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Neurotoxicity of #-keto amphetamines: deathly mechanisms elicited by methylone and MDPV in human dopaminergic SH-SY5Y cells Maria Joao Valente, Maria Lourdes Bastos, Eduarda Fernandes, Félix Carvalho, Paola Guedes de Pinho, and Marcia Carvalho ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00421 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 9, 2017
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ACS Chemical Neuroscience
Neurotoxicity of β-keto amphetamines: deathly mechanisms elicited by methylone and MDPV in human dopaminergic SHSY5Y cells
Maria J. Valente1,*, Maria de Lourdes Bastos1, Eduarda Fernandes2, Félix Carvalho1, Paola Guedes de Pinho1 and Marcia Carvalho1,3,* 1
UCIBIO-REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Porto,
Portugal 2
UCIBIO-REQUIMTE, Laboratory of Applied Chemistry, Faculty of Pharmacy, University of Porto,
Porto, Portugal 3
FP-ENAS, CEBIMED, Fundação Ensino e Cultura Fernando Pessoa, Porto, Portugal
Corresponding authors: Tel.: 00351220428596 Fax: 00351226093390 e-mail addresses:
[email protected] (M. J. Valente);
[email protected] (M. Carvalho)
Keywords: β-keto amphetamines; synthetic cathinones; neurotoxicity; oxidative stress; mitochondrial impairment; apoptosis
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Abstract Synthetic cathinones (β-keto amphetamines) act as potent CNS stimulants similarly to classical amphetamines, which raise concerns about their potential neurotoxic effects. The present in vitro study aimed to explore and compare the mechanisms underlying the neurotoxicity of two commonly abused cathinone derivatives, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxypyrovalerone (MDPV), with those of 3,4-methylenedioxymethamphetamine (MDMA), using undifferentiated and differentiated SH-SY5Y cells. Following a 24 h exposure period, methylone and MDPV induced loss of cell viability in a concentration-dependent manner, in the following order of potency: MDPV ≈ MDMA > methylone. Dopaminergic differentiated cells evidenced higher sensitivity to the neurotoxic effects of both cathinones and MDMA than the undifferentiated ones, but this effect was not inhibited by the DAT inhibitor GBR 12909. Intracellular oxidative stress mediated by methylone and MDPV was demonstrated by the increase in reactive oxygen and nitrogen species (ROS and RNS) production, depletion of intracellular reduced glutathione and increased oxidized glutathione levels. All three drugs elicited mitochondrial impairment, characterized by the mitochondrial membrane potential (∆ψm) dissipation and intracellular ATP depletion. Apoptosis was found to be a common mechanism of cell death induced by methylone and MDPV, with evident chromatin condensation and formation of pyknotic nuclei, and activation of caspases -3, -8 and -9. In conclusion, the present data shows that oxidative stress and mitochondrial dysfunction play a role in cathinones-induced neuronal damage, ultimately leading to cell death by apoptosis.
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ACS Chemical Neuroscience
Introduction Synthetic cathinones emerged this century as a novel class of recreational designer drugs and have grown to be popular drugs of abuse worldwide, with special incidence among young adults1. These substances, also known as β-keto amphetamines, are chemically related to classical amphetamines, bearing a ketone group at the β-position of the side chain2, and act essentially as stimulants of the central nervous system (CNS)3. Synthetic cathinones are currently the second largest group of new psychoactive substances (NPS) monitored globally4, 5, with 103 different derivatives identified so far5. Methylone and MDPV are two of the most commonly abused derivatives worldwide6, and belong to different chemical subgroups of cathinones: methylone is the β-keto analogue of MDMA, whereas MDPV is a pyrrolidinophenone analogue, containing a pyrrolidine group besides the 3,4-methylenedioxy ring7. Similarly to long-standing drugs of abuse such as amphetamines and cocaine, the psychostimulant effects of β-keto amphetamines arise from their interaction with monoamine membrane transporters, leading to increased levels of catecholamines in the synaptic cleft and consequent sympathetic overstimulation2. However, the type of interaction and affinity towards the different transporters vary among derivatives8. In fact, methylone acts as a non-selective substrate for monoamine transporters, like MDMA and methamphetamine, although with inferior potency9, whereas MDPV functions as a pure transporter blocker, like cocaine, strongly inhibiting the uptake of dopamine (DA) and norepinephrine (NE), but with minimal effects on serotonin (5-HT) transporter10. Subjective effects differ among derivatives. Methylone induces MDMA-like stimulating effects, such as euphoria, increased energy and openness, mood lift and empathogenic feelings11. On the other hand, due to its affinity towards DA and NE transporters (DAT and NET), MDPV induces more cocaine-like subjective effects, including increased alertness and sexual stimulation, but limited euphoria and only mild, if any, empathogenic effects12,
13
. Higher doses and long-term abuse of these substances lead to unwanted
psychological effects, including anxiety and panic attacks, hallucinations and psychosis14. Neurotoxic effects of non-keto amphetamines have been thoroughly reported, and the main mechanisms by which these substances induce neuronal damage include neuroinflammation, excitotoxicity, oxidative stress and mitochondrial damage15-17. In vitro studies regarding the ability of cathinone derivatives to induce oxidative stress and mitochondrial dysfunction have recently been published, providing some light on the mechanisms possibly involved in β-keto amphetamines cytotoxicity in hepatic18-21 and brain cells21-25. However, studies at a cellular level on the mechanisms of synthetic cathinones-induced neurotoxicity are still scarce and require clarification. Therefore, in the present work, we assessed the neurotoxic potential of methylone
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and MDPV in cultured human dopaminergic cells and investigated the mechanisms related to oxidative stress, mitochondrial dysfunction, and type of cell death.
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Results and discussion Dopaminergic phenotype renders SH-SY5Y cells more susceptible to the cytotoxicity of β-keto amphetamines Though the undifferentiated SH-SY5Y cell line expresses several dopaminergic markers, differentiated SH-SY5Y cells are more similar to primary neurons, with a mature neuron-like phenotype26. The desired phenotype determines the differentiation method. Generally, amphetamines and β-keto amphetamines present a strong affinity towards DAT, though with different potencies of binding and transporter-mediated release and/or uptake of monoamines. For instance, although the DAT binding affinity of methylone was shown to be 2.4-times higher than MDMA27, the potency of actual DAT-mediated release induced by this β-keto amphetamine appears to be much lower than its non-keto analogue9, 27. On the other hand, MDPV, which presents substantial membrane permeability and is actively transported through the blood-brain barrier (BBB), was shown to have a binding affinity to DAT 650-times higher than MDMA27 and strongly inhibits DAT-mediated DA reuptake, with a effectiveness 51-times over cocaine10 and 548-times over MDMA27. Considering this strong interaction of β-keto amphetamines with DAT, RA-TPA exposure was the selected method for the present study, which prompts cell differentiation into a more dopaminergic phenotype28. In order to evaluate the relevance of differentiation to the neurotoxicity elicited by the β-keto amphetamines, the neurotoxic potential of MDMA, methylone and MDPV was measured through the MTT reduction assay in both undifferentiated and RA-TPA differentiated cells after a 24 h exposure. Figure 1 presents the concentration–response curves for each drug in the two in vitro models, as well as the estimated EC50 for each compound. With curve fits significantly shifted to the right, undifferentiated SH-SY5Y cells proved to be more resistant to drug-induced cell death (p
