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2010 38: 1893Journal of International Medical ResearchY Hong, S Chen and J-M Zhang

StudiesHydrogen as a Selective Antioxidant: A Review of Clinical and Experimental

  

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The Journal of International Medical Research2010; 38: 1893 – 1903

1893

Hydrogen as a Selective Antioxidant: aReview of Clinical and Experimental

Studies

Y HONG*, S CHEN* AND J-M ZHANG

Department of Neurosurgery, Second Affiliated Hospital, School of Medicine and Institute ofBrain Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China

Oxidative stress is implicated in thepathogenesis of many diseases; however,currently used antioxidants have a hightoxicity that constrains administration toa narrow window of therapeutic dosage.There is a clear need for more effective andsafer antioxidants. Diatomic hydrogen(H2) was proposed as a novel antioxidantthat selectively reduces levels of toxicreactive-oxygen species. Recently, manystudies have reported that H2 (inhaled ororally ingested, typically as approximately0.8 mM H2-saturated water), can exertbeneficial effects in diverse animal modelsof ischaemia–reperfusion injury, and

inflammatory and neurological disease. Inthe clinic, oral administration of H2-saturated water is reported to improvelipid and glucose metabolism in subjectswith diabetes or impaired glucosetolerance; promising results have also beenobtained in reducing inflammation inhaemodialysis patients and treatingmetabolic syndrome. These studies suggestH2 has selective antioxidant properties,and can exert antiapoptotic, anti-inflammatory and antiallergy effects. Thisreview summarizes recent researchfindings and mechanisms concerning thetherapeutic potential of H2.

IntroductionOxidative stress is a feature of manyconditions including ischaemia–reperfusion(I/R) injury, inflammatory disorders, cancer,cardiovascular and neurological diseases,and ageing.1 Oxidative stress is generallyassociated with the production of highlyreactive free radicals including reactiveoxygen species (ROS). The mitochondrion isthe primary source of ROS, and muchresearch has focused on the identification of

naturally occurring low-toxicityantioxidants that can target mitochondrialROS. Although a relatively unexplored fieldof medicine, increasing attention is nowbeing paid to the potential use of medicinalgases as therapeutic agents.2,3 This articlereviews recent work, in pre-clinical modelsand clinical trials, on the use of diatomichydrogen (H2) – a naturally occurringbiomolecule – as a selective antioxidant.

Oxidative stressFree radicals are atoms, molecules or ions

KEY WORDS: OXIDATIVE STRESS; ANTIOXIDANT; HYDROGEN; HYDROGEN-SATURATED WATER

*Y Hong and S Chen contributed equally to this work.

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Y Hong, S Chen, J-M ZhangHydrogen as a selective antioxidant

with unpaired electrons in an open shellconfiguration; these unpaired electronsmake the radical species highly chemicallyreactive. ROS include superoxide anion (O2

–•),hydroxyl radical (•OH), hydrogen peroxide(H2O2) and singlet oxygen (1O2). Thesedifferent species interconvert via cascadereactions.

Although generally regarded as toxic by-products, physiological roles have beenidentified for molecules such as H2O2 and thenitric oxide radical (NO•),4 which have beenshown to play important roles as effectormolecules in immune defence systemsagainst pathogens, and as signallingmolecules.5,6 For this reason, the eliminationof all types of ROS through powerfulantioxidant therapies is likely to disruptimportant cellular functions. Indeed, it hasbeen reported that radical suppression ofoxidative stress can promote tumourprogression.7

Nevertheless, overproduction of severaltypes of ROS has been associated with a rangeof toxic effects. The majority of these effectshave been associated with the hydroxylradical •OH, one of the most reactive of theROS species,8 which can indiscriminatelydamage cellular components including lipid,protein, carbohydrate and nucleic acid,ultimately leading to cellular necrosis andapoptosis. This raises the prospect ofdeveloping selective antioxidants thatpreferentially remove toxic radicals such as•OH, but not others (such as H2O2 or NO•).4

The accumulation of ROS is generallycounterbalanced by a sophisticatedendogenous antioxidant defence system thatcomprises enzymes – such as superoxidedismutase (SOD), catalase and glutathioneperoxidase – and non-enzymes, such asvitamin A, vitamin C, carotene andbilirubin. Although O2

–• and H2O2 can bedetoxified by antioxidant defence enzymes,

•OH cannot be detoxified by this route. Thishas emphasized the importance ofantioxidants targeting •OH.

