A Novel Approach to Estimate ROS Origination by Hyperbaric Oxygen Exposure, Targeted Probes and Specific Inhibitors

Reactive oxygen species (ROS) are considered fundamental in various physiological/pathophysiological processes and prevention/treatment measures such as hyperbaric oxygen (HBO) therapy. In this study, the origination of ROS in human umbilical vein endothelial cells was investigated under basal and HBO conditions.

Whole cell or mitochondria-targeted fluorescent probes were applied to mark superoxide anion (O2-), and the ROS produced from mitochondrial respiratory chain (MRC), NADPH oxidase (NOX) and xanthine oxidase (XO) were identified by flow cytometry, confocal imaging and microplate fluorometry with or without specific inhibitors. An algorithm was established to calculate ROS proportion of each source.

HBO increased ROS to about 2.14-2.44 fold in mitochondria and 1.32-1.42 fold in whole cell. Then ROS levels were significantly decreased by MRC inhibition about 30% and 16%, respectively. NOX or XO inhibition did not affect HBO-induced ROS generation. Based on these data, it could be further estimated that mitochondrial ROS accounted for 32%-39% of basal whole-cell ROS including 3% from MRC complex II, and NOX accounted for at least 24%-29%. Following HBO treatment, almost all increased ROS originated from mitochondria, and MRC complex II contributed at least 45%-60%.

This study provided a simple but effective method to estimate the origination of intracellular ROS and found that MRC were the main source of HBO-induced ROS generation.