In order to correlate the EtOH-induced ROS production with oxidative damage and neuronal injury, we detected the marker for lipid peroxidation product 4-hydroxynonenal (4-HNE), and neuronal injury marker protein neurofilament. Treatment of neurons with EtOH/Ach for 72 h significantly elevated the level of 4-HNE by 43 or 48%, respectively, in neurons compared with control (Figs. 7A and B). Interestingly, this increase in 4-HNE levels in neurons after exposure to EtOH/Ach paralleled a 40% decrease in the light-chain neurofilament protein (70 kDa) or a 36% decrease in neuronal viability as compared with respective controls (Figs. 7C-E). Pretreatment of 4-MP, AP+APC, or ALC prevented the EtOH/Ach-induced increase in 4-HNE, and decrease in neurofilament protein or neuronal death. These results suggest that EtOH/Ach-induced neuronal injury can be respectively reversed by restoring mitochondrial function (by ALC), or by inhibiting the activation of NOX/XOX (by AP+APC), or by inhibiting alcohol metabolism (by 4-MP). The potential mechanisms of alcohol-induced mitochondrial impairment for producing excess ROS and the putative role of ROS for initiation of neuroinflammation are currently under investigation, thus will be described in a future communication.
