2. Academic Validation
  3. Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

  • Arch Toxicol. 2019 Jun;93(6):1585-1608. doi: 10.1007/s00204-019-02473-y.
Johannes Delp 1 2,  Melina Funke 1,  Franziska Rudolf 1,  Andrea Cediel 3,  Susanne Hougaard Bennekou 4,  Wanda van der Stel 5,  Giada Carta 6,  Paul Jennings 6,  Cosimo Toma 7,  Iain Gardner 8,  Bob van de Water 5,  Anna Forsby 3 9,  Marcel Leist 10
Affiliations

Affiliations

  • 1 Chair for In Vitro Toxicology and Biomedicine, Department of Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, Universitaetsstr. 10, 78457, Constance, Germany.
  • 2 Cooperative Doctorate College InViTe, University of Konstanz, Constance, Germany.
  • 3 Swetox Unit for Toxicological Sciences, Karolinska Institutet, Stockholm, Sweden.
  • 4 National Food Institute, Technical University of Denmark (DTU), Lyngby, Denmark.
  • 5 Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
  • 6 Division of Molecular and Computational Toxicology, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
  • 7 Laboratory of Environmental Chemistry and Toxicology, Department of Environmental Health Sciences, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via la Masa 19, 20156, Milan, Italy.
  • 8 Certara UK Limited, Sheffield, UK.
  • 9 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
  • 10 Chair for In Vitro Toxicology and Biomedicine, Department of Inaugurated by the Doerenkamp-Zbinden Foundation, University of Konstanz, Universitaetsstr. 10, 78457, Constance, Germany. [email protected].
PMID: 31190196 DOI: 10.1007/s00204-019-02473-y
Abstract

Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.

Keywords

High content imaging; High-throughput toxicity screening; Mechanistic safety assessment; Metabolic reprogramming; Mitotoxicity; Neurotoxicity.

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