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 NOX inhibitor apocynin (APC) or the XOX inhibitor allopurinol (AP). Our results indicated dose-dependent increments of 38-69% in ROS production by Ach stimulation (5-50 μM) in human neurons, and AP/APC individually or in combination significantly inhibited (27-43%, P<0.03) the increased ROS production caused by 50 μM Ach (Fig. 3B). Taken together, these findings suggest that EtOH metabolism by ADH/CYP2E1 generated ROS and Ach, and Ach subsequently activated NOX and XOX, exacerbating oxidative stress level in the CNS.
