We propose that alcohol-induced ROS production derived from FWH reaction via the CYP2E1-mediated EtOH metabolism is modest. It appears that ROS and NO are derived mostly from aftermath activation of NOX, XOX, or NOS by EtOH metabolite, resulting in exacerbation of oxidative stress in the CNS. This claim is supported by the findings that Ach dose dependently increases the ROS levels in neuronal culture, and APC (NOX inhibitor) or AP (XOX inhibitor) reduces the Ach-induced ROS production, suggesting a direct involvement of the NOX/XOX activation pathway. Interestingly, activation of NOX/XOX by EtOH/Ach seems to be regulated by a transcriptional factor because actinomycin D (RNA synthesis inhibitor) effectively reduces both mRNA and protein levels of NOX/XOX. Since we used the p47phox peptide sequence for NOX mRNA analysis, we propose that tyrosine phosphorylation of p47phox initiated the downstream stimulation of transcription factor with a subsequent activation of NOX at the cytoplasmic membrane, similar to protein tyrosine kinase-mediated activation of matrix metalloproteinases [13,16]. It is plausible because the complex enzyme NOX is active only when the cytosolic components of the regulatory subunits p47phox
