Abstract
Methylone (3,4-methylenedioxymethcathinone) is one of the most popular new psychoactive drugs worldwide. Although advertised as a safe drug, its use has been associated to several cases of liver damage. In this work, a metabolomics approach based on gas chromatography–mass spectrometry (GC–MS) combined with chemometric analyses was used to characterize the disturbances occurring in the intra- and extracellular metabolome of primary mouse hepatocytes exposed to two subtoxic concentrations (LC01 and LC10) of methylone to better understand the early hepatotoxic events. Results showed a characteristic metabolic fingerprint for methylone, where aspartate, cysteine, 2-methyl-1-pentanol, 4-methylheptane, dodecane, 2,4-dimethyl-1-heptene, 1,3-di-tert-butylbenzene, acetophenone, formaldehyde and glyoxal levels were significantly changed at both concentrations tested. Furthermore, subtoxic concentrations of methylone caused profound changes in several biochemical pathways, suggesting adaptations in energy production processes (TCA cycle, amino acids metabolism and pyruvate metabolism), cellular antioxidant defenses (glutamate, cysteine and glutathione metabolism) and hepatic enzymes (associated to hydrocarbons, alcohols, aldehydes and ketones metabolism). This metabolic response to the initial methylone challenge most probably reflects the activation of protective mechanisms to restore cellular homeostasis. Overall, this study highlights the potential of untargeted metabolomic analysis to reveal the hepatic metabolic signature of methylone at subtoxic concentrations, and also provides clues to clarify the early mechanisms underlying the toxicity triggered by this new psychoactive substance, opening a new perspective for the study of toxicity mechanisms of new xenobiotics.
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References
Araujo AM, Bastos ML, Fernandes E, Carvalho F, Carvalho M, Guedes de Pinho P (2018a) GC-MS metabolomics reveals disturbed metabolic pathways in primary mouse hepatocytes exposed to subtoxic levels of 3,4-methylenedioxymethamphetamine (MDMA). Arch Toxicol. https://doi.org/10.1007/s00204-018-2314-9
Araujo AM, Moreira N, Lima AR et al (2018b) Analysis of extracellular metabolome by HS-SPME/GC-MS: optimization and application in a pilot study to evaluate galactosamine-induced hepatotoxicity. Toxicol Lett 295:22–31. https://doi.org/10.1016/j.toxlet.2018.05.028
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. https://doi.org/10.1007/s00414-015-1212-4
Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 57(1):289–300
Berben L, Sereika SM, Engberg S (2012) Effect size estimation: methods and examples. Int J Nurs Stud 49(8):1039–1047. https://doi.org/10.1016/j.ijnurstu.2012.01.015
Berg JM, Tymoczko JL, Stryer L (2002) Carbon atoms of degraded amino acids emerge as major metabolic intermediates. In: Biochemistry. 5th edn, New York
Bouhifd M, Hartung T, Hogberg HT, Kleensang A, Zhao L (2013) Review: toxicometabolomics. J Appl Toxicol 33(12):1365–1383. https://doi.org/10.1002/jat.2874
Carbone PN, Carbone DL, Carstairs SD, Luzi SA (2013) Sudden cardiac death associated with methylone use. Am J Forensic Med Pathol 34(1):26–28. https://doi.org/10.1097/PAF.0b013e31827ab5da
Carvalho M, Pontes H, Remiao F, Bastos ML, Carvalho F (2010) Mechanisms underlying the hepatotoxic effects of ecstasy. Curr Pharm Biotechnol 11(5):476–495
Carvalho M, Carmo H, Costa VM et al (2012) Toxicity of amphetamines: an update. Arch Toxicol 86(8):1167–1231. https://doi.org/10.1007/s00204-012-0815-5
