Abstract
Autophagy has an essential role in neuronal homeostasis and its dysregulation has been recently linked to neurotoxic effects of a growing list of psychoactive drugs, including amphetamines. However, the role of autophagy in β-keto amphetamine (β-KA) designer drugs-induced neurotoxicity has hitherto not been investigated. In the present study, we show that two commonly abused cathinone derivatives, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxypyrovalerone (MDPV), elicit morphological changes consistent with autophagy and neurodegeneration, including formation of autophagic vacuoles and neurite retraction in dopaminergic SH-SY5Y cells. Methylone and MDPV prompted the formation of acidic vesicular organelles (AVOs) and lead to increased expression of the autophagy-associated protein LC3-II in a concentration- and time-dependent manner. Electron microscopy confirmed the presence of autophagosomes with typical double membranes and autolysosomes in cells exposed to both β-KA. The autophagic flux was further confirmed using bafilomycin A1, a known inhibitor of the late phase of autophagy. Moreover, we showed that autophagy markers were activated before the triggering of cell death and caspase 3 activation, suggesting that β-KA-induced autophagy precedes apoptotic cell death. To address the role of oxidative stress in autophagy induction, we also investigated the effects of antioxidant treatment with N-acetyl-l-cysteine (NAC) on autophagy and apoptotic markers altered by these drugs. NAC significantly attenuated methylone- and MDPV-induced cell death by completely inhibiting the generation of reactive oxygen and nitrogen species, and hampering both apoptotic and autophagic activity, suggesting that oxidative stress plays an important role in mediating autophagy and apoptosis elicited by these drugs.
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References
Adam A, Gerecsei LI, Lepesi N, Csillag A (2014) Apoptotic effects of the ‘designer drug’ methylenedioxypyrovalerone (MDPV) on the neonatal mouse brain. Neurotoxicology 44:231–236. doi:10.1016/j.neuro.2014.07.004
Anneken JH, Angoa-Perez M, Kuhn DM (2015) 3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: beta-ketoamphetamine modulation of neurotoxicity by the dopamine transporter. J Neurochem 133(2):211–222. doi:10.1111/jnc.13048
Barrios L, Grison-Hernando H, Boels D, Bouquie R, Monteil-Ganiere C, Clement R (2016) Death following ingestion of methylone. Int J Legal Med 130(2):381–385. doi:10.1007/s00414-015-1212-4
Baumann MH, Ayestas MA Jr, Partilla JS et al (2012) The designer methcathinone analogs, mephedrone and methylone, are substrates for monoamine transporters in brain tissue. Neuropsychopharmacology 37(5):1192–1203. doi:10.1038/npp.2011.304
Baumann MH, Partilla JS, Lehner KR et al (2013) Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive ‘bath salts’ products. Neuropsychopharmacology 38(4):552–562. doi:10.1038/npp.2012.204
Borek HA, Holstege CP (2012) Hyperthermia and multiorgan failure after abuse of “bath salts” containing 3,4-methylenedioxypyrovalerone. Ann Emerg Med 60(1):103–105. doi:10.1016/j.annemergmed.2012.01.005
Cao L, Walker MP, Vaidya NK, Fu M, Kumar S, Kumar A (2016) Cocaine-mediated autophagy in astrocytes involves sigma 1 receptor, PI3 K, mTOR, Atg5/7, beclin-1 and induces type ii programed cell death. Mol Neurobiol 53(7):4417–4430. doi:10.1007/s12035-015-9377-x
Carvalho M, Carmo H, Costa VM et al (2012) Toxicity of amphetamines: an update. Arch Toxicol 86(8):1167–1231. doi:10.1007/s00204-012-0815-5
Chakrabarti S, Munshi S, Banerjee K, Thakurta IG, Sinha M, Bagh MB (2014) Mitochondrial dysfunction during brain aging: role of oxidative stress and modulation by antioxidant supplementation. Aging Dis 2(3):242–256