Approximately 4000 antioxidants havebeen described to date, most of which areelectron donors that react with ROS to formharmless end-products such as water.Although many have given promisingresults in animal models of oxidative stress,in most cases the beneficial effects seen inanimal studies have not been reiterated inclinical trials.9 Barriers to the utilization ofexogenous antioxidants include lowmembrane permeability and high toxicity,which constrain administration to a narrowwindow of therapeutic dosage.10 Theidentification of a novel and more effectiveantioxidant is, therefore, of high priority.

Rationale behind H2: aselective antioxidantIt has long been known that H2 – acolourless, odourless and tasteless gas – hasantioxidant properties, but the potentialexploitation of H2 as a therapeutic agent hasonly recently been explored in animalmodels and in the clinic. The first study, byDole et al.11 reported that there wassignificant cancer regression in patients withsquamous cell carcinoma exposed tohyperbaric H2 for 2 weeks. In 2001,hyperbaric H2 was reported to be beneficialin the treatment of schistosomiasis-associated chronic liver inflammation, andits therapeutic properties were ascribed toscavenging of •OH.12 These studies were not,however, extended by other researchers,perhaps in view of the explosion hazardsassociated with hydrogen. Nevertheless, it isimportant to note that such risks areeliminated when used in H2/air mixtures of < 4.6% (v/v).12 Ohsawa et al.13 studied theantioxidant properties of molecular H2 andreported that it selectively reduces •OH and

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Y Hong, S Chen, J-M ZhangHydrogen as a selective antioxidant

ONOO– but does not affect physiologicalROS. Subsequent studies14 – 20 confirmed thatthe beneficial effects of H2 are principallymediated by •OH scavenging (Fig. 1).

H2 as a therapeutic agent:review of animal modelsand clinical trialsThe discovery by Ohsawa et al.13 thatinhalation of H2 gas can protect the brainagainst oxidative stress associated with I/Rprompted a series of studies in diverse models,exploring the potential of H2 inhalation (or

oral ingestion of H2-saturated water) to reduceoxidative damage. These studies, summarizedin Table 1,14 – 43 have targeted a diverse rangeof disorders and organ systems including thenervous, digestive, cardiovascular andrespiratory systems. In the following sections,reports on H2 therapy in different diseaseclasses are reviewed.

ISCHAEMIA–REPERFUSION INJURYOxidative stress is thought to play a majorrole in cell damage following I/R injury andseveral studies have addressed whether H2

administration can reduce I/R-induced cell

FIGURE 1: The reactive oxygen species (ROS) production pathway and the selectivereduction of •OH and ONOO– by hydrogen, showing that ROS are produced invarious ways; one type of ROS can be converted into another type by electrontransmission. •OH and ONOO– are much more toxic than other ROS. Hydrogenselectively reduces •OH (b) and ONOO– (a), but does not influence physiologicalROS, including O2

–• and H2O2 (COX, cyclo-oxygenase; LOX, 5-lipo-oxygenase; NO•,nitic oxide radical; NAPDH, reduced nicotinamide adenine dinucleotide phosphate;SOD, superoxide dismutase; GPX, glutathione peroxidase 1; MPO, myeloperoxidase;HOCI, hypochlorous acid; O2

–• superoxide anion; •OH, hydroxyl radical; H2O2,hydrogen peroxide; ONOO–, peroxynitrite)

Xanthine

1. Lipid peroxidation

a b

2. DNA mutation3. Protein oxidation4. Mitochondrial depolarization

Apoptosis and necrosis

O2

O2

–•

NO•

SOD MPO

Cat

alas

e

GPX

+

ONOO–

e–

e–

O2

Arachidonic acid

Mitochondrial respiratory chainXanthine oxidase

CO

X LO

X

Selectivereduction

Selectivereduction

NADPH– oxidase

•OH

H2O2 HOCI

H2O

H2

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TABLE 1: Evidence for therapeutic use of diatomic hydrogen (H2) in various diseases and its effectson various markers

