Molecular Hydrogen as an Emerging Therapeutic Medical Gas for Neurodegenerative and Other Diseases

Abstract

Effects of molecular hydrogen on various diseases have been documented for 63 disease models and human diseases in the past four and a half years. Most studies have been performed on rodents including two models of Parkinson's disease and three models of Alzheimer's disease. Prominent effects are observed especially in oxidative stress-mediated diseases including neonatal cerebral hypoxia; Parkinson's disease; ischemia/reperfusion of spinal cord, heart, lung, liver, kidney, and intestine; transplantation of lung, heart, kidney, and intestine. Six human diseases have been studied to date: diabetes mellitus type 2, metabolic syndrome, hemodialysis, inflammatory and mitochondrial myopathies, brain stem infarction, and radiation-induced adverse effects. Two enigmas, however, remain to be solved. First, no dose-response effect is observed. Rodents and humans are able to take a small amount of hydrogen by drinking hydrogen-rich water, but marked effects are observed. Second, intestinal bacteria in humans and rodents produce a large amount of hydrogen, but an addition of a small amount of hydrogen exhibits marked effects. Further studies are required to elucidate molecular bases of prominent hydrogen effects and to determine the optimal frequency, amount, and method of hydrogen administration for each human disease.

Full text

Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longe vity
Volume 2012, Article ID 353152, 11 pages
doi:10.1155/2012/353152
Review A rticle
Molecular Hydrogen as an Emerging Therapeutic Medical Gas for
Neurodegenerative and Other Diseases
Kinji Ohno,
1
Mikako Ito,
1
Masatoshi Ichihara,
2
and Masafumi Ito
3
1
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine,
65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan
2
Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Aichi 487-8501, Japan
3
Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
Correspondence should be addressed to Kinji Ohno, ohnok@med.nagoya-u.ac.jp
Received 11 January 2012; Revised 24 March 2012; Accepted 13 April 2012
Academic Editor: Marcos Dias Pereira
Copyright © 2012 Kinji Ohno et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Effects of molecular hydrogen on various diseases have been documented for 63 disease models and human diseases in the past four
and a half years. Most studies have been performed on rodents including two models of Parkinson’s disease and three models of
Alzheimer’s disease. Prominent effects are observed especially in oxidative stress-mediated diseases including neonatal cerebral
hypoxia; Parkinson’s disease; ischemia/reperfusion of spinal cord, heart, lung, liver, kidney, and intestine; transplantation of
lung, heart, kidney, and intestine. Six human diseases have been studied to date: diabetes mellitus type 2, metabolic syndrome,
hemodialysis, inflammatory and mitochondrial myopathies, brain stem infarction, and radiation-induced adverse effects. Two
enigmas, however, remain to be solved. First, no dose-response effect is observed. Rodents and humans are able to take a small
amount of hydrogen by drinking hydrogen-rich water, but marked effects are observed. Second, intestinal bacteria in humans and
rodents produce a large amount of hydrogen, but an addition of a small amount of hydrogen exhibits marked effects. Further
studies are required to elucidate molecular bases of prominent hydrogen effects and to determine the optimal frequency, amount,
and method of hydrogen administration for each human disease.
1. Introduction
Molecular hydrogen (H
2
) is the smallest gas molecule made
of two protons and two electrons. Hydrogen is combustible
when the concentration is 4–75%. Hydrogen, however, is a
stable gas that can react only with oxide radical ion (
•O
−
)
and hydroxyl radical (
•OH) in water with low reaction rate
constants [1]:
•O
−
+H
2
−→ H• +OH
−
k = 8.0 × 10
7
M
−1
· s
−1
•OH + H
2
−→ H• +H
2
O k = 4.2 × 10
7
M
−1
· s
−1
H• + • OH −→ H
2
O k = 7.0 × 10
9
M
−1
· s
−1
.
(1)
Thereactionrateconstantsof
•O
−
and •OH with other
molecules are mostly in the orders of 10
9
to 10
10
M
−1
·s
−1
,
whereas those with H
2
are in the order of 10
7
M
−1
·s
−1
.
Hydrogen, however, is a small molecule that can easily
dissipate throughout the body and cells, and the collision
ratesofhydrogenwithothermoleculesareexpectedtobe
very high, which is likely to be able to overcome the low
reaction rate constants [2]. Hydrogen is not easily dissolved
in water, and 100%-saturated hydrogen water contains
1.6 ppm or 0.8 mM hydrogen at room temperature.
In 1995, hydrogen was first applied to human to over-
come high-pressure nervous syndrome in deep sea diving
[3]. Hydrogen was used to reduce nitrogen (N
2
) toxicity
and to reduce breathing resistance in the deep sea. In
2001, being prompted by the radical-scavenging activity of
hydrogen, Gharib and colleagues examined an effect of
molecular hydrogen on a mouse model of schistosomiasis-
associated chronic liver inflammation [4]. Mice were placed
in a chamber with 70% hydrogen gas for two weeks. The
mice exhibited decreased fibrosis, improvement of hemo-
dynamics, increased nitric oxide synthase (NOS) II activity,

