Enhanced hydrogen peroxide generation accompanies the beneficial bioenergetic effects of methylene blue in isolated brain mitochondria

The redox dye methylene blue (MB) is proven to have beneficial effects in various models of neurodegenerative diseases. Here we investigated the effects of MB (100 nM, 300 nM, and 1 μM) on key bioenergetic parameters and on H2O2 production/elimination in isolated guinea pig brain mitochondria under normal as well as respiration-impaired conditions. As measured by high-resolution Oxygraph the rate of resting oxygen consumption was increased, but the ADP-stimulated respiration was unaffected by MB with any of the substrates (glutamate malate, succinate, or α-glycerophosphate) used for supporting mitochondrial respiration. In mitochondria treated with inhibitors of complex I or complex III MB moderately but significantly increased the rate of ATP production, restored ΔΨm, and increased the rate of Ca2+ uptake. The effects of MB are consistent with transferring electrons from upstream components of the electron transport chain to cytochrome c, which is energetically favorable when the flow of electrons in the respiratory chain is compromised. On the other hand, MB significantly increased the production of H2O2 measured by Amplex UltraRed fluorimetry under all conditions, in resting, ATP-synthesizing, and respiration-impaired mitochondria, with each substrate combination supporting respiration. Furthermore, it also decreased the elimination of H2O2. Generation of H2O2 without superoxide formation, observed in the presence of MB, is interpreted as a result of reduction of molecular oxygen to H2O2 by the reduced MB. The elevated generation and impaired elimination of H2O2 should be considered for the overall oxidative state of mitochondria treated with MB