Cawrse BM, Levine B, Jufer RA et al (2012) Distribution of methylone in four postmortem cases. J Anal Toxicol 36(6):434–439. https://doi.org/10.1093/jat/bks046
Cerretani D, Bello S, Cantatore S et al (2011) Acute administration of 3,4-methylenedioxymethamphetamine (MDMA) induces oxidative stress, lipoperoxidation and TNFalpha-mediated apoptosis in rat liver. Pharmacol Res 64(5):517–527. https://doi.org/10.1016/j.phrs.2011.08.002
Chen Y, Dong H, Thompson DC, Shertzer HG, Nebert DW, Vasiliou V (2013) Glutathione defense mechanism in liver injury: insights from animal models. Food Chem Toxicol 60:38–44. https://doi.org/10.1016/j.fct.2013.07.008
Chong J, Soufan O, Li C et al (2018) MetaboAnalyst 4.0: towards more transparent and integrative metabolomics analysis. Nucleic Acids Res 46(W1):W486–W494. https://doi.org/10.1093/nar/gky310
Coppola M, Mondola R (2012) Synthetic cathinones: chemistry, pharmacology and toxicology of a new class of designer drugs of abuse marketed as “bath salts” or “plant food”. Toxicol Lett 211(2):144–149. https://doi.org/10.1016/j.toxlet.2012.03.009
Cunha-Oliveira T, Rego AC, Oliveira CR (2013) Oxidative stress and drugs of abuse: an update. Mini Rev Org Chem 10(4):321–334
El-Tawil OS, Abou-Hadeed AH, El-Bab MF, Shalaby AA (2011) d-Amphetamine-induced cytotoxicity and oxidative stress in isolated rat hepatocytes. Pathophysiology 18(4):279–285. https://doi.org/10.1016/j.pathophys.2011.04.001
EMCDDA (2015) New psychoactive substances in Europe: an update from the EU Early Warning System. http://www.emcdda.europa.eu/publications/rapid-communications/2015/new-psychoactive-substances_en
EMCDDA (2018) European Drug Report 2018: Trends and Developments. http://www.emcdda.europa.eu/publications/edr/trends-developments/2018_en
Fiehn O (2002) Metabolomics–the link between genotypes and phenotypes. Plant Mol Biol 48(1–2):155–171
German CL, Fleckenstein AE, Hanson GR (2014) Bath salts and synthetic cathinones: an emerging designer drug phenomenon. Life Sci 97(1):2–8. https://doi.org/10.1016/j.lfs.2013.07.023
Godoy P, Hewitt NJ, Albrecht U et al (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87(8):1315–1530. https://doi.org/10.1007/s00204-013-1078-5
Hakim M, Broza YY, Barash O et al (2012) Volatile organic compounds of lung cancer and possible biochemical pathways. Chem Rev 112(11):5949–5966. https://doi.org/10.1021/cr300174a
Kovacs K, Toth AR, Kereszty EM (2012) A new designer drug: methylone related death. Orv Hetil 153(7):271–276. https://doi.org/10.1556/OH.2012.29310
Lange JN, Wood KD, Knight J, Assimos DG, Holmes RP (2012) Glyoxal formation and its role in endogenous oxalate synthesis. Adv Urol 2012:819202. https://doi.org/10.1155/2012/819202
Leon Z, Garcia-Canaveras JC, Donato MT, Lahoz A (2013) Mammalian cell metabolomics: experimental design and sample preparation. Electrophoresis 34(19):2762–2775. https://doi.org/10.1002/elps.201200605
Lu SC (2013) Glutathione synthesis. Biochim Biophys Acta 1830(5):3143–3153. https://doi.org/10.1016/j.bbagen.2012.09.008
Luethi D, Liechti ME, Krahenbuhl S (2017) Mechanisms of hepatocellular toxicity associated with new psychoactive synthetic cathinones. Toxicology 387:57–66. https://doi.org/10.1016/j.tox.2017.06.004
Mastrangelo A, Ferrarini A, Rey-Stolle F, Garcia A, Barbas C (2015) From sample treatment to biomarker discovery: a tutorial for untargeted metabolomics based on GC-(EI)-Q-MS. Anal Chim Acta 900:21–35. https://doi.org/10.1016/j.aca.2015.10.001
Nowotny K, Jung T, Hohn A, Weber D, Grune T (2015) Advanced glycation end products and oxidative stress in type 2 diabetes mellitus. Biomolecules 5(1):194–222. https://doi.org/10.3390/biom5010194
