In order to correlate the induction of CYP2E1/ADH activity with reactive metabolite production, we detected ROS levels in cultured neurons after EtOH/Ach exposure with or without 4-MP, DDC (diethydithiocarbamate; inhibitor of superoxide dismutase), or ALC (acetyl-L-carnitine; antioxidant). Increase in ROS production (by 68%) after EtOH treatment was significantly inhibited by 4-MP (Fig. 3A), suggesting that alcohol metabolism resulted in ROS generation, in part, via a Fenton-Weiss-Haber (FWH) reaction as direct EtOH metabolite (see schematic pathway). Inhibition of superoxide dismutase (by DDC) further enhanced the EtOH-induced ROS level (P<0.05), indicating a key role of SOD regulating the oxidants levels. Interestingly, Ach exposure also led to a 68% increase in ROS levels, suggesting the potential involvement of Ach in ROS generation. Since Ach is not an ROS donor, and Ach metabolism is not capable of producing ROS, increased levels of reactive species might be due to Ach-mediated activation of NOX or XOX. In order to address whether Ach increases ROS level via NOX/XOX activation (besides FWH reactions), we studied the dose-dependent effects of Ach on ROS production in cultured neurons using the