Chandramani Shivalingappa P, Jin H, Anantharam V, Kanthasamy A, Kanthasamy A (2012) N-acetyl cysteine protects against methamphetamine-induced dopaminergic neurodegeneration via modulation of redox status and autophagy in dopaminergic cells. Parkinsons Dis 2012:424285. doi:10.1155/2012/424285
Chen G, Ke Z, Xu M et al (2012) Autophagy is a protective response to ethanol neurotoxicity. Autophagy 8(11):1577–1589. doi:10.4161/auto.21376
den Hollander B, Sundstrom M, Pelander A et al (2014) Keto amphetamine toxicity-focus on the redox reactivity of the cathinone designer drug mephedrone. Toxicol Sci 141(1):120–131. doi:10.1093/toxsci/kfu108
den Hollander B, Sundstrom M, Pelander A et al (2015) Mitochondrial respiratory dysfunction due to the conversion of substituted cathinones to methylbenzamides in SH-SY5Y cells. Sci Rep 5:14924. doi:10.1038/srep14924
Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A (2009) Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ 16(7):966–975. doi:10.1038/cdd.2009.33
EMCDDA-Europol (2016) EU drug markets report: in-depth analysis. EMCDDA—Europol joint publications. European monitoring centre for drugs and drug addiction and Europol, Luxembourg
Filomeni G, Desideri E, Cardaci S, Rotilio G, Ciriolo MR (2010) Under the ROS: thiol network is the principal suspect for autophagy commitment. Autophagy 6(7):999–1005. doi:10.4161/auto.6.7.12754
Filomeni G, De Zio D, Cecconi F (2015) Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ 22(3):377–388. doi:10.1038/cdd.2014.150
Firuzi O, Miri R, Tavakkoli M, Saso L (2011) Antioxidant therapy: current status and future prospects. Curr Med Chem 18(25):3871–3888
Galluzzi L, Vicencio JM, Kepp O, Tasdemir E, Maiuri MC, Kroemer G (2008) To die or not to die: that is the autophagic question. Curr Mol Med 8(2):78–91
Guo ML, Liao K, Periyasamy P et al (2015) Cocaine-mediated microglial activation involves the ER stress-autophagy axis. Autophagy 11(7):995–1009. doi:10.1080/15548627.2015.1052205
Halpin LE, Collins SA, Yamamoto BK (2014) Neurotoxicity of methamphetamine and 3,4-methylenedioxymethamphetamine. Life Sci 97(1):37–44. doi:10.1016/j.lfs.2013.07.014
He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93. doi:10.1146/annurev-genet-102808-114910
Huang YN, Yang LY, Wang JY, Lai CC, Chiu CT, Wang JY (2017) l-ascorbate protects against methamphetamine-induced neurotoxicity of cortical cells via inhibiting oxidative stress, autophagy, and apoptosis. Mol Neurobiol 54(1):125–136. doi:10.1007/s12035-015-9561-z
Jaeger PA, Wyss-Coray T (2009) All-you-can-eat: autophagy in neurodegeneration and neuroprotection. Mol Neurodegener 4:16. doi:10.1186/1750-1326-4-16
Kanthasamy A, Anantharam V, Ali SF, Kanthasamy AG (2006) Methamphetamine induces autophagy and apoptosis in a mesencephalic dopaminergic neuronal culture model: role of cathepsin-D in methamphetamine-induced apoptotic cell death. Ann N Y Acad Sci 1074:234–244. doi:10.1196/annals.1369.022
King HE, Wetzell B, Rice KC, Riley AL (2014) 3,4-Methylenedioxypyrovalerone (MDPV)-induced conditioned taste avoidance in the F344/N and LEW rat strains. Pharmacol Biochem Behav 126:163–169. doi:10.1016/j.pbb.2014.09.021
Kongsuphol P, Mukda S, Nopparat C, Villarroel A, Govitrapong P (2009) Melatonin attenuates methamphetamine-induced deactivation of the mammalian target of rapamycin signaling to induce autophagy in SK-N-SH cells. J Pineal Res 46(2):199–206. doi:10.1111/j.1600-079X.2008.00648.x
Larsen KE, Fon EA, Hastings TG, Edwards RH, Sulzer D (2002) Methamphetamine-induced degeneration of dopaminergic neurons involves autophagy and upregulation of dopamine synthesis. J Neurosci 22(20):8951–8960