Marker Models of disease H2 effect References

Antioxidase SOD Radiation complication, metabolic Qian et al.,14 Nakao et sepsis, obstructive jaundice al.,15 Xie et al.,16 Liu

et al.17

CAT Sepsis, obstructive jaundice Xie et al.,16 Liu et al.17

GSH Radiation complication Qian et al.14

Oxidative MDA I/R (hepatic, intestinal, neonatal Fukuda et al.,18 Zheng etstress hypoxia, cardiac, renal), Alzheimer’s al.,19 Mao et al.,20 Cai et

disease, cognitive disorders, al.,21 Chen et al.,22 Sun pancreatitis, atherosclerosis, et al.,23 Nakao et al.,24

obstructive jaundice, radiation and Cardinal et al.,25 Li et al.,26

chemotherapy complication, spinal Nagata et al.,27 Chen et cord injury al.,28 Ohsawa et al.,29 Liu

et al.,17 Qian et al.,14

Nakashima-Kamimura etal.,30 Chen et al.31

4HNE Atherosclerosis, Parkinson’s disease, Ohsawa et al.,29 Fu et Alzheimer’s disease, cognitive al.,32 Fujita et al.,33 Li et disorders, I/R (renal and retinal), al.,26 Nagata et al.,27

haemorrhagic Cardinal et al.,25

Oharazawa et al.,34 Chenet al.35

8-OHdG I/R (cerebral, myocardium, retinal, Ohsawa et al.,13 Hayashidarenal), Parkinson’s disease, et al.,36 Sun et al.,23

haemorrhagic, metabolic syndrome, Oharazawa et al.,34

radiation complication Shingu et al.,37 Fu et al.,32

Chen et al.,35 Nakao etal.,15 Qian et al.14

MPO Intestinal I/R injury, sepsis, Zheng et al.,19 Chen et pancreatitis, haemodialysis, al.,22 Chen et al.,28 Xie et obstructive jaundice, spinal cord al.,16 Nakayama et al.,38

injury Liu et al.,17 Chen et al.31

8-oxoG Parkinson’s disease Fujita et al.33

8-isoPGF2α Sepsis, metabolic syndrome Xie et al.,16 Nakao et al.15

DAO Intestinal I/R injury Zheng et al.19

TBARs Metabolic syndrome Nakao et al.15

NADPH Allergic Itoh et al.39

Inflammation IL-1β Intestinal I/R, colon inflammation, Buchholz et al.,40 Zheng etfactor obstructive jaundice al.,19 Mao et al.,20 Kajiya

et al.,41 Liu et al.17

IL-6 Intestinal I/R, Alzheimer’s disease, Buchholz et al.,40 Zheng et renal transplantation, obstructive al.,19 Li et al.,26 Cardinal et jaundice al.,25 Liu et al.17

TNF-α Intestinal I/R, Alzheimer’s disease, Zheng et al.,19 Mao et colon inflammation, renal al.,20 Li et al.,26 Kajiya et transplantation, obstructive al.,41 Cardinal et al.,25 Liu jaundice, hepatitis et al.,17 Kajiya et al.42

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Y Hong, S Chen, J-M ZhangHydrogen as a selective antioxidant

loss in animal models. H2 was reported toprotect against I/R injury in models ofcerebral,13,21,43 myocardial,24,36 hepatic,18

intestinal,22,40 retinal34 and renal37 I/R. H2

was also reported to reduce infarct size in ratmodels of focal cerebral and myocardialischaemia,13,36 maintain the integrity of theblood–brain barrier and reducehaemorrhagic transformation in a rat model

of focal ischaemia.35

The beneficial effects of H2 treatment arelikely to be due to direct scavenging of •OH,although the kinetic favourability of thisdirect reaction has not been established. Itwas observed, however, that in biochemicalstudies of I/R injury, H2 treatment was able toreduce the levels of multiple markers ofoxidative stress. Notably, H2 decreased the

TABLE 1 (continued): Evidence for therapeutic use of diatomic hydrogen (H2) in various diseases and its effectson various markers

