Recent Progress Toward Hydrogen Medicine: Potential of Molecular Hydrogen for Preventive and Therapeutic Applications

Persistent oxidative stress is one of the major causes of most lifestyle-related diseases, cancer and the aging process. Acute oxidative stress directly causes serious damage to tissues. Despite the clinical importance of oxidative damage, antioxidants have been of limited therapeutic success. We have proposed that molecular hydrogen (H2) has potential as a “novel” antioxidant in preventive and therapeutic applications [Ohsawa et al., Nat Med. 2007: 13; 688-94]. H2 has a number of advantages as a potential antioxidant: H2 rapidly diffuses into tissues and cells, and it is mild enough neither to disturb metabolic redox reactions nor to affect reactive oxygen species (ROS) that function in cell signaling, thereby, there should be little adverse effects of consuming H2. There are several methods to ingest or consume H2, including inhaling hydrogen gas, drinking H2-dissolved water (hydrogen water), taking a hydrogen bath, injecting H2-dissolved saline (hydrogen saline), dropping hydrogen saline onto the eye, and increasing the production of intestinal H2 by bacteria. Since the publication of the first H2 paper in Nature Medicine in 2007, the biological effects of H2 have been confirmed by the publication of more than 38 diseases, physiological states and clinical tests in leading biological/medical journals, and several groups have started clinical examinations. Moreover, H2 shows not only effects against oxidative stress, but also various anti-inflammatory and anti-allergic effects. H2 regulates various gene expressions and protein-phosphorylations, though the molecular mechanisms underlying the marked effects of very small amounts of H2 remain elusive.


INTRODUCTION
Oxidative stress arises from the strong cellular oxidizing potential of excess reactive oxygen species (ROS) [1]. Acute oxidative stress arises from a variety of situations, including ischemia reperfusion [2]. Persistent oxidative stress is widely accepted as one of the causes of most lifestyle-related diseases, cancer and the aging process [3][4][5][6][7]; however, many antioxidant supplements could not prevent cancer, myocardial farction and atherosclerosis, but rather conversely increase mortality [8][9][10][11]; thus, it is very important to be aware of side effects when developing an effective antioxidant for the prevention of oxidative stress-related diseases.
We found that molecular hydrogen (H 2 ) has roles as a "novel" antioxidant in preventive and therapeutic applications [12]. H 2 has advantages as a potential antioxidant without adverse effects: it is mild enough neither to disturb metabolic redox reactions nor to affect ROS, which function in cell signaling [13][14][15] and has favorable distribution characteristics in its own physical ability to penetrate biomembranes and diffuse through barriers into cellular components.
Here, we review the recent progress toward therapeutic and preventive applications of H 2 in widespread fields.

ROS AS ONE OF THE MAJOR CAUSES OF ACUTE AND CHRONIC DISEASES 2.1. Persistent Oxidative Stress
ROS are generated inside the body throughout our daily lives, such as during hard exercise, smoking, exposure to ultraviolet rays or air pollution, aging, physical or psychological stress, and so on [16][17][18][19]. Inside every aerobic organism, ROS are generated when breathing consumes oxygen.
These ROSs are generated under the condition of excessively high membrane potential to leak electrons from the electron transport chain [28]. In fact, uncoupling proteins control the membrane potential to suppress the production of ROS and then consequently to repress diabetes [29][30][31].
Mitochondrial aldehyde hydydrogenase 2 (ALDH2) functions as a protector against oxidative stress by detoxifying cytotoxic aldehydes, such as 4-hydroxy-2-nonenal [4,5,32]. Thus, a defect of ALDH2 sufficiently induces phenotypes of age-dependent dementia by accumulating such cytotoxic aldehydes [32]. Paradoxically, such aldehydes stimulate protective systems against oxidative stress [33]. Thus, oxidative stress has two faces, to damage tissues and to enhance protective systems.  [49] and in the spin trap signals. Notably, H 2 decreased •OH levels even in the nuclear region [12].

