Accordingly, we observed a cumulative increase of the inflammasome-related markers Casp1 and NLRP3 (Fig. 7), of Casp3+ cells (Fig. 6a and 8a), and of LC3B puncta (Fig. 7b and 8b) in iPS cells that had undergone the double challenge compared to the single challenge (Fig. 6 and 7), showing that ethanol treatment induces long-term and long-lasting metabolic changes in the cell that can drive an enhanced response to any additional damage. On the contrary, while an increase in the number of Casp3+ cells was evident with peroxide or ethanol treatment alone in NPCs, no significant difference was detectable between cells that had undergone the double challenge compared to a single challenge (Fig. 9). This suggests that NPCs are more resilient than iPS cells to cumulative damages and/or that in our cell-based system the range of sensitivity is too narrow to reach statistical significance. Consistently, LC3B puncta appeared increased by each single challenge, but a quantitative evaluation in double challenged cells was impaired by the altered cell morphology. These data suggest that ethanol exposure in iPS cells and NPCs results in greater sensitivity to oxidative stress, which may contribute to the pathophysiology of neurogenesis in humans.
