Factors affecting liver mitochondrial hydrogen peroxide emission
2022
Okoye, Chidozie N. | Chinnappareddy, Nirmala | Stevens, Don | Kamunde, Collins
Mitochondria are key cellular sources of reactive oxygen species (ROS) and contain at least 12 known sites on multiple enzymes that convert molecular oxygen to superoxide and hydrogen peroxide (H₂O₂). Quantitation of site-specific ROS emission is critical to understand the relative contribution of different sites and the pathophysiologic importance of mitochondrial ROS. However, factors that affect mitochondrial ROS emission are not well understood. We characterized and optimized conditions for maximal total and site-specific H₂O₂ emission during oxidation of standard substrates and probed the source of the high H₂O₂ emission in unenergized rainbow trout liver mitochondria. We found that mitochondrial H₂O₂ emission capacity depended on the substrate being oxidized, mitochondrial protein concentration, and composition of the ROS detection system. Contrary to our expectation, addition of exogenous superoxide dismutase reduced H₂O₂ emission. Titration of conventional mitochondrial electron transfer system (ETS) inhibitors over a range of conditions revealed that one size does not fit all; inhibitor concentrations evoking maximal responses varied with substrate and were moderated by the presence of other inhibitors. Moreover, the efficacy of suppressors of electron leak (S1QEL1.1 and S3QEL2) was low and depended on the substrate being oxidized. We found that H₂O₂ emission in unenergized rainbow trout liver mitochondria was suppressed by GKT136901 suggesting that it is associated with NADPH oxidase activity. We conclude that optimization of assay conditions is critical for quantitation of maximal H₂O₂ emission and would facilitate more valid comparisons of mitochondrial total and site-specific H₂O₂ emission capacities between studies, tissues, and species.
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