, H 2 O 2 and NO in Cultured
After antimycin A treatment, H 2 prevented the decline of the mitochondrial membrane potential. This suggested that H 2 protected mitochondria from •OH. Along with this protective effect, H 2 also prevented a decrease in the cellular level of ATP synthesized in mitochondria. The fact that H 2 protected mitochondria and nuclear DNA provided evidence that H 2 penetrated most membranes and diffused into organelles. Consequently, H 2 protected cultured cells against oxidative stress [12].

Other Effects Shown by Using Culture Systems
H 2 dissolved in medium protected cultured auditory hair cells from free radicals [50] and is suggested to decrease •OH, as judged by the decrease in HPF fluorescence in vestibular tissue [51].
•OH causes most ionizing radiation-induced cellular damage. H 2 exhibited protective effects against radiation-induced damage in cultured cells and mice [52]. Cosmic radiation is known to induce DNA and lipid damage associated with increased oxidative stress and remains a major concern in space travel. It is expected that space mission activities will increase in coming years both in number and duration. It is therefore important to estimate and prevent the risks encountered by astronauts due to oxidative stress prior to developing clinical symptoms of disease. Schoenfeld et al. hypothesized that H 2 administration to astronauts by either inhalation or drinking hydrogen water may potentially yield a novel and feasible preventative/therapeutic strategy to prevent radiation-induced adverse events [53].
On the other hand, H 2 treatment prolonged the replicable lifespan of bone marrow multipotential stromal cells in vitro while preserving differentiation and paracrine potentials. Cell therapy with bone marrow multipotential stromal cells/mesenchymal stem cells represents a promising approach in the field of regenerative medicine. Low frequency of mesenchymal stem cells in adult bone marrow necessitates ex vivo expansion of mesenchymal stem cells after harvest; however, such manipulation causes cellular senescence with loss of differentiation, proliferative, and therapeutic potentials of mesenchymal stem cells. As oxidative stress is one of the key insults promoting cell senescence in vivo as well as in vitro, H 2 prevented the senescent process during mesenchymal stem cell expansion. Notably, 3% hydrogen gas treatment did not decrease •OH, protein carbonyl, and 8-hydroxydeoxyguanosine, suggesting that scavenging •OH might not be responsible for these effects of hydrogen gas in this study [54].

ADVANTAGES OF HYDROGEN 5.1. Rapid Diffusion
H 2 has a number of advantages as a potential antioxidant. First, it has favorable distribution characteristics with its own physical ability to penetrate biomembranes and diffuse into the cytosol.
Excessive oxidative damage is a major factor because the mitochondrial respiratory chain is a significant source of damaging reactive oxygen species; however, despite the clinical importance of mitochondrial oxidative damage, antioxidants have been of limited therapeutic success. This may be because antioxidants are not selectively taken up by mitochondria [55][56][57]. As H 2 effectively reaches the nucleus and mitochondria, the protection of nuclear DNA and mitochondria suggests preventive effects on lifestyle-related diseases, cancer and the aging process [12]. Moreover, H 2 passes through the blood brain barrier, although most antioxidant compounds cannot; this is also an advantage of H 2 .
Monitoring H 2 concentration inside various tissues can prove gaseous diffusion [58].

No Direct Elimination of Functionally Important ROS
Despite their cytotoxic effects, low concentrations of ROS, such as •O 2 and H 2 O 2 , function as signaling molecules and regulate apoptosis, cell proliferation, and differentiation [14,15]. As mentioned, unexpectedly and notably, recent studies have suggested that excessive antioxidants increased mortality and rates of cancer [9,11,[59][60][61][62] because they may interfere with some essential defensive mechanisms [13,60,[63][64][65][66][67]. At higher concentrations, H 2 O 2 is converted to hypochlorous acid by myeloperoxidase to defend against bacterial invasion [68]. Additionally, NO functions as a neurotransmitter and is essential for the dilation of blood vessels [69].
Since H 2 reduces •OH but does not affect •O 2 and H 2 O 2 having physiological roles [12], we propose that the adverse effects of H 2 are very small compared to other antioxidants.

