Ikuroh Ohsawa

Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals

Acute oxidative stress induced by ischemia-reperfusion or inflammation causes serious damage to tissues, and persistent oxidative stress is accepted as one of the causes of many common diseases including cancer. We show here that hydrogen (H2) has potential as an antioxidant in preventive and therapeutic applications. We induced acute oxidative stress in cultured cells by three independent methods. H2 selectively reduced the hydroxyl radical, the most cytotoxic of reactive oxygen species (ROS), and effectively protected cells; however, H2 did not react with other ROS, which possess physiological roles. We used an acute rat model in which oxidative stress damage was induced in the brain by focal ischemia and reperfusion. The inhalation of H2 gas markedly suppressed brain injury by buffering the effects of oxidative stress. Thus H2 can be used as an effective antioxidant therapy; owing to its ability to rapidly diffuse across membranes, it can reach and react with cytotoxic ROS and thus protect against oxidative damage.

Protection against ischemic brain injury by protein therapeutics

Preventing massive cell death is an important therapeutic strategy for various injuries and disorders. Protein therapeutics have the advantage of delivering proteins in a short period. We have engineered the antiapoptotic bcl-x gene to generate the super antiapoptotic factor, FNK, with a more powerful cytoprotective activity. In this study, we fused the protein transduction domain (PTD) of the HIV/Tat protein to FNK and used the construct in an animal model of ischemic brain injury. When added into culture media of human neuroblastoma cells and rat neocortical neurons, PTD-FNK rapidly transduced into cells and localized to mitochondria within 1 h. It protected the neuroblastomas and neurons against staurosporine-induced apoptosis and glutamate-induced excitotoxicity, respectively. The cytoprotective activity of PTD-FNK was found at concentrations as low as 0.3 pM. Additionally, PTD-FNK affected the cytosolic movement of calcium ions, which may relate to its neuroprotective action. Immunohistochemical analysis revealed that myc-tagged PTD-FNK (PTD-myc-FNK) injected i.p. into mice can have access into brain neurons. When injected i.p. into gerbils, PTD-FNK prevented delayed neuronal death in the hippocampus caused by transient global ischemia. These results suggest that PTD-FNK has a potential for clinical utility as a protein therapeutic strategy to prevent cell death in the brain.

Consumption of molecular hydrogen prevents the stress-induced impairments in hippocampus-dependent learning tasks during chronic physical restraint in mice.

We have reported that hydrogen (H2) acts as an efficient antioxidant by gaseous rapid diffusion. When water saturated with hydrogen (hydrogen water) was placed into the stomach of a rat, hydrogen was detected at several μM level in blood. Because hydrogen gas is unsuitable for continuous consumption, we investigated using mice whether drinking hydrogen water ad libitum, instead of inhaling hydrogen gas, prevents cognitive impairment by reducing oxidative stress. Chronic physical restraint stress to mice enhanced levels of oxidative stress markers, malondialdehyde and 4-hydroxy-2-nonenal, in the brain, and impaired learning and memory, as judged by three different methods: passive avoidance learning, object recognition task, and the Morris water maze. Consumption of hydrogen water ad libitum throughout the whole period suppressed the increase in the oxidative stress markers and prevented cognitive impairment, as judged by all three methods, whereas hydrogen water did not improve cognitive ability when no stress was provided. Neural proliferation in the dentate gyrus of the hippocampus was suppressed by restraint stress, as observed by 5-bromo-2′-deoxyuridine incorporation and Ki-67 immunostaining, proliferation markers. The consumption of hydrogen water ameliorated the reduced proliferation although the mechanistic link between the hydrogen-dependent changes in neurogenesis and cognitive impairments remains unclear. Thus, continuous consumption of hydrogen water reduces oxidative stress in the brain, and prevents the stress-induced decline in learning and memory caused by chronic physical restraint. Hydrogen water may be applicable for preventive use in cognitive or other neuronal disorders.

Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice.

Oxidative stress is implicated in atherogenesis; however most clinical trials with dietary antioxidants failed to show marked success in preventing atherosclerotic diseases. We have found that hydrogen (dihydrogen; H(2)) acts as an effective antioxidant to reduce oxidative stress [I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, K. Katsura, Y. Katayama, S, Asoh, S. Ohta, Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med. 13 (2007) 688-694]. Here, we investigated whether drinking H(2)-dissolved water at a saturated level (H(2)-water) ad libitum prevents arteriosclerosis using an apolipoprotein E knockout mouse (apoE(-/-)), a model of the spontaneous development of atherosclerosis. ApoE(-/-) mice drank H(2)-water ad libitum from 2 to 6 month old throughout the whole period. Atherosclerotic lesions were significantly reduced by ad libitum drinking of H(2)-water (p=0.0069) as judged by Oil-Red-O staining series of sections of aorta. The oxidative stress level of aorta was decreased. Accumulation of macrophages in atherosclerotic lesions was confirmed. Thus, consumption of H(2)-dissolved water has the potential to prevent arteriosclerosis.

Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of Parkinson's disease

Molecular hydrogen serves as an antioxidant that reduces hydroxyl radicals, but not the other reactive oxygen and nitrogen species. In the past year, molecular hydrogen has been reported to prevent or ameliorate eight diseases in rodents and one in human associated with oxidative stress. In Parkinsons disease, mitochondrial dysfunction and the associated oxidative stress are major causes of dopaminergic cell loss in the substantia nigra. We examined effects of approximately 50%-saturated molecular hydrogen in drinking water before or after the stereotactic surgery on 6-hydroxydopamine-induced nigrostrital degeneration in a rat model of Parkinsons disease. Methamphetamine-induced behavioral analysis showed that molecular hydrogen prevented both the development and progression of the nigrostrital degeneration. Tyrosine hydroxylase staining of the substantia nigra and striatum also demonstrated that pre- and post-treatment with hydrogen prevented the dopaminergic cell loss. Our studies suggest that hydrogen water is likely able to retard the development and progression of Parkinsons disease.

