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

Johannes Delp, Melina Funke, Franziska Rudolf, Andrea Cediel, Susanne Hougaard Bennekou, Wanda van der Stel, Giada Carta, Paul Jennings, Cosimo Toma, Iain Gardner, Bob van de Water, Anna Forsby, Marcel Leist*

*Corresponding author for this work

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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.
Original languageEnglish
JournalArchives of Toxicology
Volume93
Issue number6
Pages (from-to)1585-1608
ISSN0340-5761
DOIs
Publication statusPublished - 2019

Keywords

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

Cite this

Delp, Johannes ; Funke, Melina ; Rudolf, Franziska ; Cediel, Andrea ; Bennekou, Susanne Hougaard ; van der Stel, Wanda ; Carta, Giada ; Jennings, Paul ; Toma, Cosimo ; Gardner, Iain ; van de Water, Bob ; Forsby, Anna ; Leist, Marcel. / Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants. In: Archives of Toxicology. 2019 ; Vol. 93, No. 6. pp. 1585-1608.
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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",
author = "Johannes Delp and Melina Funke and Franziska Rudolf and Andrea Cediel and Bennekou, {Susanne Hougaard} and {van der Stel}, Wanda and Giada Carta and Paul Jennings and Cosimo Toma and Iain Gardner and {van de Water}, Bob and Anna Forsby and Marcel Leist",
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doi = "10.1007/s00204-019-02473-y",
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Delp, J, Funke, M, Rudolf, F, Cediel, A, Bennekou, SH, van der Stel, W, Carta, G, Jennings, P, Toma, C, Gardner, I, van de Water, B, Forsby, A & Leist, M 2019, 'Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants', Archives of Toxicology, vol. 93, no. 6, pp. 1585-1608. https://doi.org/10.1007/s00204-019-02473-y

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants. / Delp, Johannes; Funke, Melina; Rudolf, Franziska; Cediel, Andrea; Bennekou, Susanne Hougaard; van der Stel, Wanda; Carta, Giada; Jennings, Paul; Toma, Cosimo; Gardner, Iain; van de Water, Bob; Forsby, Anna; Leist, Marcel.

In: Archives of Toxicology, Vol. 93, No. 6, 2019, p. 1585-1608.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

AU - Delp, Johannes

AU - Funke, Melina

AU - Rudolf, Franziska

AU - Cediel, Andrea

AU - Bennekou, Susanne Hougaard

AU - van der Stel, Wanda

AU - Carta, Giada

AU - Jennings, Paul

AU - Toma, Cosimo

AU - Gardner, Iain

AU - van de Water, Bob

AU - Forsby, Anna

AU - Leist, Marcel

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - High content imaging

KW - High-throughput toxicity screening

KW - Mechanistic safety assessment

KW - Metabolic reprogramming

KW - Mitotoxicity

KW - Neurotoxicity

U2 - 10.1007/s00204-019-02473-y

DO - 10.1007/s00204-019-02473-y

M3 - Journal article

VL - 93

SP - 1585

EP - 1608

JO - Archives of Toxicology

JF - Archives of Toxicology

SN - 0340-5761

IS - 6

ER -