No toxicity Even at Higher Concentration.
Several medical gasses are expected to provide more effective therapeutic interventions and preventive medicine despite their severe toxicity. Gas inhalation as disease therapy has received recent interest [70]. In past decades, there has been extraordinary, rapid growth in our knowledge of gaseous molecules, including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H 2 S), which have been known to play important roles in biological systems [71,72].
In pre-clinical experimental models of disease, including ischemia-reperfusion injury, the inhalation of exogenous CO or H 2 S has produced a favorable outcome for most vital organs [73][74][75][76]. In particular, NO has been approved as a therapeutic agent in clinical practice [77]. The inherent toxicity of these gasses must be investigated for gas inhalation to be considered an effective therapeutic strategy because these gasses are highly toxic at considerable concentrations. Additionally, NO enhances oxidative stress via the reaction with •O 2 by the production of highly oxidative peroxynitrite (NO + •O 2 -ONOO -). It is unknown if the therapeutically effective threshold for CO or H 2 S can be attained locally in target organs without delivering a potentially toxic level of the gasses via the lungs.
In contrast, H 2 has more advantages from the aspect of toxicity: H 2 has no cytotoxicity even at high concentration [78][79][80][81]. Furthermore, safety standards have been established for high concentrations of hydrogen gas for inhalation since high pressure hydrogen gas is used in deep diving gas mixes to prevent decompression sickness and arterial gas thrombi [81]. The safety of H 2 for humans is demonstrated by its application in Hydreliox, an exotic, breathing gas mixture of 49% H 2 , 50% helium and 1% O 2 , which is used to prevent decompression sickness and nitrogen narcosis during very deep technical diving [78][79][80][81].

METHODS OF INGEST HYDROGEN I: INHALATION OF HYDROGEN GAS 6.1. Inhalation of Hydrogen Gas
Inhalation of hydrogen gas is a straightforward therapeutic method. Hydrogen gas can be inhaled by delivering hydrogen gas through a ventilator circuit, facemask or nasal cannula. Since inhaled hydrogen gas acts more rapidly, it may be suitable for defense against acute oxidative stress. In particular, inhalation of gas does not affect blood pressure [12]; blood pressure increased by infusion may cause serious obstacles during the treatment of myocardial infarction. Hydrogen gas poses no risk of explosion in air and in pure oxygen when present at concentrations < 4%, as mentioned earlier; however, safety could be a concern and the desired concentration of H 2 must be monitored and maintained with an approved and commercially available tool.
Rats inhaled hydrogen gas in a mix of nitrous oxide (N 2 O) (for anesthesia), O 2 , and N 2 . The inhalation of H 2 actually increased H 2 dissolved in arterial blood depending upon the hydrogen gas concentrations, and H 2 levels in venous blood were lower than in arterial blood; the different level between arterial and venous blood indicates the amount of H 2 incorporated into tissues [12].

Direct Demonstration of Rapid Diffusion of Hydrogen as a Medical Gas
Gasses possess the ability to diffuse readily in different materials and become uniformly distributed within a defined space. "Biologic gasses" are assumed to diffuse freely across biologic membranes, acting in a variety of functional capacities [70]; hydrogen gas is an example of this. PC12 cells were placed in culture media containing H2 (0.6 mM) and O2 (0.24 mM), and then oxidative stress was induced by adding antimycin A (10 μg/mL), an inhibitor of the electron transport chain of mitochondria, and maintained for 1 day. Two markers of oxidative stress were detected by immunostaining with anti-8-hydroxy-Guanine (Nucleus) and anti-4-hydoroxy-2-nonenal (Membrane). Thirty minutes after adding antimycin A with or without H2, 100 nM tetramethylrhodamine methyl ester (TMRM), a fluorescent detector of the membrane potential of mitochondrion, were added, incubated for 10 min, and cells were imaged with a laser scanning confocal microscope. These results indicate that H2 reach the nucleus and mitochondria and protects them.
The gaseous diffusion of H 2 is indeed proven by monitoring its concentration inside various tissues. H 2 can be detected with specific electrodes. H 2 concentration has been monitored within a rat myocardium. The electrode was inserted into the non-ischemic myocardium of the left ventricle. The incremental rate of H 2 saturation for the non-ischemic myocardium and arterial blood was similar. Then, the electrode was inserted into the 'at risk' area for infarction to investigate the diffusion of H 2 into the ischemic myocardium, induced by coronary artery occlusion. Notably, H 2 concentration was increased even in the ischemic myocardium. Although the incremental rate of H 2 saturation was slower in the ischemic myocardium than in the non-ischemic myocardium, the peak level of H 2 in the ischemic myocardium was approximately two thirds of the value observed for the non-ischemic myocardium [58].

