Hypoxia decreases mitochondrial ROS production in cells

We re-examined the reported increase in mitochondrial ROS production during acute hypoxia in cells. Using the Amplex Ultrared/horseradish peroxidase assay we found a decrease, not increase, in hydrogen peroxide release from HEK293 cells under acute hypoxia, at times ranging from 1 min to 3 h. The rates of superoxide/hydrogen peroxide production from each of the three major sites (site I_Q in complex I and site III_Qo in complex III in mitochondria, and NADH oxidases (NOX) in the cytosol) were decreased to the same extent by acute hypoxia, with no change in the cells' ability to degrade added hydrogen peroxide. A similar decrease in ROS production under acute hypoxia was found using the diacetyldichlorofluorescein assay. Using a HIF1α reporter cell line we confirmed earlier observations that suppression of superoxide production by site III_Qo decreases HIF1α expression, and found similar effects of suppressing site I_Q or NOX. We conclude that increased mitochondrial ROS do not drive the response of HIF1α to acute hypoxia, but suggest that cytosolic H₂O₂ derived from site I_Q, site III_Qo and NOX in cells is necessary to permit HIF1α stabilization by other signals.