LaVoie MJ, Hastings TG (1999) Dopamine quinone formation and protein modification associated with the striatal neurotoxicity of methamphetamine: evidence against a role for extracellular dopamine. J Neurosci 19(4):1484–1491
Lee J, Giordano S, Zhang J (2012) Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem J 441(2):523–540. doi:10.1042/BJ20111451
Li Y, Hu Z, Chen B et al (2012) Taurine attenuates methamphetamine-induced autophagy and apoptosis in PC12 cells through mTOR signaling pathway. Toxicol Lett 215(1):1–7. doi:10.1016/j.toxlet.2012.09.019
Li IH, Ma KH, Weng SJ, Huang SS, Liang CM, Huang YS (2014) Autophagy activation is involved in 3,4-methylenedioxymethamphetamine (‘ecstasy’)—induced neurotoxicity in cultured cortical neurons. PLoS One 9(12):e116565. doi:10.1371/journal.pone.0116565
Li L, Tan J, Miao Y, Lei P, Zhang Q (2015) ROS and autophagy: interactions and molecular regulatory mechanisms. Cell Mol Neurobiol 35(5):615–621. doi:10.1007/s10571-015-0166-x
Li IH, Ma KH, Kao TJ et al (2016) Involvement of autophagy upregulation in 3,4-methylenedioxymethamphetamine (‘ecstasy’)-induced serotonergic neurotoxicity. Neurotoxicology 52:114–126. doi:10.1016/j.neuro.2015.11.009
Ma J, Wan J, Meng J, Banerjee S, Ramakrishnan S, Roy S (2014) Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor. Cell Death Dis 5:e1099. doi:10.1038/cddis.2014.64
Macleod MR, Allsopp TE, McLuckie J, Kelly JS (2001) Serum withdrawal causes apoptosis in SHSY 5Y cells. Brain Res 889(1–2):308–315
Marino G, Niso-Santano M, Baehrecke EH, Kroemer G (2014) Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol 15(2):81–94. doi:10.1038/nrm3735
Mercer LD, Higgins GC, Lau CL, Lawrence AJ, Beart PM (2017) MDMA-induced neurotoxicity of serotonin neurons involves autophagy and rilmenidine is protective against its pathobiology. Neurochem Int. doi:10.1016/j.neuint.2017.01.010
Mizushima N (2007) Autophagy: process and function. Genes Dev 21(22):2861–2873. doi:10.1101/gad.1599207
Monks TJ, Jones DC, Bai F, Lau SS (2004) The role of metabolism in 3,4-(+)-methylenedioxyamphetamine and 3,4-(+)-methylenedioxymethamphetamine (ecstasy) toxicity. Ther Drug Monit 26(2):132–136
Nakagawa Y, Suzuki T, Tayama S, Ishii H, Ogata A (2009) Cytotoxic effects of 3,4-methylenedioxy-N-alkylamphetamines, MDMA and its analogues, on isolated rat hepatocytes. Arch Toxicol 83(1):69–80. doi:10.1007/s00204-008-0323-9
Nopparat C, Porter JE, Ebadi M, Govitrapong P (2010) The mechanism for the neuroprotective effect of melatonin against methamphetamine-induced autophagy. J Pineal Res 49(4):382–389. doi:10.1111/j.1600-079X.2010.00805.x
Paglin S, Hollister T, Delohery T et al (2001) A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res 61(2):439–444
Pedersen AJ, Petersen TH, Linnet K (2013) In vitro metabolism and pharmacokinetic studies on methylone. Drug Metab Dispos 41(6):1247–1255. doi:10.1124/dmd.112.050880
Periyasamy P, Guo ML, Buch S (2016) Cocaine induces astrocytosis through ER stress-mediated activation of autophagy. Autophagy 12(8):1310–1329. doi:10.1080/15548627.2016.1183844
Prosser JM, Nelson LS (2012) The toxicology of bath salts: a review of synthetic cathinones. J Med Toxicol 8(1):33–42. doi:10.1007/s13181-011-0193-z
Rosas-Hernandez H, Cuevas E, Lantz SM et al (2016a) 3,4-Methylenedioxypyrovalerone (MDPV) induces cytotoxic effects on human dopaminergic SH-SY5Y cells. J Drug Alcohol Res 5:1–6. doi:10.4303/jdar/235991
Rosas-Hernandez H, Cuevas E, Lantz SM et al (2016b) Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells. Neurosci Lett 629:125–130. doi:10.1016/j.neulet.2016.06.029
