Down-regulated genes affected by intermittent ethanol exposure in high-PRS and low-PRS microglial cells have similar predicted functions. They are significantly enriched in cell cycle regulation, including chromosome separation and mitotic sister chromatid separation (Fig. 4, C and D). To better visualize the gene expression changes in ethanol-responsive DEGs and cell cycle phases in both low-PRS and high-PRS microglial cells, we generated a heatmap summarizing the expression of cell cycle–related genes (fig. S6A). Upon ethanol exposure, the pathway analyses for the up-regulated genes showed enrichment in the G1-S and G2-M DNA damage checkpoint markers (figs. S6B and S7). This suggests that ethanol may cause cell cycle arrest and/or DNA damage in high-PRS and low-PRS microglial cells. An increase in γ-H2AX [a sensitive molecular marker of DNA damage (53)] after exposure to 75 mM ethanol was found by immunostaining in both high-PRS and low-PRS microglial cells (fig. S6, C and D). Consistent with a previous report in mouse neurons (54), these results indicated that intermittent ethanol exposure may cause DNA damage in human microglia, potentially interfering with microglial proliferation.