Protective Effects on Ischemia Reperfusion Model by Rat Cerebral Infarction
Hydrogen gas was applied to a rat model of ischemiareperfusion as an acute model [82]. We produced focal ischemia by occlusion of the rat middle cerebral artery with subsequent reperfusion. One day after middle cerebral artery occlusion, infarct volumes decreased in a H 2 -dependent manner. One week after middle cerebral artery occlusion, the difference in infarct volumes between non-treated and H 2 -treated rats increased. H 2 -treated rats also showed improvements in body weight and temperature and movement defects vs. untreated rats. Thus, H 2 suppressed not only the initial brain injury, but also the progression of injury. H 2 markedly decreased several oxidative stress markers. In this experiment, H 2 was demonstrated to have the potential to markedly decrease oxidative stress and suppress brain injury [12].

Protective Effects on Hepatic and Cardiac Ischemia Reperfusion Injury
Next, inhalation of hydrogen gas was also applied to a hepatic ischemia reperfusion injury model [83]. Inhalation of H 2 clearly attenuated the degeneration induced by hepatic ischemia reperfusion and increased the protective effect in an H 2 -dependent manner. In contrast, helium gas (He) exhibited no effect, indicating that H 2 clearly has a specific protective effect [84].
The degree of cardioprotection against ischemia-reperfusion injury was evaluated by measuring oxidative damage and infarct size after left anterior descending coronary artery occlusion and reperfusion. Inhalation of an incombustible level of hydrogen gas (2%) before reperfusion significantly reduced oxidative stressinduced myocardial injury and infarct size without affecting hemodynamic parameters, and thereby prevented deleterious left ventricle remodeling [58].

Protective Effects in Organ Transplantation
H 2 inhalation significantly ameliorated intestinal and pulmonary transplant injury and prevented remote organ inflammation via its antioxidant effects [85,86]. Ischemia/reperfusion injury during small intestinal and lung transplantation frequently causes complications, including dysmotility, inflammation and organ failure.
H 2 treatment resulted in significantly improved gastrointestinal transit, as well as jejunal smooth muscle contractility in response to bethanechol [86]. Graft lipid peroxidation was significantly reduced in the presence of H 2 , demonstrating antioxidant effects of H 2 in the transplanted lungs. Exposure to 2% hydrogen gas significantly blocked the production of several pro-inflammatory mediators and reduced apoptosis with induction of the anti-apoptotic molecules Bcell lymphoma-2 and B-cell lymphoma-extra large.
Rat cardiac cold ischemia reperfusion injury was ameliorated with inhaled H 2 or carbon monoxide (CO), or both. Combined therapy with H 2 and CO demonstrated enhanced therapeutic efficacy via both anti-oxidant and anti-inflammatory mechanisms, and may be a clinically feasible approach for preventing cold ischemia reper-fusion injury of the myocardium [87]. Inhaled hydrogen gas effectively reduced ventilator-induced lung injury-associated inflammatory responses, at both a local and systemic level, via its antioxidant, anti-inflammatory and anti-apoptotic effects [88].

Protective Effects in Infectious Diseases and antiinflammatory Effects
Sepsis, a multiple organ dysfunction syndrome, is the leading cause of death in critically ill patients [89]. Hydrogen gas inhalation significantly improved the survival rate and organ damage of septic mice with moderate or severe cecal ligation and puncture by reducing levels of early and late pro-inflammatory cytokines in serum and tissues [90].
The effects of 2% H 2 treatment was investigated on the survival rate and organ damage in zymosan-induced generalized inflammation model. The beneficial effects of H 2 treatment zymosan-induced organ damage were associated with decreased levels of oxidative product, increased activities of antioxidant enzyme, and reduced levels of early and late pro-inflammatory cytokines in serum and tissues. H 2 treatment protected against multiple organ damage in a zymosan-induced generalized inflammation model, suggesting the potential use of H 2 as a therapeutic agent in the therapy of conditions associated with inflammation-related multiple organ dysfunction syndrome [91].

