Student Research: Joshua Frederick Robinson

MS, , 2008
Faculty Advisor: Elaine M. Faustman

Differential Sensitivity and Response to Metal-induced Neural Tube Defects Using a Comparative Mouse Model


Abstract

Neural tube defects (NTDs) are the most common of all serious human birth defects. Studies have identified genetic components that may be linked to increased susceptibility to environmentally-induced NTDs. The SWV and C57BL/J6 mice strains have shown to have a large disparity in sensitivity to environmentally-induced NTDs. When exposed to metals during neurogenesis, the C57BL/J6 has been shown to be more sensitive than the SWV. Due to its complex status of proliferation, differentiation and cell turnover, the developing embryo is highly susceptible to metal-induced alterations in cell cycle and apoptosis. We hypothesize that differential metal-induced impacts within cell cycle and apoptosis pathways contribute to observed differing sensitivities between these two strains. To investigate our hypothesis, a primary in vitro model was established for each strain by isolating murine embryonic fibroblasts (MEFs) at day 14 of gestation. At passage 4-6, MEFs were treated with environmentally relevant concentrations of cadmium (Cd) and methyl mercury (MeHg). A cytotoxicity profile was generated after 24hrs using the neutral red assay. To examine changes in apoptotic pathways, caspase 3/7 and 8 activities were measured after 24hrs and further confirmed by Western blot. To characterize metal induced-alterations within cell cycle pathways: 1) cell cycle kinetics were assessed using flow cytometry (brdu/Hoechst) after 8, 24, and 48hrs, 2) gene transcriptional changes of key cell cycle regulators p21 and cyclin B1 were measured using RTPCR (8 and 24hrs), and 3) metal-induced translational changes of p53 and cdc 20 were quantified using Western blot (24hr only). We observed a dose dependent decrease in cell viability with increasing concentrations of both metals. The SWV exhibited higher sensitivity to Cd, confirmed by a statistically significant shift in cell viability compared to the C57BL/J6. A dose dependent increase of caspase 3/7 activity was observed for both strains across both metals. An increase in caspase 8 activity was also detected at the highest concentrations of MeHg and Cd (C57BL/J6 only). The C57BL/J6 displayed a higher increase in caspase 3 (p£0.05) and caspase 8 (p£0.07) activity compared to the SWV at the highest dose of cadmium. At relatively low cytotoxic levels (£20% reduction in cell viability after 24hrs) both Cd and MeHg significantly repressed cell cycle progression in both SWV and C57BL/J6 MEFs. In summary, in SWV and C57BL/J6 MEFs, we observed dose dependent metal-induced alterations in cell cycle and apoptotic pathways. Furthermore, differences in the magnitude of effect within these pathways were observed between the SWV and C57BL/J6 upon treatment of Cd. These differences may contribute to differing sensitivities observed in these two strains.