2 Oxidative Me dicine and Cellular Longevity
40
30
20
10
0
2007 2008 2009 2010 2011
Year
Number of papers
Figure 1: Number of papers that report effects of molecular hydro-
gen since 2007 shown in Tabl e 1.
increased antioxidant enzyme ac tivity, decreased lipid perox-
ide levels, and decreased circulating tumor-necrosis-factor-
(TNF-) α levels. Although helium gas also exerted some
protective effects in their model, the effect of helium gas was
not recapitulated in a mouse model of ischemia/reperfusion
injury of the liver [5].
2. Effects of Hydrogen Have Been Reported in
63 Disease Models and Human Diseases
A major breakthrough in hydrogen research occurred after
Ohsawa and colleagues reported a prominent effect of
molecular hydrogen on a rat model of cerebral infarction
in June 2007 [6]. Rats were subjected to left middle cerebral
artery occlusion. Rats placed in 2–4% hydrogen ga s chamber
showed significantly smal ler infarction volumes compared
to controls. They attributed the hydrogen effect to the
specific scavenging activ ity of hydroxyl radical (
•OH). They
also demonstrated that hydrogen scavenges peroxynitrite
(ONOO
−
) but to a lesser extent.
As have been previously reviewed [7, 8], effects of
molecular hydrogen on various diseases have been reported
since then. The total number of disease models and human
diseases for which molecular hydrogen has been proven to
be effective has reached 63 (Table 1). The number of papers
is increasing each year (Figure 1). Among the 87 papers
cited in Tabl e 1,21papersshowedaneffect with inhalation
of hydrogen gas, 23 with drinking hydrogen-rich water,
27 with intraperitoneal administration or drip infusion of
hydrogen-rich saline, 10 with hydrogen-rich medium for
cell or tissue culture, and 6 with the other administration
methods including instillation and dialysis solution. In
addition, among the 87 papers, 67 papers showed an effect
in rodents, 7 in humans, 1 in rabbits, 1 in pigs, and 11 in
cultured cells or cultured tissues.
Two papers, however, showed that hydrogen was inef-
fective for two disease models (Table 2 ). One such disease
was moderate to severe neonatal brain hypoxia [9], although
marked effects of hydrogen gas [10, 11] and intraperitoneal
administration of hydrogen-rich saline [12] on neonatal
brain hypoxia have been reported in rats [10, 12] and pigs
[11]. We frequently observe that therapeutic intervention
that is effective for mild cases has little or no effect on
severe cases, and hydrogen is unlikely to be an exception.
Another disease is muscle disuse atrophy [13]. Although
oxidative stress is involved in the development of muscle
disuse at rophy, oxidative stress may not be a major driving
factor causing atrophy and thus attenuation of oxidative
stress by hydrogen may not be able to exhibit a beneficial
effect.
Effects of molecular hydrogen have been observed essen-
tially in all the tissues and disease states including the brain,
spinal cord, eye, ear, lung, heart, liver, kidney, pancreas, intes-
tine, blood vessel, muscle, cartilage, metabolism, perinatal
disorders, and inflammation/allergy. Among them, marked
effects are observed in ischemia/reperfusion disorders as
well as in inflammatory disorders. It is interesting to note,
however, that only three papers addressed effects on cancers.
First, molecular hydrogen caused growth inhibition of
human tongue carcinoma cells HSC-4 and human fibrosar-
coma cells HT-1080 but did not compromise growth of nor-
mal human tongue epithelial-like cells DOK [14]. Second,
hydrogen suppressed the expression of vascular endothelial
growth factor (VEGF), a key m ediator of tumor angiogen-
esis, in human lung adenocarcinoma cells A549, which was
mediated by downregulation of extracellular signal-regulated
kinase (ERK) [ 15 ]. Third, hydrogen protected BALB/c mice
from developing radiation-induced thymic lymphoma [16].
Elimination of radical oxygen species by hydrogen should
reduce a probability of introducing somatic mutations.
Unlike other disease models, cancer studies were performed
only with cells in two of the three papers. Hydrogen is
likely to have a beneficial effect on cancer de velopment
by suppressing somatic mutations, but an effect on cancer
growth and invasion needs to be analyzed further in detail.
3. Effects of Molecular Hydrogen on Rodent
Models of N eurodegenerative Diseases
Parkinson’s disease is caused by death of dopaminergic neu-
rons at the substantia nigra pars compact of the midbrain
and is the second most common neurodegenerative disease
after Alzheimer’s disease. Parkinson’s disease is caused by
two mechanisms: excessive oxidative stress and abnormal
ubiquitin-proteasome system [17]. The neurotransmitter,
dopamine, is a prooxidant by itself and dopaminergic cells
are destined to be exposed to high concentrations of radical
oxygen species. An abnormal ubiquitin-proteasome system
also causes aggregation of insoluble α-synuclein in the
neuronal cell body that leads to neuronal cell death. We made
a rat model of hemi-Parkinson’s disease by stereotactically
injecting catecholaminergic neurotoxin 6-hydroxydopamine
(6-OHDA) in the right striatum [ 18]. Ad libitum administra-
tion of hydrogen-rich water starting one week before surgery
completely abolished the de velopment of hemi-Parkinson’s
symptoms. The number of dopaminergic neurons on the
toxin-injected side was reduced to 40.2% of that on the