Others
Other reports had the following titles: Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model [92]; hydrogen gas reduced acute hyperglycemia-enhanced hemorrhagic transformation in a focal ischemia rat model [93]; hydrogen is neuroprotective and preserves cerebrovascular reactivity in asphyxiated newborn pigs [94]; beneficial effects of hydrogen gas in a rat model of traumatic brain injury via reducing oxidative stress [95]; beneficial effects of hydrogen gas against spinal cord ischemiareperfusion injury in rabbits [96]; and hydrogen protects vestibular hair cells from free radicals [97].

METHODS OF INGEST HYDROGEN II: ORAL INGES-TION OF HYDROGEN WATER 7.1. Oral Ingestion by Drinking Hydrogen Water
Since inhaled hydrogen gas acts more rapidly, it may be suitable for defense against acute oxidative stress. In particular, inhalation of gas does not affect blood pressure; blood pressure increased by infusion may be serious in myocardial infarction; however, inhalation of hydrogen gas may be unsuitable or not practical as continuous H 2 consumption in daily life for preventive use. In contrast, solubilized H 2 (H 2 -dissolved water; namely, hydrogen water) may be beneficial since it is a portable, easily administered and a safe means of delivering H 2 [98]. H 2 can be dissolved in water up to 0.8 mM under atmospheric pressure at room temperature as mentioned earlier. Unexpectedly, drinking hydrogen water had effects comparable to hydrogen gas inhalation [99].
Hydrogen water can be made by several methods, including dissolving hydrogen gas in water under high pressure, dissolving electrolyzed H 2 in water, and by the reaction of magnesium metal with water. The method of dissolving hydrogen gas under high pressure has an advantage because it is applicable not only using water but also any other solvents.
When water saturated with H 2 was placed into the stomach of a rat, H 2 was detected at several μM level in blood [98,99]. Moreover, hepatic H 2 was monitored with a needle-type hydrogen electrode, and H 2 accumulated after oral administration of hydrogen water, partly explaining why consumption of even a small amount of H 2 over a short dwell time could efficiently improve various disease models. An additional in vitro experiment confirmed that polymers of carbohydrates, including glycogen and starch, have an affinity for H 2 [99].

Prevention of Cognitive Decline
Chronic physical restraint stress on mice enhanced levels of oxidative stress in the brain, and impaired learning and memory [100,101]. Consumption of hydrogen water ad libitum suppressed the increase in oxidative stress, and prevented cognitive impairment. Neural proliferation in the dentate gyrus of the hippocampus was suppressed by restraint stress [101]. The consumption of hydrogen water ameliorated the reduced proliferation; however, a mechanistic link between H 2 -dependent changes in neurogenesis and cognitive impairments remains unclear. Thus, continuous consumption of hydrogen water reduced oxidative stress in the brain and prevented the stress-induced decline in learning and memory [98].

Preventive and Therapeutic Affects on Parkinson Disease Model
In Parkinson's disease, mitochondrial dysfunction and the associated oxidative stress are major causes of dopaminergic cell loss in the substantia nigra [102]. H 2 in drinking water was given before or after stereotactic surgery for 6-hydroxydopamine-induced nigrostrital degeneration in a rat model of Parkinson's disease. Hydrogen water prevented both the development and progression of nigrostriatal degeneration. Hydrogen water likely retards the development and progression of Parkinson's disease [103].

Prevention of Atherosclerosis Model
Oxidative stress is involved in atherosclerosis [105,106]; however most clinical trials of dietary antioxidants failed to show marked success in preventing atherosclerotic diseases [8,107,108]. Drinking hydrogen water ad libitum decreased the aorta oxidative stress level and prevented arteriosclerosis in an apolipoprotein E knockout mouse [109]. Thus, consumption of hydrogen water has potential to prevent arteriosclerosis more effectively than other antioxidants [110].

