Category: Preclinical Development
Purpose: Our purpose is to demonstrate that the actin cytoskeleton plays a role in regulating the toxicity of chlorpyrifos in SH-SY5Y cells.
The mechanical properties of a cell, governed by cytoskeletal proteins and cues from the extracellular matrix (ECM), are a constitutional guide of cell fate. The stiffness of cells and their environment affect the cells morphology, differentiation, endocytosis, and gene expression. Therefore, it is reasonable to expect cell mechanics to play a role in toxicity as well. However, the role of cell mechanics in toxicity is rarely examined.
Chlorpyrifos, an organophosphate pesticide, inhibits acetylcholinesterase and has been shown to present a risk to human health. Exposure to chlorpyrifos has been shown to have detrimental neurological effects and has the potential disrupt healthy brain development in children. The neurological effects of chlorpyrifos are exacerbated in children and fetuses, which may be due to the rapid development and restructuring of the brain that occurs during this period. One of the changes the brain undergoes during this period is rapid stiffening (Chatelin, Vappou, Roth, Raul, & Willinger, 2012), changing the mechanical properties of the cells and ECM.
Our hypothesis is that cell mechanics, through changes in the actin cytoskeleton, plays a role in chlorpyrifos cytotoxicity in SH-SY5Y cells and that, by changing the actin cytoskeleton through substrate stiffness or through chemical modification, we will alter the cytotoxic response.
Methods: Cell Culture - Cells were plated at 40K/well in 24-well plates and grown for two days in OptiMEM (Gibco) media with 10% FBS (Gibco), 1% pen strep, 1% MEM NEAA, and 1% sodium pyruvate. The cells were dosed with chlorpyrifos in serum-free media.
Preparation of Polyacrylamide (PA) Gels - Glass coverslips (12-mm) were coated with a 0.1M NaOH solution and then treated with APTES and glutaraldehyde. Gel solutions of varying acrylamide and bis-acrylamide were prepared and allowed to polymerize on the prepared coverslips. The coverslips were then exposed to sulfo-SANPAH under UV light. A 10 μg/mL collagen solution was then placed on the gels overnight.
Atomic Force Microscopy - Cells were grown at low density (100K/mL) overnight. The cells were probed with a 4.5 μm polystyrene bead on a gold coated cantilever with a spring constant of 0.06 N/m (Novascan).
Confocal Microscopy - Cells were grown on 12-mm glass coverslips and on PA gels and fixed with a 4% formaldehyde solution. Cells were then made permeable with a 0.1% Triton X-100 solution and washed. Cells were stained with phalloidin and DAPI and imaged with a Zeiss LSM 710 confocal microscope.
Results: Atomic force microscopy (AFM) studies performed on SH-SY5Y indicated that chlorpyrifos exposure induces cell stiffening. Cells dosed for four hours with 10 μM chlorpyrifos had a young’s modulus of 0.62 ± 0.17 kPa whereas undosed cells measured at 0.41 ± 0.05 kPa.
Confocal images of SH-SY5Y cells dosed with increasing amounts of chlorpyrifos for four hours show a change in cell morphology. Cells dosed with chlorpyrifos at 1 μM and 10 μM doses show an increase in stress fibrils and the presence of many pointed protrusions at the edges of the cells.
A cell viability assay (MTS) was used to assess the effect of chlorpyrifos on SH-SY5Y cells plated on soft (1.1 kPa), stiff (7.9 kPa), and glass substrates. At a 100 μM dose of chlorpyrifos, cells plated on the soft substrate had significantly higher cell viability (62%) than cells plated on the stiff substrate (46%) or on glass (32 %) versus an un-dosed control.
Conclusion: Exposure to a subtoxic dose of chlorpyrifos results in cell stiffening, an increase in stress fibrils and a change in cell morphology in SH-SY5Y cells, indicating that chlorpyrifos exposure increases actin polymerization and alters the cell’s mechanical properties. The cell’s mechanical properties can futher be changed through substrate stiffness, which we did by plating our cells on PA gel substrates of varying stiffness and on glass. Stiffer substrates result in stiffer cells and higher actin polymerization. With this, we saw that cells on stiffer substrates had lower cell viability upon chlorpyrifos exposure than cells plated on softer substrates. When we disrupted actin polymerization through pretreatment with cytocalasin D, we likewise saw an increase in cell viability, therefore demonstrating that actin polymerization and actin cytoskeletal remodeling is involved in the decrease in cell viability on stiffer substrates with chlorpyrifos exposure.
References: Chatelin, S., Vappou, J., Roth, S., Raul, J. S., & Willinger, R. (2012). Towards child versus adult brain mechanical properties. Journal of the Mechanical Behavior of Biomedical Materials, 6, 166-173. doi:10.1016/j.jmbbm.2011.09.013