Marker Models of disease H2 effect References

HMGB1 Sepsis, myocardium I/R, obstructive Xie et al.,16 Nakao et al.,24

jaundice Liu et al.17

PCNA Pancreatitis, intestinal I/R Chen et al.,28 Chen et al.22

IFN-γ Hepatitis, renal transplantation Kajiya et al.,42 Cardinal etal.25

CCL2 Intestinal I/R injury Buchholz et al.40

ICAM-1 Renal transplantation Cardinal et al.25

iNOS Myocardium I/R injury Nakao et al.24

IL-12 Colon inflammation Kajiya et al.41

Iba1 Neonatal hypoxia–ischaemia Cai et al.21

Inflammation MAPK Renal transplantation, obstructive Cardinal et al.,25 Liu et signals jaundice al.17

MEK-1 Renal transplantation Cardinal et al.25

Lyn-P Allergic Itoh et al.39

NF-κβ Pancreatitis Chen et al.28

Apoptosis TUNEL I/R (myocardium, intestinal, Hayashida et al.,36 Chen etneonatal hypoxia), chemotherapy al.,22 Cai et al.,21 Cai et complication, spinal cord injury, al.,43 Nakashima-pancreatitis Kamimura et al.,30 Chen et

al.,31 Chen et al.28

Annexin V Radiation complication Qian et al.14

Caspase-3 Neonatal hypoxia, myocardium Cai et al.,43 Cai et al.,21

I/R injury, spinal cord injury Sun et al.,23 Nakao et al.,24

Chen et al.31

Caspase-12 Neonatal hypoxia, spinal cord injury Cai et al.,43 Chen et al.31

, increase of the marker; , decrease of the marker; SOD, superoxide dismutase; CAT, catalase; GSH,glutathione; MDA, malondialdehyde; I/R, ischaemia–reperfusion; 4HNE, 4-hydroxynonenal; 8-OHdG, 8-hydroxydeoxyguanosine; MPO, myeloperoxidase; 8-oxoG, 8-oxoguanine; 8-isoPGF2α, 8-isoprostane; DAO,diamine oxidase; TBARs, thiobarbituric acid reactive substances; NADPH, reduced nicotinamide adeninedinucleotide phosphate; IL, interleukin; TNF-α, tumour necrosis factor α; HMGB1, high-mobility group box 1;PCNA, proliferating cell nuclear antigen; IFN-γ, interferon-γ; CCL2, chemokine (C–C motif) ligand 2; ICAM-1,intercellular adhesion molecule-1; iNOS, inducible nitric oxide synthase; Iba1, ionized calcium bindingadaptor molecule 1; Lyn-P, phosphorylated Lyn tyrosine kinase; MAPK, mitogen-activated protein kinase;MEK-1, MAP-extracellular signal regulated protein kinase-1; NF-κβ, nuclear factor-κβ; TUNEL, terminaldeoxynucleotidyl transferase deoxyuridine triphosphate nick-end labelling.

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Y Hong, S Chen, J-M ZhangHydrogen as a selective antioxidant

levels of: malondialdehyde (MDA), anindicator of ROS-mediated lipidperoxidation;18 – 20,23 4-hydroxynonenal, aproduct of lipid peroxidation;18,34

myeloperoxidase, a marker of oxidativestress;20 and 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA oxidation.23,34,36

These results argue that H2 has significantantioxidant effects in I/R injury. Furthermore,I/R injury is commonly associated with celldeath in the ischaemic region as a result ofapoptosis and/or necrosis. Importantly, it hasbeen reported that H2 treatment cansignificantly reduce the numbers of terminaldeoxynucleotidyl transferase deoxyuridinetriphosphate nick-end labelling (TUNEL)-positive cells in parallel with reductions inthe levels of activated caspases 3 and 12,23,34

arguing that the protective effects of H2 aremediated in part through inhibition ofpathways of programmed cell death.

The above studies, which were all double-blind and placebo-controlled, suggest thatthe use of H2 for I/R is effective. Clinicalapplication of H2 during I/R is slight, so wespeculated whether clinical studies wouldalso show encouraging results. In ouropinion, the therapeutic window for I/Rdisease is usually very short. Whentranslated into a clinical application, H2

must be most frequently applied as early aspossible in the treatment of patients withacute infarction. It is particularly importantto make sure that H2 can rapidly reach ‘at-risk’ ischaemic areas before blood flow in theoccluded infarct-related artery is re-established. Consequently, further research iswarranted to enhance the application of H2

for curing I/R injury.