Improvement of Metabolic Syndrome
Increased oxidative stress in obesity affects metabolic syndrome [111]. Long-term drinking of hydrogen water significantly controlled fat and body weights, despite no change in the consumption of food and water. Moreover, drinking hydrogen water decreased levels of plasma glucose, insulin and triglyceride, the effect of which on hyperglycemia was similar to diet restriction [112]. A mechanistic study revealed that the gene expression of the hepatic hormone, fibroblast growth factor 21 (FGF21) was enhanced, which should function to enhance fatty acid and glucose expenditure. Indeed, drinking hydrogen water stimulated energy metabolism, as measured by O 2 consumption and CO 2 expiration. These results suggest the potential benefit of H 2 in improving obesity, diabetes and metabolic syndrome [112].

Prevention of Adverse Effects by an Anti-tumor Drug
Cisplatin is a widely used anti-cancer drug in the treatment of a wide range of tumors; however, its application is limited by causing nephrotoxicity, which may be mediated by oxidative stress [113]. Inhalation of hydrogen gas (1% H 2 in air) or drinking hydrogen water improved mortality and body-weight loss caused by cisplatin, and alleviated nephrotoxicity. Consumption of hydrogen water improved metamorphosis accompanying decreased apoptosis in the kidney. Despite its protective effects against cisplatin-induced toxicity, H 2 did not impair the anti-tumor activity of cisplatin against cancer cell lines in vitro and in tumor-bearing mice in vivo. Thus, H 2 , whether hydrogen gas or hydrogen water, could improve the quality of life of patients during chemotherapy [99]. This finding was confirmed by another group [114].

Anti-allergic Reactions
It was demonstrated using a mouse model that drinking hydrogen water could attenuate an immediate-type allergic reaction by suppressing the phosphorylation of Fc RI-associated Lyn and its downstream signaling molecules, which subsequently inhibited NADPH oxidase activity and reduced the generation of hydrogen peroxide [115]. These findings imply that the beneficial effects of H 2 are not only imparted by its radical scavenging activity, but also by modulating a specific signaling pathway.

Effects on Transplantation
ROS contributes to the development of interstitial fibrosis and tubular atrophy seen in chronic allograft nephropathy. Nakao's group tested the effect of treatment with hydrogen water in a model of kidney transplantation, in which allografts from Lewis rats were orthotopically transplanted into Brown Norway recipients that had undergone bilateral nephrectomy. Drinking hydrogen water improved allograft function, slowed the progression of chronic allograft nephropathy, reduced oxidant injury and inflammatory mediator production, and improved overall survival. Inflammatory signaling pathways, such as mitogen-activated protein kinases, were less activated in renal allografts from hydrogen water-treated rats as compared with normal water-treated rats. Thus, oral hydrogen water is an effective antioxidant and anti-inflammatory agent that reduced chronic allograft nephropathy, improving the survival of rat renal allografts [116].

Others
It has been shown that drinking hydrogen water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/GNL knockout mice [117], that H 2 in drinking water attenuates noiseinduced hearing loss in guinea pigs [118], that drinking hydrogen water ameliorated cognitive impairment in senescence-accelerated mice [119], and that H 2 exhibited potential cardioprotective effects in irradiated mice [120].

METHODS OF INGEST HYDROGEN III: INJECTION OF HYDROGEN SALINE 8.1. Advantage of injection
Even though oral administration is safe and convenient, H 2 in water tends to escape over time and some H 2 is lost in the stomach or intestine, making it difficult to control the concentration of H 2 administered. Administration of H 2 via an injectable hydrogen saline (H 2 -dissolved saline) vehicle may allow the delivery of more accurate concentrations of H 2 [121].

Effects of Hydrogen Saline on Various Disease Models
Sun's group administered H 2 -saturated saline by peritoneal injection to various model animals with great success. Thus, hydrogen saline has potential in actual clinical treatment. For example, injection of hydrogen saline showed neuroprotective effects in a neonatal hypoxia-ischemia rat model [121]. Moreover, H 2 saline was applied to an Alzheimer's disease model mouse, which was generated by intracerebroventricular injection of the A 1-42 peptide. H 2 treatment decreased the level of oxidative stress and inflammation markers and prevented memory dysfunction and motor dysfunction, respectively [122].

