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Archives of Toxicology

, Volume 93, Issue 10, pp 2979–2992 | Cite as

Biophysical and biomechanical properties of neural progenitor cells as indicators of developmental neurotoxicity

  • Gautam Mahajan
  • Moo-Yeal Lee
  • Chandrasekhar KothapalliEmail author
Reproductive Toxicology
  • 175 Downloads

Abstract

Conventional in vitro toxicity studies have focused on identifying IC50 and the underlying mechanisms, but how toxicants influence biophysical and biomechanical changes in human cells, especially during developmental stages, remain understudied. Here, using an atomic force microscope, we characterized changes in biophysical (cell area, actin organization) and biomechanical (Young’s modulus, force of adhesion, tether force, membrane tension, tether radius) aspects of human fetal brain-derived neural progenitor cells (NPCs) induced by four classes of widely used toxic compounds, including rotenone, digoxin, N-arachidonoylethanolamide (AEA), and chlorpyrifos, under exposure up to 36 h. The sub-cellular mechanisms (apoptosis, mitochondria membrane potential, DNA damage, glutathione levels) by which these toxicants induced biochemical changes in NPCs were assessed. Results suggest a significant compromise in cell viability with increasing toxicant concentration (p < 0.01), and biophysical and biomechanical characteristics with increasing exposure time (p < 0.01) as well as toxicant concentration (p < 0.01). Impairment of mitochondrial membrane potential appears to be the most sensitive mechanism of neurotoxicity for rotenone, AEA and chlorpyrifos exposure, but compromise in plasma membrane integrity for digoxin exposure. The surviving NPCs remarkably retained stemness (SOX2 expression) even at high toxicant concentrations. A negative linear correlation (R2 = 0.92) exists between the elastic modulus of surviving cells and the number of living cells in that environment. We propose that even subtle compromise in cell mechanics could serve as a crucial marker of developmental neurotoxicity (mechanotoxicology) and therefore should be included as part of toxicology assessment repertoire to characterize as well as predict developmental outcomes.

Keywords

Developmental neurotoxicity Neural stem cells Insecticides AEA Chlorpyrifos Multi-variable logistic regression Mechanotoxicology 

Notes

Acknowledgements

The authors acknowledge help from Soo-Yeon Kang with cell passaging and high-content imaging equipment. This work was partially supported by funds from National Institutes of Health (NIEHS R01ES025779) to C.K. and M.Y.L., National Science Foundation (CBET, Award # 1337859) to C.K., and Graduate Student Research Award to G.M from Cleveland State University.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The manuscript does not contain clinical studies or patient data.

Supplementary material

204_2019_2549_MOESM1_ESM.docx (25 kb)
Supplementary material 1 (DOCX 24 kb)
204_2019_2549_MOESM2_ESM.tiff (8.4 mb)
Supplementary Figure 1 Representative immunofluorescence images of SOX2 expression in ReNcells VM exposed to various concentrations (0, 0.25 × IC50, 0.5 × IC50, IC50) of rotenone, digoxin, AEA, and chlorpyrifos for 24 h. Cultures were counterstained with DAPI for cell identification. Primary antibody for SOX2 was used in combination with appropriate secondary antibody. Scale bar: 200 µm. (TIFF 8554 kb)
204_2019_2549_MOESM3_ESM.tiff (407 kb)
Supplementary Figure 2 Average (± standard error) elastic modulus (A), adhesion force (B) and tether force (C) of human NPCs in control cultures for 4, 12, 24 and 36 h (n ≥ 45 cells in each case). No significant changes were observed in the biomechanical properties in control cultures over time. (TIFF 407 kb)
204_2019_2549_MOESM4_ESM.tiff (347 kb)
Supplementary Figure 3 The IC50 values from the four assays were compared to identify the most sensitive mechanism by which rotenone, digoxin, AEA, or chlorpyrifos induces toxicity in human NPCs. All assays were done at the 24 h time point. (TIFF 346 kb)

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Copyright information

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

Authors and Affiliations

  1. 1.Department of Chemical and Biomedical EngineeringWashkewicz College of Engineering, Cleveland State UniversityClevelandUSA

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