Correlating Biochemical Impact of Environmental Toxicants on Human Neural Stem Cells to Biophysical Changes

Today one out of six children are diagnosed with developmental disorders and there is a critical need to screen compounds such as environmental toxicants and pharmaceutical drugs for their potential developmental toxicity. It is widely accepted that developing central nervous system is highly susceptible to damage by exposure to toxicants due to the still immature blood-brain barrier. With current in vitro tests focusing mainly on cytotoxicity, many important biochemical and biophysical characteristics are not being explored. Such biochemical and biophysical cues play an integral role in regulating cell morphogenesis, movement, and metabolism during fetal development. In this study, we investigated the biophysical and biochemical effects of rotenone (an active ingredient of insecticides) on human neural stem cells (NSCs). NSCs were exposed to a wide range of rotenone concentrations and using dose-response curves, toxic concentration (IC 50 = 0.24 μM) was flagged. IC 50 and lower concentrations were tested for biophysical assessment of rotenone-exposed NSCs. Using a MFP-3D-BIO AFM and customized culture platform, the biomechanical characteristics were quantified from force-deflection curves obtained at various locations on live NSCs cultured under exposure conditions (n > 50 cells/ dose/ time). Cytoskeleton marker (actin) expression was assessed to correlate to changes in biomechanics. Results showed a negative dose-dependent effect on cell viability (p < 0.001) and a significant dose-time dependent reorganization of cytoskeleton (p < 0.05). NSC stiffness varied from 450 Pa to 5 kPa in the presence and absence of rotenone, respectively. The changes in membrane tension suggests a strong correlation between dosage and duration of rotenone exposure (p < 0.01). Adhesive forces between AFM probe and NSCs were strongly dependent on exposure duration and dosage of rotenone (p < 0.01), demonstrating the alterations of cell membrane with rotenone exposure.