Metabolic signature of methylone in primary mouse hepatocytes, at subtoxic concentrations

  • Ana Margarida AraújoEmail author
  • Márcia CarvalhoEmail author
  • Maria de Lourdes Bastos
  • Félix Carvalho
  • Paula Guedes de PinhoEmail author
Organ Toxicity and Mechanisms


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.


Primary mouse hepatocytes Methylone Metabolomics GC–MS Hepatotoxicity Intracellular and extracellular metabolite profiling 



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.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.

Supplementary material

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Supplementary material 5 (PDF 129 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.UCIBIO, REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of PortoPortoPortugal
  2. 2.UFP Energy, Environment and Health Research Unit (FP-ENAS), University Fernando PessoaPortoPortugal

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