INFLAMMATORY DISEASEInflammatory processes are known to beclosely linked with oxidative stress, andseveral studies have addressed the potential

of H2 as an anti-inflammatory therapeutic.In inflammatory conditions, H2 treatment

was found significantly to reduce levels ofinterleukin (IL)-6 and tumour necrosisfactor-α (TNF-α), as well as levels of otherinflammation-associated molecules includingintercellular adhesion molecule-1, IL-12,high-mobility group box 1 and interferon-γ16,23,25,28,40 – 42 in rodent models. Importantly,H2 partly inhibited mitogen-activatedprotein kinase signalling pathways,including c-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated proteinkinase (ERK1/2), as well as upstream kinasecascades.25

In animal models of inflammatorydisorders, it has been reported that H2 canattenuate inflammation in hepatitis,42

colitis,41 pancreatitis,28 obstructive jaundice17

and sepsis.16 H2 treatment was alsoassociated with the downregulation of theexpression of proinflammatory cytokinesand suppression of inflammatory cellinfiltration (summarized in Table 1).

NEUROLOGICAL DISEASEIn a rat model of Parkinson’s disease, 6-hydroxydopamine (6-OHDA) inducedoxidative stress in dopaminergic neurons,leading to nigrostriatal degeneration.32 Fu etal.32 first examined the effects of oraladministration of 50% H2-saturated waterbefore intrastriatal injection of 6-OHDA, andfound that H2 significantly reduced theextent of degeneration. More recently, alower concentration of orally administeredH2 (5% saturated H2 in drinking water) wasfound to prevent DNA damage and lipidperoxidation, and to reduce dopaminergicneuron loss in a MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxinmodel of Parkinson’s disease.33

In Alzheimer’s disease it is thought thatamyloid β (Aβ), the major component of

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Y Hong, S Chen, J-M ZhangHydrogen as a selective antioxidant

senile plaques, plays a causal role in diseasedevelopment, and exerts its toxic effects inpart via the induction of oxidative stress.26

Oral administration of H2-saturated waterhas been reported to attenuate Aβ-induceddeficits, in a rat model.26 It was also reportedthat H2 could improve spatial recognitionand memory, and that this wasaccompanied by reduced levels ofproinflammatory cytokines, lipidperoxidation products and glial fibrillaryacidic protein immunoreactivity.26

Furthermore, in a different animal model(restraint-induced stress) H2 administrationwas found to alleviate stress-induced deficitsin learning and memory.27

Oxidative stress, inflammation andapoptosis are also central features of cell lossfollowing acute spinal cord contusion. Usingan animal model, H2 treatment led toimproved rates of functional locomotorrecovery after spinal cord injury; thisrecovery was associated with reducedoxidative stress and elevated brain-derivedneurotrophic factor levels.31 Gu et al.44

reported that daily consumption of H2-saturated water was effective in preventingage-related learning and memoryimpairments; this effect was attributed toremoval of ROS and increased SOD activity.

METABOLIC DISORDERSMetabolic syndrome refers to a commondisorder characterized by a combination ofobesity, dyslipidaemia, hypertension andinsulin resistance. These metabolic disordersare suitable for clinical research with H2

because of their chronic characteristics.Consequently, clinical studies of H2 are, atpresent, mostly limited to metabolic diseases.Nakao et al.,15 in an open-label pilot studyinvolving 20 subjects with potentialmetabolic syndrome, demonstrated thatconsumption of 1.5 – 2.0 l/day of H2-

saturated water for 8 weeks reduced thelevels of oxidative stress indicators.Moreover, the treated subjects showedsignificant improvements in liver and kidneyfunction. In addition to direct antioxidanteffects, it was reported that H2 enhancedSOD levels, thereby increasing endogenousantioxidant defence against O2