Salazar M, Carracedo A, Salanueva ÍJ et al (2009) Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells. J Clin Investig 119(5):1359–1372
Scherz-Shouval R, Elazar Z (2011) Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci 36(1):30–38. doi:10.1016/j.tibs.2010.07.007
Strano-Rossi S, Cadwallader AB, de la Torre X, Botre F (2010) Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MDPV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry. Rapid Commun Mass Spectrom RCM 24(18):2706–2714. doi:10.1002/rcm.4692
Tanida I, Ueno T, Kominami E (2008) LC3 and Autophagy. Methods Mol Biol 445:77–88. doi:10.1007/978-1-59745-157-4_4
Valente MJ, Guedes de Pinho P, de Lourdes Bastos M, Carvalho F, Carvalho M (2014) Khat and synthetic cathinones: a review. Arch Toxicol 88(1):15–45. doi:10.1007/s00204-013-1163-9
Valente MJ, Araujo AM, Bastos ML et al (2016a) Characterization of hepatotoxicity mechanisms triggered by designer cathinone drugs (beta-Keto amphetamines). Toxicol Sci 153(1):89–102. doi:10.1093/toxsci/kfw105
Valente MJ, Araujo AM, Silva R et al (2016b) 3,4-Methylenedioxypyrovalerone (MDPV): in vitro mechanisms of hepatotoxicity under normothermic and hyperthermic conditions. Arch Toxicol 90(8):1959–1973. doi:10.1007/s00204-015-1653-z
Valente MJ, Bastos ML, Fernandes E, Carvalho F, Guedes de Pinho P, Carvalho M (2017) Neurotoxicity of beta-Keto amphetamines: deathly mechanisms elicited by methylone and MDPV in human dopaminergic SH-SY5Y Cells. ACS Chem Neurosci. doi:10.1021/acschemneuro.6b00421
von Haefen C, Sifringer M, Menk M, Spies CD (2011) Ethanol enhances susceptibility to apoptotic cell death via down-regulation of autophagy-related proteins. Alcohol Clin Exp Res 35(8):1381–1391. doi:10.1111/j.1530-0277.2011.01473.x
Wojcieszak J, Andrzejczak D, Woldan-Tambor A, Zawilska JB (2016) Cytotoxic activity of pyrovalerone derivatives, an emerging group of psychostimulant designer cathinones. Neurotox Res 30(2):239–250. doi:10.1007/s12640-016-9640-6
Xu HD, Wu D, Gu JH et al (2013) The pro-survival role of autophagy depends on Bcl-2 under nutrition stress conditions. PLoS One 8(5):e63232. doi:10.1371/journal.pone.0063232
Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y (1998) Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 23(1):33–42
Zaitsu K, Katagi M, Tatsuno M, Sato T, Tsuchihashi H, Suzuki K (2011) Recently abused β-keto derivatives of 3, 4-methylenedioxyphenylalkylamines: a review of their metabolisms and toxicological analysis. Forensic Toxicol 29(2):73–84
Zhao L, Zhu Y, Wang D et al (2010) Morphine induces Beclin 1-and ATG5-dependent autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus. Autophagy 6(3):386–394
Acknowledgements
This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013. The study is a result of the project NORTE-01-0145-FEDER-000024, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement (DESignBIOtecHealth—New Technologies for three Health Challenges of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases), through the European Regional Development Fund (ERDF). M.J.V. and C.A. thank Fundação para a Ciência e Tecnologia (FCT), Portugal, for their PhD (SFRH/BD/89879/2012) and Post-Doc grants (SFRH/BPD/98304/2013), respectively.
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Valente, M.J., Amaral, C., Correia-da-Silva, G. et al. Methylone and MDPV activate autophagy in human dopaminergic SH-SY5Y cells: a new insight into the context of β-keto amphetamines-related neurotoxicity. Arch Toxicol 91, 3663–3676 (2017). https://doi.org/10.1007/s00204-017-1984-z
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DOI: https://doi.org/10.1007/s00204-017-1984-z