METHODS OF INGEST HYDROGEN IV: DIRECT AB-SORPTION OF HYDROGEN 9.1. Improvement of Glaucoma Model
Alternatively, H 2 -loaded eye drops were prepared by dissolving H 2 in saline and directly administering to the ocular surface [131,132].
In acute glaucoma of the eyes, transient elevation of intraocular pressure causes significant reductions in the thickness of the retina by ischemia-reperfusion injury mediated through the generation of reactive oxygen species [133]. The direct application of eye drops containing H 2 ameliorated ischemia-reperfusion injury of the retina in a rat model. When H 2 eye drops were continuously administered, the H 2 concentration increased in the vitreous body and the •OH level decreased during retinal ischemia-reperfusion. H 2 eye drops reduced the number of apoptotic and oxidative stress markerpositive cells 1 day after ischemia-reperfusion injury, and reduced retinal thinning with accompanying activation of Müller glia, astrocytes and microglia at 7 days after ischemia-reperfusion injury, improving the recovery of inner retinal layer thickness to >70%.
Moreover, we devised eye drops with dissolved H 2 to directly administer H 2 to the retina, and monitored the time course of changes in H 2 levels using a needle-shaped hydrogen sensor electrode inserted through the sclera to the vitreous body in rats. H 2 was able to reach the vitreous body by administering H 2 saturated in normal saline. When H 2 eye drops were administered continuously, approximately 70% H 2 was detected on the ocular surface. Two minutes after the start of administration, H 2 concentration in the vitreous body started to increase and reached a maximum level after 15 min. At that time, H 2 concentration was approximately 20% of H 2 in the eye-drops. The maximum concentration of H 2 in the vitreous body reached approximately one third of the value observed on the ocular surface [131].

Hydrogen Bath
H 2 easily penetrates the skin and distributes throughout the body via blood flow. Thus, taking a warm water bath with dissolved H 2 is a method of incorporating H 2 into the body in daily life, especially in Japan. It takes only 10 minutes to distribute throughout the whole body, as judged by measuring hydrogen gas in expiration (unpublished results).

METHODS OF INGEST HYDROGEN V: INCREASE IN INTESTINAL HYDROGEN 10.1. Production of Hydrogen in Intestinal Bacteria
Other medical gasses, CO, NO and H 2 S, are generated by endogenous enzymatic systems. Pharmaceutical development has taken advantage of these systems to design exogenous molecules to simulate those generated endogenously; however, mammals lack their own enzyme to produce H 2 [70].
Instead of endogenous enzymatic systems, the spontaneous production of hydrogen gas in the human body occurs via the fermentation of undigested carbohydrates by resident enterobacterial flora [134]. H 2 is transferred to the portal circulation and excreted through the breath in significant amounts [135]. For this reason, measurement of H 2 levels in expired air is used to detect carbohydrate malabsorption [76]; however, there have been few studies on the physiological function of gastrointestinal tract-derived hydrogen gas as an antioxidant.

Are -glucosidase Inhibitors an Indirect Antioxidant?
-Glucosidase inhibitors are pharmacological agents that specifically reduce postprandial hyperglycemia through retardation of disaccharide digestion, thereby reducing glucose absorption. A large scale epidemiologic trial has demonstrated that the treatment of patients with impaired glucose tolerance with an -glucosidase Fig. (2). Measurement of the accumulation of H2 in rat liver.
The concentration of H2 in the liver was monitored using a needle-type hydrogen sensor inserted into fed-or overnight fasted-rat liver. Rat received hydrogen water (0.8 mM H2 in water) orally by stomach gavage at 15 ml/kg. Arrow indicates the time point when rat was administered hydrogen water.
inhibitor was associated with a 25% reduction in the risk of progression to diabetes, a 34% reduction in the risk of developing de novo hypertension, and a 49% risk reduction of cardiovascular events [136]. Furthermore, meta-analysis of seven long-term studies suggested that acarbose reduced the risk of myocardial infarction for patients with type 2 diabetes [137]. Such risk reduction for coronary heart disease events in patients with type 2 diabetes was not observed by improved glycemic control achieved by intensified treatment with insulin and glibenclamid [138]. Actually, acarbose, which is an -glucosidase inhibitor, markedly increased H 2 production in volunteers. Thus, we propose that H 2 produced by intestinal bacteria acts as a unique antioxidant and prevents cardiovascular events [139].