–•. Thesample in this study was, however, so smallthat the finding should be accepted withcaution.15 In a randomized, double-blind,placebo-controlled, crossover study,Kajiyama et al.45 reported thatsupplementation with 900 ml/day of H2-saturated water for 8 weeks reduced thelevels of several biomarkers of oxidativestress, including plasma oxidized low-densitylipoprotein cholesterol and urinary 8-isoprostanes, and also improved glucosemetabolism in patients with either type 2diabetes or impaired glucose tolerance;however, their breath hydrogen levels werenot reported in detail after consumption.Supplementation with H2-saturated waternormalized the oral glucose tolerance test infour out of six patients with impaired glucosetolerance.45 Furthermore, consumption of H2

ad libitum was reported to prevent thedevelopment of atherosclerosis inapolipoprotein E knockout mice, partlythrough its ability to limit oxidative stress inblood vessels.29 The efficacy of H2-saturatedwater in these studies appeared to besignificantly greater than other testedantioxidants including folic acid, vitamin E,iron and α-lipoic acid.

CHEMOTHERAPY, HAEMODIALYSISAND RADIOTHERAPYCOMPLICATIONSReagents such as cisplatin, which targetrapidly proliferating cells, are widely used totreat aggressive neoplastic conditions.Nevertheless, cisplatin use is restricted

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Y Hong, S Chen, J-M ZhangHydrogen as a selective antioxidant

because of its major side-effects duringtherapy, some of which have been ascribedto oxidative stress and/or inflammation.Using an animal model, Nakashima-Kamimura et al.30 reported that both oralconsumption of H2-saturated water, as wellas H2 administration by inhalation, couldalleviate cisplatin-induced nephrotoxicitywithout compromising antitumour activity.

In a non-randomized study with aconcurrent control, Nakayama et al.38 foundthat adding H2 to haemodialysis solutionsafter a 1-month run-in period amelioratedinflammatory reactions, decreased plasmaoxidative markers and improved bloodpressure control during a 6-month trial. Thestudy period was, however, short and theinflammatory conditions of the subjects werenot very serious. Thus, long-term studies onpatients with more severe inflammation maybe optimal for clarifying the impact of H2.

Excess exposure to ionizing radiationcauses a spectrum of tissue damage knownas acute radiation syndrome. Importantly, ithas been estimated that 60% – 70% ofionizing radiation-induced cellular damageis caused by •OH.46 However, levels ofapoptotic cells, plasma MDA and intestinal8-OHdG were significantly decreased whencells or mice were treated with H2 afterirradiation.14 This suggests that H2

administration could also be of benefit incancer radiotherapy.

In summary, H2 can counter side-effectscommonly seen during chemo therapy,radiotherapy and haemodialysis, therebyimproving the patient’s quality of life.

ALLERGIC DISEASESImmediate-type allergic reactions includepollinosis, bronchial asthma and urticaria,which are conditions that have not beencausally associated with oxidative stress.Nevertheless, it is possible that H2 exerts

therapeutic effects that are independent ofthe removal of •OH. Itoh et al.39 reported thatH2 attenuated degranulation by suppressingFcεRI-mediated signal transduction in amast-cell culture model. Administration of H2

down-regulated antigen-induced phos -phoryla tion of FcεRI-associated Lyn – a keyregulatory step in the pathway – andsuppressed activation of downstream signaltargets including Syk, phospholipase C (PLC)-γ1, PLCγ2, Akt, ERK1/2, JNK, p38 andcytosolic phospholipase A2, without affectingthe levels of the immunoglobulin E receptorFcεRIβ. It is, therefore, possible that H2 canintervene in specific signal transductionpathways.

H2: advantages overpharmaceutical drugsDiatomic hydrogen has several potentialadvantages, compared with pharmaceuticaldrugs (Table 2). The administration of H2,either as a low percentage H2/air mixture or asa solution in water or saline, has severaladvantages over conventional antioxidants.First, H2 has not been reported to be toxic ateffective dosages, and overdosing is unlikelybecause excess H2 is expired via the lungs.44

This contrasts with antioxidants, such asvitamins C and E, where the effective dosagein humans is higher than the upper limit oftolerated intake. Secondly, H2 selectivelyscavenges the most aggressive ROS, •OH, butis far less effective against O2

