Molecular hydrogen suppresses free-radical-induced cell death by mitigating fatty acid peroxidation and mitochondrial dysfunction

Molecular hydrogen (H2) was believed to be an inert and non-functional molecule in mammalian cells; however, we overturned the concept by reporting the therapeutic effects of H2 against oxidative stress. Subsequently, extensive studies revealed multiple functions of H2 by exhibiting the efficacies of H2 in various animal models and clinical studies. Here, we investigated the effect of H2 on free radical-induced cytotoxicity using tert-butyl hydroperoxide (TBHP) in a human acute monocytic leukemia cell line, THP-1. Cell membrane permeability was determined using lactate dehydrogenase (LDH) release assay and Hoechst 33342/propidium iodide (PI) staining. Fatty acid peroxidation and mitochondrial viability were measured using two kinds of fluorescent dyes, Liperfluo and C11-BODIPY, and using the alamarBlue assay based on the reduction of resazurin to resorufin by mainly mitochondrial succinate dehydrogenase, respectively. Mitochondrial membrane potential (MMP) was evaluated using tetramethylrhodamine methyl ester. As a result, H2 protected the cultured cells against the cytotoxic effects induced by TBHP; H2 suppressed cellular fatty acid-peroxidation and cell membrane permeability, H2 mitigated the decline in mitochondrial oxidoreductase activity and MMP, and protected cells against cell death evaluated using PI-staining. These results suggested that H2 suppresses free radical-induced cell death through protection against fatty acid-peroxidation and mitochondrial dysfunction.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author