Molecular hydrogen suppresses superoxide generation in the mitochondrial complex I and reduced mitochondrial membrane...

Molecular hydrogen (H₂) is recognized as a medical gas applicable to numerous diseases including neurodegenerative diseases, metabolic disorders, and rheumatoid arthritis. Although the efficacy of H₂ is reportedly attributed to its scavenging capability against the hydroxyl radical, the mechanisms underlying its therapeutic efficacy are not fully understood. Herein, we estimated the role of H₂ in the energy converting system of the mitochondria, the source of reactive oxygen species. To investigate the effects of H₂ on mitochondrial function, direction of electron flow, superoxide generation, and mitochondrial membrane potential were investigated. Forward electron transport (FET) or reverse electron transport (RET) was assessed by monitoring the decrease or increase of β-nicotinamide adenine dinucleotide hydrate (NADH, - or +, μM, respectively) in the presence of β-nicotinamide adenine dinucleotide (NAD⁺) and/or succinate in the isolated mitochondria. H₂O₂ converted from superoxide by superoxide dismutase (SOD) was measured to estimate electron leakage in the mitochondria. The effects of H₂ on mitochondrial membrane potential were observed by staining cells with the fluorescence probe, teramethylrhodamine ethyl ester (TMRE). Despite the absence of succinate, a distinct RET was observed (from +0.0313 ± 0.0106 μM to +1.20 ± 0.302 μM) by adding 25 μM H₂. In the presence of 5 μM NADH, RET by succinate inverted to FET from +1.62 ± 0.358 μM to -1.83 ± 0.191 μM, accompanied by a suppression of superoxide generated predominantly from complex I by 51.1%. H₂ solely reduced mitochondrial membrane potential of the cultured cells by 11.3% as assessed by TMRE. The direction of electron flow was altered by H₂ depending on the NAD⁺/NADH ratio, accompanied by suppression of superoxide generation H₂ could suppress superoxide generation in complex I in vitro and reduce membrane potential in vivo. H₂ may also neutralize semiquinone radicals to reduce superoxide produced in complex III. H₂ may function as a rectifier of the electron flow affecting the mitochondrial membrane potential to suppress oxidative damage in mitochondria.