Anti-inflammation Effects by Intestinal Bacteria via Hydrogen
Escherichia coli can produce a considerable amount of H 2 by catalyzing with hydrogenase. Kawai et al. examined whether H 2 released from intestinally colonized bacteria could affect concanavalin A-induced mouse hepatitis. Reconstitution of intestinal flora with H 2 -producing E. coli, but not hydrogenase-deficient mutant E. coli, down-regulated concanavalin A-induced liver inflammation. These results indicate that H 2 released from intestinal bacteria can suppress inflammation [140]. H 2 also mediates the suppression of colon inflammation induced by dextran sodium sulfate [141].

Others
Dietary turmeric induced H 2 production from the intestinal bacteria [142], and lactulose was shown to be an indirect antioxidant ameliorating inflammatory bowel disease [143].

CLINICAL TESTS
Several groups have started clinical examinations. Clinical tests have revealed that drinking hydrogen water reduced oxidative stress markers in patients with type 2 diabetes [144] or subjects with potential metabolic syndrome [145] and influenced glucose [144] and cholesterol metabolism [145].
Hemodialysis using dialysis solution with H 2 significantly decreased the levels of plasma monocyte chemoattractant protein 1 and myeloperoxidase [146].

REGULATION OF GENE EXPRESSIONS AND PRO-TEIN PHOSPHORYLATIONS
It has been reported that H 2 acts as an anti-inflammatory and anti-allergic regulator by inducing inflammatory cytokines and inhibiting phosphorylating signal factors, respectively; however, the transcriptional factors and kinases involved in the effects afforded by H c have not been identified.
H 2 is involved in the regulation of various factors; up-regulation of PCNA, bFGF, HGF, IFN-, and down-regulation of i-NOS [87] and VEGF [54].
Heme oxygenase-1 (HO-1), a microsomal enzyme degrading heme to carbon monoxide, free iron, and biliverdin, participates in the cell defense against oxidative stress and has been speculated to be a new therapeutic target [150]. Notably, H 2 modulates HO-1 expression, which is commonly up-regulated by these medical gasses [48,151]. Additionally, H 2 up-reguated the expression of FGF21, which is a regulator of energy metabolism [112].

CLOSING REMARKS: ISSUES TO BE DISSOLVED IN THE FUTURE
In our first report published in 2007, we indicated that H 2 reacted with strong reactive oxygen/nitrogen species, including •OH and ONOOin cell-free reactions. Cells cultured in H 2 -rich medium were protected against oxidative stress by the •OH-scavenging activity of H 2 , depending upon the decrease of •OH [12]; however, recent evidence shows that the scavenging property is not the only explanation for the potent beneficial effects of H 2 . When model animals and human subjects consumed H 2 by drinking water with dissolved H 2 , even a very small amount of H 2 was extensively effective. It may be difficult to explain that direct reduction of •OH by a very small amount of H 2 reveals all the functions of H 2 , because the saturated level of H 2 is only 0.8 mM and the dwelling time of •OH is very short in the body.
We have recently shown that H 2 can be accumulated with hepatic glycogen; this finding indicates the possible accumulation of H 2 in a specific region; however, it is unlikely that the amount of H 2 is sufficient to exhibit all of its functions [112]. Additionally, drinking 0.04 or 0.08 mM H 2 was shown to be effective [104,112]. The amount of administered H 2 seems to be, in many cases, independent of the magnitude of effects. Intestinal bacteria produce more than 1 liter of hydrogen gas per day, whereas the amount of H 2 originating from drinking hydrogen water is less than 50 ml. Nevertheless, additional H 2 in drinking hydrogen water is unambiguously effective.
Many additional issues of hydrogen therapy including the molecular mechanisms underlying the marked effects of a very small amount of H 2 remain elusive. The primary molecular target of H 2 remains unknown. Although H 2 regulates various gene expressions and protein-phosphorylations, it remains unclear whether such regulations are the cause or consequence of the effects against oxidative stress. One of the open questions is how H 2 involves the cross-talk among anti-oxidation, anti-inflammation and anti-allergy. Thus, it should not be fair to classify the roles of H 2 by outward effects at this stage.
Finally, the author summarizes the reports showing the effects of H 2 by the classification of target organs ( Table 1) [152].

DISCLOSURE
The author declares no conflicts of interest.