O’Brien PJ, Siraki AG, Shangari N (2005) Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Crit Rev Toxicol 35(7):609–662
Owen OE, Kalhan SC, Hanson RW (2002) The key role of anaplerosis and cataplerosis for citric acid cycle function. J Biol Chem 277(34):30409–30412. https://doi.org/10.1074/jbc.R200006200
Pearson JM, Hargraves TL, Hair LS et al (2012) Three fatal intoxications due to methylone. J Anal Toxicol 36(6):444–451. https://doi.org/10.1093/jat/bks043
Pluskal T, Castillo S, Villar-Briones A, Oresic M (2010) MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinform 11:395. https://doi.org/10.1186/1471-2105-11-395
Prosser JM, Nelson LS (2012) The toxicology of bath salts: a review of synthetic cathinones. J Med Toxicol 8(1):33–42. https://doi.org/10.1007/s13181-011-0193-z
Reinke H, Asher G (2016) Circadian clock control of liver metabolic functions. Gastroenterology 150(3):574–580. https://doi.org/10.1053/j.gastro.2015.11.043
Rowan DD (2011) Volatile metabolites. Metabolites 1(1):41–63. https://doi.org/10.3390/metabo1010041
Rui L (2014) Energy metabolism in the liver. Compr Physiol 4(1):177–197. https://doi.org/10.1002/cphy.c130024
Seheult J, Fitzpatrick G, Boran G (2017) Lactic acidosis: an update. Clin Chem Lab Med 55(3):322–333. https://doi.org/10.1515/cclm-2016-0438
Shangari N, O’Brien PJ (2004) The cytotoxic mechanism of glyoxal involves oxidative stress. Biochem Pharmacol 68(7):1433–1442. https://doi.org/10.1016/j.bcp.2004.06.013
Song BJ, Moon KH, Upreti VV, Eddington ND, Lee IJ (2010) Mechanisms of MDMA (ecstasy)-induced oxidative stress, mitochondrial dysfunction, and organ damage. Curr Pharm Biotechnol 11(5):434–443
Srivastava S (2016) Emerging therapeutic roles for NAD(+) metabolism in mitochondrial and age-related disorders. Clin Transl Med 5(1):25. https://doi.org/10.1186/s40169-016-0104-7
Sumner LW, Amberg A, Barrett D et al (2007) Proposed minimum reporting standards for chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics 3(3):211–221. https://doi.org/10.1007/s11306-007-0082-2
Valente MJ, Araujo AM, Bastos Mde L et al (2016a) Editor’s Highlight: characterization of Hepatotoxicity Mechanisms Triggered by Designer Cathinone Drugs (beta-Keto Amphetamines). Toxicol Sci 153(1):89–102. https://doi.org/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. https://doi.org/10.1007/s00204-015-1653-z
Warrick BJ, Wilson J, Hedge M, Freeman S, Leonard K, Aaron C (2012) Lethal serotonin syndrome after methylone and butylone ingestion. J Med Toxicol 8(1):65–68. https://doi.org/10.1007/s13181-011-0199-6
Wheelock AM, Wheelock CE (2013) Trials and tribulations of ‘omics data analysis: assessing quality of SIMCA-based multivariate models using examples from pulmonary medicine. Mol BioSyst 9(11):2589–2596. https://doi.org/10.1039/c3mb70194h
Yuan L, Kaplowitz N (2009) Glutathione in liver diseases and hepatotoxicity. Mol Aspects Med 30(1–2):29–41. https://doi.org/10.1016/j.mam.2008.08.003
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 a 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 Program (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). A. M. Araújo thanks to FCT for her PhD fellowship (SFRH/BD/107708/2015) and M. Carvalho also acknowledges FCT through the UID/MULTI/04546/2019 project.
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Araújo, A.M., Carvalho, M., Bastos, M.d.L. et al. Metabolic signature of methylone in primary mouse hepatocytes, at subtoxic concentrations. Arch Toxicol 93, 3277–3290 (2019). https://doi.org/10.1007/s00204-019-02566-8
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DOI: https://doi.org/10.1007/s00204-019-02566-8