Amino-terminal region of secreted form of amyloid precursor protein stimulates proliferation of neural stem cells

β‐Amyloid precursor protein (APP) has been reported to be expressed in the CNS from the early stages of development. However, the functional role of APP during early development remains unclear. In the present study, we found that the secreted form of APP (sAPP) significantly enhanced proliferation of neural stem cells. Cells were prepared from 13‐day embryonic rat neocortex, which was dissected with a Pasteur pipette to make cell clusters. After 12 h of cultivation in the medium without serum, cells around the centre of the cluster were still nestin‐positive proliferative cells, i.e. neural stem cells. To determine whether the proliferation of cells was regulated by sAPP, cultures were treated with recombinant sAPP695, the secreted form of human APP695 produced by yeast. Both DNA synthesis and expression of proliferating cell nuclear antigen markedly increased after 5 h of sAPP695 addition. The enhancement of DNA synthesis by sAPP695 stimulation was blocked by the 22C11 monoclonal antibody specific for the amino‐terminal region of sAPP. Then, we examined the effect of the amino‐terminal fragment of sAPP and the epitope peptide of 22C11 antibody, and found that both of them also promoted DNA synthesis, suggesting that the amino‐terminal region of sAPP is responsible for the biological activity. Our findings indicate the possibility that sAPP enhances proliferation of neural stem cells in vivo and plays an important role during the early CNS development.

Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late‐onset Alzheimers disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2‐deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2‐deficient transfectants were highly vulnerable to exogenous 4‐hydroxy‐2‐nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2‐deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4‐hydroxy‐2‐nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.

Metabolic Remodeling Induced by Mitochondrial Aldehyde Stress Stimulates Tolerance to Oxidative Stress in the Heart

Rationale: Aldehyde accumulation is regarded as a pathognomonic feature of oxidative stress–associated cardiovascular disease. Objective: We investigated how the heart compensates for the accelerated accumulation of aldehydes. Methods and Results: Aldehyde dehydrogenase 2 (ALDH2) has a major role in aldehyde detoxification in the mitochondria, a major source of aldehydes. Transgenic (Tg) mice carrying an Aldh2 gene with a single nucleotide polymorphism (Aldh2*2) were developed. This polymorphism has a dominant-negative effect and the Tg mice exhibited impaired ALDH activity against a broad range of aldehydes. Despite a shift toward the oxidative state in mitochondrial matrices, Aldh2*2 Tg hearts displayed normal left ventricular function by echocardiography and, because of metabolic remodeling, an unexpected tolerance to oxidative stress induced by ischemia/reperfusion injury. Mitochondrial aldehyde stress stimulated eukaryotic translation initiation factor 2&agr; phosphorylation. Subsequent translational and transcriptional activation of activating transcription factor-4 promoted the expression of enzymes involved in amino acid biosynthesis and transport, ultimately providing precursor amino acids for glutathione biosynthesis. Intracellular glutathione levels were increased 1.37-fold in Aldh2*2 Tg hearts compared with wild-type controls. Heterozygous knockout of Atf4 blunted the increase in intracellular glutathione levels in Aldh2*2 Tg hearts, thereby attenuating the oxidative stress–resistant phenotype. Furthermore, glycolysis and NADPH generation via the pentose phosphate pathway were activated in Aldh2*2 Tg hearts. (NADPH is required for the recycling of oxidized glutathione.) Conclusions: The findings of the present study indicate that mitochondrial aldehyde stress in the heart induces metabolic remodeling, leading to activation of the glutathione–redox cycle, which confers resistance against acute oxidative stress induced by ischemia/reperfusion.

Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress

Mitochondria combine the production of energy with an efficient chain of reduction-oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.

Protection of the Retina by Rapid Diffusion of Hydrogen: Administration of Hydrogen-Loaded Eye Drops in Retinal Ischemia–Reperfusion Injury

PURPOSE Retinal ischemia-reperfusion (I/R) injury by transient elevation of intraocular pressure (IOP) is known to induce neuronal damage through the generation of reactive oxygen species. Study results have indicated that molecular hydrogen (H(2)) is an efficient antioxidant gas that selectively reduces the hydroxyl radical (*OH) and suppresses oxidative stress-induced injury in several organs. This study was conducted to explore the neuroprotective effect of H(2)-loaded eye drops on retinal I/R injury. METHODS Retinal ischemia was induced in rats by raising IOP for 60 minutes. H(2)-loaded eye drops were prepared by dissolving H(2) gas into a saline to saturated level and administered to the ocular surface continuously during the ischemia and/or reperfusion periods. One day after I/R injury, apoptotic cells in the retina were quantified, and oxidative stress was evaluated by markers such as 4-hydroxynonenal and 8-hydroxy-2-deoxyguanosine. Seven days after I/R injury, retinal damage was quantified by measuring the thickness of the retina. RESULTS When H(2)-loaded eye drops were continuously administered, H(2) concentration in the vitreous body immediately increased and I/R-induced *OH level decreased. The drops reduced the number of retinal apoptotic and oxidative stress marker-positive cells and prevented retinal thinning with an accompanying activation of Müller glia, astrocytes, and microglia. The drops improved the recovery of retinal thickness by >70%. CONCLUSIONS H(2) has no known toxic effects on the human body. Thus, the results suggest that H(2)-loaded eye drops are a highly useful neuroprotective and antioxidative therapeutic treatment for acute retinal I/R injury.