C8-D1A

Exposure To MeHg Induces Mortality and Stress Oxidative in C8-D1A Astrocytic Cells

MeHg poisons astrocytes despite early Nrf2 activation. Using C8-D1A cells, Ahmed et al. blocked STAT3 with AG490/C188-9 and applied ROS scavengers NAC/Trolox to ask if STAT3 curbs acute toxicity and how ROS modulate its activation.

C8-D1A astrocytic cells were exposed to various MeHg concentrations, and cytotoxicity was evaluated with LDH assay. After 24 h, 20 µM MeHg significantly increased LDH release (H=19.926, p=0.003; post hoc p=0.006 vs non-exposed, Fig. 1A); shorter exposures or lower concentrations had no effect (Fig. 2A-C). MTT assay corroborated these findings: 24 h exposure to 20 µM MeHg significantly reduced cell viability in a concentration-dependent manner (H=18.351, p=0.005, Fig. 1B), whereas lower concentrations and shorter times did not (Fig. 2D-F).

MeHg is known to induce oxidative stress; therefore, ROS production was measured with CM-H2DCFDA. Twenty µM MeHg elevated ROS at 3 h (H=21.927, p=0.001; post hoc p=0.006, Fig. 1C) and also at 30 min (Fig. 2G) and 6 h (Fig. 2I). Ten µM MeHg significantly increased ROS at both 3 h and 6 h as well. Because MeHg disrupts mitochondrial function and increases mitochondrial superoxide, they used MitoSOX Red. Twenty µM MeHg significantly raised mitochondrial superoxide at 3 h (H=33.564, p<0.001, Fig. 1D), with similar results at 30 min, 1 h and 6 h (Fig. 2J-L); 5 µM was already effective at 30 min. To further assess oxidative stress, intracellular glutathione (GSH) was measured. Although MeHg is reported to deplete GSH, 10 µM MeHg did not significantly alter total GSH levels after 6 h (Fig. 2M).