Use of human neuroblastoma SH-SY5Y cells to evaluate glyphosate-induced effects on oxidative stress, neuronal development and cell death signaling pathways

María Aránzazu Martínez, José Luis Rodríguez, Bernardo Lopez-Torres, Marta Martínez, María Rosa Martínez-Larrañaga, Jorge Enrique Maximiliano, Arturo Anadón, Irma Ares

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22 Scopus citations

Abstract

Glyphosate-containing herbicides are the most used agrochemicals in the world. Their indiscriminate application raises some concerns regarding the possible health and environmental hazards. In this study, we investigated in human neuroblastoma cell line SH-SY5Y if oxidative stress, altered neurodevelopment and cell death pathways are involved in response to glyphosate and its metabolite aminomethylphosphonic acid (AMPA) exposures. MTT and LDH assays were carried out to assess the glyphosate and AMPA cytotoxicity. Lipid peroxides measured as malondialdehyde (MDA), nitric oxide (NO) and reactive oxygen species (ROS) production, and caspase-Glo 3/7 activity were evaluated. The neuroprotective role of melatonin (MEL), Trolox, N-acetylcysteine (NAC) and Sylibin against glyphosate- and AMPA-induced oxidative stress was examined. Glyphosate and AMPA effects on neuronal development related gene transcriptions, and gene expression profiling of cell death pathways by Real-Time PCR array were also investigated. Glyphosate (5 mM) and AMPA (10 mM) induced a significant increase in MDA levels, NO and ROS production and caspase 3/7 activity. Glyphosate exposure induced up-regulation of Wnt3a, Wnt5a, Wnt7a, CAMK2A, CAMK2B and down-regulation of GAP43 and TUBB3 mRNA expression involved in normal neural cell development. In relation to gene expression profiling of cell death pathways, of the 84 genes examined in cells a greater than 2-fold change was observed for APAF1, BAX, BCL2, CASP3, CASP7, CASP9, SYCP2, TNF, TP53, CTSB, NFκB1, PIK3C3, SNCA, SQSTMT, HSPBAP1 and KCNIPI mRNA expression for glyphosate and AMPA exposures. These gene expression data can help to define neurotoxic mechanisms of glyphosate and AMPA. Our results demonstrated that glyphosate and AMPA induced cytotoxic effects on neuronal development, oxidative stress and cell death via apoptotic, autophagy and necrotic pathways and confirmed that glyphosate environmental exposure becomes a concern. This study demonstrates that SH-SY5Y cell line could be considered an in vitro system for pesticide screening.

Original languageEnglish
Article number105414
JournalEnvironmental International
Volume135
DOIs
StatePublished - Feb 2020

Bibliographical note

Funding Information:
This work was supported by Project Ref. RTA2015-00010-C03-03 from Ministerio de Economía, Industria y Competitividad , Spain. María-Aránzazu Martínez received her DPharm in Complutense University, Madrid, Spain, and obtained her PhD in 2000. She is currently Pharmacology and Toxicology Professor and a leader researcher at Department of Pharmacology and Toxicology, Complutense University, Madrid, Spain. José-Luis Rodríguez received his DVM degree in National University of San Marcos, Lima, Peru, in 2008 and obtained his PhD at 2018 from Complutense University, Madrid, Spain. He works as researcher in Pharmacology and Toxicology Department, Complutense University, Madrid. Bernardo Lopez-Torres received his DVM degree in National University of San Marcos, Lima, Peru, in 2016. Since 2017, he was admitted into the Department of Pharmacology and Toxicology, Complutense University, Madrid, Spain, for his undergraduate studies and joined to the research group of Prof. Arturo Anadón. Marta Martínez received her DPharm in the Complutense University, Madrid, Spain, and obtained her PhD at 2004. She is currently an Associate Professor and a researcher at the Department of Pharmacology and Toxicology, Complutense University, Madrid, Spain. María-Rosa Martínez-Larrañaga received her DSci and obtained her PhD at 1974 from Complutense University, Madrid, Spain. She is currently Full Professor at the Department of Pharmacology and Toxicology, Complutense University, Madrid, Spain. Jorge-Enrique Maximiliano received his DVM degree in National University of San Marcos, Lima, Peru, in 2017. Since 2018, he was admitted into the Department of Pharmacology and Toxicology, Complutense University, Madrid, Spain, for his undergraduate studies and joined to the research group of Prof. Arturo Anadón. Arturo Anadón was awarded his DVM and obtained his PhD at 1974 from Complutense University, Madrid, Spain. He worked as researcher at Medical Research Council, Department of Applied Physiology of the Royal College of Surgeons of England, London, U.K., and Fellow of Real Colegio Complutense at Harvard University, Cambridge MA, USA. He is currently Full Professor at Pharmacology and Toxicology Department, Complutense University, Madrid. Irma Ares received her DPharm in the Complutense University, Madrid, Spain, and obtained her PhD at 2010. She is currently an Associate Professor and a researcher at the Department of Pharmacology and Toxicology, Complutense University, Madrid, Spain.

Funding Information:
This work was supported by Project Ref. RTA2015-00010-C03-03 from Ministerio de Econom?a, Industria y Competitividad, Spain.

Publisher Copyright:
© 2019 The Authors

Keywords

  • Cell death pathways
  • Glyphosate
  • Neurotoxicity
  • Oxidative stress
  • Risk factors
  • SH-SY5Y cells

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