–• and H2O2,which play physiological roles. Moreover,there is no evidence that H2, a mildantioxidant, is able to disturb metabolic redoxreactions or to disrupt cell signalling mediatedby less potent ROS. Indeed, H2 does notinfluence physiological parameters such astemperature, blood pressure, pH or pO2.Thirdly, because of its low molecular weight,H2 can diffuse extremely rapidly into tissueand is likely to reach important target

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Y Hong, S Chen, J-M ZhangHydrogen as a selective antioxidant

subcellular compartments, includingmitochondria and the nucleus. This isparticularly important because mitochondriaare the primary sites of the generation of ROSafter I/R and are notoriously difficult to target.Nuclear targeting could also be important inprotecting DNA from oxidative damage.Finally, it seems likely that H2 offers significantadvantages in terms of cost compared withconventional pharmaceuticals.

Future prospects: manyunsolved problemsAlthough multiple studies havedemonstrated that H2 administration can beeffective against a range of disorders, both inanimal models and in the clinic, manyquestions remain. At the molecular level, it isunclear how H2 achieves chemical selectivityin view of the enormous diversity of potentialmolecular targets in vivo. Moreover, thepublished rate constant for the reaction of•OH with H2 to form H2O and H• is markedlyslower than for most radical–radicalreactions. Intriguingly, there are someindications that H2 might not act exclusivelyas an antioxidant and could also interactdirectly with specific signalling pathways,39

as previously discovered for other simplegases including NO, CO and H2S. Furtherwork will be required to determine the in vivo

molecular targets for H2. The protective effects of H2 have been

established in both animal models andclinical trials; however, none of thepublished studies has examined theconcentration of H2 in the active site. Furthermonitoring of H2 concentrations in injurywould be helpful to understand thepharmacokinetics in more detail. In Japan,H2 therapy has been used clinically indiabetes, the metabolic syndrome andhaemodialysis, but further appropriatelydesigned, large-scale, placebo-controlledtrials using different dosages and routes ofadministration will be required to evaluatethe therapeutic efficacy of H2 treatmentrigorously in these and other conditions.

Delivery of high concentrations of H2 gasby inhalation has major drawbacks in view ofthe risks associated with its flammability andthe complex apparatus required for safedelivery. Nagata et al.27 first reported thatconsumption of H2-saturated water couldafford a safe and effective alternative toinhalation. Nevertheless, the pharmaco -kinetics of different routes of administrationhave not been studied in depth, although Fuet al.32 reported that arterial H2 concentrationsfollowing consumption of 0.4 mM hydrogenwater were similar to those achieved oninhalation of 2% (v/v) hydrogen gas. It seems

TABLE 2: Summary of the therapeutic advantages of the antioxidant, diatomic hydrogen

1 Easily diffuses across the cellular membrane to reach sub-cellular compartments2 Does not influence physiological parameters in the blood (temperature, blood pressure, pH, pO2)3 Does not disturb metabolic oxidation–reduction reactions and cell signalling4 High concentrations of hydrogen are well tolerated; fewer systemic side-effects5 Low cost6 Multiple mechanisms of action:

(i) antioxidant effect (ii) anti-inflammatory effect (iii) inhibition of apoptosis (iv) antiallergic effect

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• Received for publication 11 June 2010 • Accepted subject to revision 2 July 2010 • Revised accepted 11 October 2010

Copyright © 2010 Field House Publishing LLP

likely that oral administration of H2 solutionwill be the route of choice in further studies onthe antioxidant properties of H2.

We have reviewed a large number ofstudies, principally in animal models, inwhich H2 administration reduced tissue injuryassociated with oxidative stress andinflammation. Despite important differencesbetween animal models and human disease,we argue that the therapeutic potential of H2

– a non-toxic, convenient, safe and effective

antioxidant – warrants further clinicalevaluation in oxidative stress-related diseases.

AcknowledgementWe acknowledge the support of the NaturalScience Foundation of Zhejiang (No.Z2090200).

Conflicts of interestThe authors had no conflicts of interest todeclare in relation to this article.

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Author’s address for correspondenceProfessor Jian-Min Zhang

Department of Neurosurgery, Second Affiliated Hospital, School of Medicine and Institute ofBrain Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, Zhejiang

Province, China.E-mail: zjm135vip@sina.com

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