Y cells, while 4-FA depleted ATP content, increased ROS formation, and decreased mitochondrial membrane potential in undifferentiated SH-SY5Y cells. This study further supported the toxicity rank of para-halogenated amphetamines and cathinones (Cl > F > H). In the last study, we investigated the effects of hyperthermia (40.5 °C) on the neurotoxicity of MC, 4-MMC and 4-CMC in SH-SY5Y cells. We found that 4-MMC and 4-CMC caused cell membrane damage, decreased intracellular ATP content, impaired the function of ETC, and increased ROS levels at both thermic conditions. At the hyperthermic condition (40.5 °C), SH-SY5Y cells exposed to test drugs were more sensitive than at the normothermic condition (37 °C). MC also induced an increase of ROS and inhibited ETC, however only at the hyperthermic condition. Moreover, hyperthermia reduced drug-induced apoptosis by promoting the expression of the 70 kilodalton heat shock proteins (Hsp70), but was associated with late autophagy and cell death. In conclusion, hyperthermic conditions increased the neurotoxic properties of methcathinones due to enhanced impairment of mitochondrial function and induced late autophagy and cell death when early protective measures were overwhelmed.