The idea that alcohol abuse causes neuronal injury is well accepted; however, its underlying mechanisms are still poorly understood. Previously, we demonstrated that EtOH metabolism in primary human brain endothelial cells resulted in ROS generation and intracellular calcium release leading to BBB dysfunction and enhanced leukocyte migration across the BBB. We hypothesized that EtOH metabolism in primary human neurons causes ROS/NO production leading to neurodegeneration. To verify this idea, we determined the dose- and time-dependent neurotoxic effects of EtOH, Ach, or S-nitroso-N-acetylpenicillamine (an NO donor) on neuronal cell culture. Using broad range concentrations of EtOH (10-100 mM), Ach (10-100 μM), or SNAP (10-100 μM), we observed that EtOH higher than 20 mM or Ach/SNAP higher than 20 μM adversely affects cell viability as detected by live and dead assay after a 24-h treatment period. For each batch of primary neurons isolated from human fetal tissue, MAP-2 or α-tubulin immunostainings assessed the purity of neurons in cultured neuronal cells demonstrating 75-85% of cells positive for neuronal markers.
