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Combination of multiple neural crest migration assays to identify environmental toxicants from a proof-of-concept chemical library

Abstract

Many in vitro tests have been developed to screen for potential neurotoxicity. However, only few cell function-based tests have been used for comparative screening, and thus experience is scarce on how to confirm and evaluate screening hits. We addressed these questions for the neural crest cell migration test (cMINC). After an initial screen, a hit follow-up strategy was devised. A library of 75 compounds plus internal controls (NTP80-list), assembled by the National Toxicology Program of the USA (NTP) was used. It contained some known classes of (developmental) neurotoxic compounds. The primary screen yielded 23 confirmed hits, which comprised ten flame retardants, seven pesticides and six drug-like compounds. Comparison of concentration–response curves for migration and viability showed that all hits were specific. The extent to which migration was inhibited was 25–90%, and two organochlorine pesticides (DDT, heptachlor) were most efficient. In the second part of this study, (1) the cMINC assay was repeated under conditions that prevent proliferation; (2) a transwell migration assay was used as a different type of migration assay; (3) cells were traced to assess cell speed. Some toxicants had largely varying effects between assays, but each hit was confirmed in at least one additional test. This comparative study allows an estimate on how confidently the primary hits from a cell function-based screen can be considered as toxicants disturbing a key neurodevelopmental process. Testing of the NTP80-list in more assays will be highly interesting to assemble a test battery and to build prediction models for developmental toxicity.

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Abbreviations

AOP:

Adverse outcome pathways

AraC:

Cytosine arabinoside

cMINC:

Circular MINC

CytoD:

Cytochalasin D

DMSO:

Dimethyl sulfoxide

DNT:

Developmental neurotoxicity

EC:

Effective concentration

EC90V:

EC90 of viability

EC75M:

EC75 of migration

EGCG:

Epigallocatechin gallate

EGF:

Epidermal growth factor

FBS:

Foetal bovine serum

FGF:

Fibroblast growth factor

FR:

Flame retardant

GRIND2:

Grid-independent descriptors

hESC:

Human embryonic stem cell

logP :

Octanol–water distribution coefficient

MeHgCl:

Methylmercury(II) chloride

MINC:

Migration of neural crest cell

MW:

Molecular weight

NCC:

Neural crest cell

NHBA:

Number of hydrogen bond acceptors

NOAELV :

No observed adverse effect level for viability

NRB:

Number of rotatable bonds

NTP:

National Toxicology Program

PAH:

Polycyclic aromatic hydrocarbon

PBDE:

Polybrominated diphenyl ether

PBS:

Phosphate buffered saline

PCA:

Principal component analysis

QSAR:

Quantitative structure-activity relationship

ROI:

Region of interest

TB-BPA:

Tetrabromobisphenol A

TPSA:

Polar surface

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Acknowledgements

This work was supported by the Land BW, the Doerenkamp-Zbinden Foundation, the DFG (RTG1331, KoRS-CB) and the European Project EU-ToxRisk. We are grateful to M. Kapitza, H. Leisner and the staff of the bioimaging center (BIC) and the flow cytometry center (FlowKon) for invaluable experimental support.

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Correspondence to Marcel Leist.

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Nyffeler, J., Dolde, X., Krebs, A. et al. Combination of multiple neural crest migration assays to identify environmental toxicants from a proof-of-concept chemical library. Arch Toxicol 91, 3613–3632 (2017). https://doi.org/10.1007/s00204-017-1977-y

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Keywords

  • Cell migration
  • Cell tracking
  • Cytotoxicity
  • High content imaging
  • Developmental toxicity
  • Human stem cells