Oxidative Medicine and Cellular Longevity 3
Table 1: Sixty-three disease models and human diseases for which beneficial effects of hydrogen have been documented.
Diseases Species Administration
Brain
Cerebral infarction [6, 30, 55, 56] Rodent, human Gas, saline
Cerebral superoxide production [75] Rodent Water
Restraint-induced dementia [22] Rodent Water
Alzheimer’s disease [23, 24] Rodent Saline
Senile dementia in senescence-accelerated mice [25] Rodent Water
Parkinson’s disease [18, 19] Rodent Water
Hemorrhagic infarction [34] Rodent Gas
Brain trauma [76] Rodent Gas
Carbon monoxide intoxication [52] Rodent Saline
Transient global cerebral ischemia [66] Rodent Gas
Deep hypothermic circulatory arrest-induced brain damage [57] Rodent Saline
Surgically induced brain injury [77] Rodent Gas
Spinal Cord
Spinal cord injury [78] Rodent Saline
Spinal cord ischemia/reperfusion [51] Rabbit Gas
Eye
Glaucoma [79] Rodent Instillation
Corneal alkali-burn [61] Rodent Instillation
Ear
Hearing loss [80–82] Tissue, rodent Medium, water
Lung
Oxygen-induced lung injury [53, 60, 83, 84] Rodent Saline
Lung transplantation [85] Rodent Gas
Paraquat-induced lung injury [86] Rodent Saline
Radiation-induced lung injury [87–89] Rodent Water
Burn-induced lung injury [90] Rodent Saline
Intestinal ischemia/reperfusion-induced lung injury [44] Rodent Saline
Heart
Acute myocardial infarction [36, 65, 91
] Rodent Gas, saline
Cardiac transplantation [46] Rodent Gas
Sleep apnea-induced cardiac hypoxia [48] Rodent Gas
Liver
Schistosomiasis-associated chronic liver inflammation [ 4] Rodent Gas
Liver ischemia/reperfusion [5] Rodent Gas
Hepatitis [43] Rodent Intestinal gas
Obstructive jaundice [47] Rodent Saline
Carbon tetrachloride-induced hepatopathy [62] Rodent Saline
Radiation-induced adverse effects for liver tumors [31] Human Water
Kidney
Cisplatin-induced nephropathy [92–94] Rodent Gas, water
Hemodialysis [20, 28] Human Dialysis solution
Kidney transplantation [95] Rodent Water
Renal ischemia/reperfusion [54] Rodent Saline
Melamine-induced urinary stone [96] Rodent Water
Chronic kidney disease [37] Rodent Water

4 Oxidative Me dicine and Cellular Longevity
Table 1: Continued.
Diseases Species Administration
Pancreas
Acute pancreatitis [97] Rodent Saline
Intestine
Intestinal transplantation [41, 45, 59] Rodent Gas, medium, saline
Ulcerative colitis [42] Rodent Gas
Intestinal ischemia/reperfusion [63] Rodent Saline
Blood vessel
Atherosclerosis [98] Rodent Water
Muscle
Inflammatory and mitochondrial myopathies [29] Human Water
Cartilage
NO-induced cartilage toxicity [38] Cells Medium
Metabolism
Diabetes mellitus type I [32] Rodent Water
Diabetes mellitus type II [26] Human Water
Metabolic syndrome [27, 99] Human, rodent Water
Diabetes/obesity [33] Rodent Water
Perinatal disorders
Neonatal cerebral hypoxia [10–12] Rodent, pig Gas, saline
Preeclampsia [58] Rodent Saline
Inflammation/allergy
Type I allergy [64] Rodent Water
Sepsis [100] Rodent Gas
Zymosan-induced inflammation [101] Rodent Gas
LPS/IFNγ-induced NO production [67] Cells Gas
Cancer
Growth of tongue carcinoma cells [14] Cells Medium
Lung cancer cells [15] Cells Medium
Radiation-induced thymic lymphoma [16] Rodent Saline
Others
UVB-induced skin injury [49] Rodent Bathing
Decompression sickness [102] Rodent Saline
Viability of pluripotent stromal cells [103] Cells Gas
Radiation-induced cell damage [104, 105] Cells Medium
Oxidized low density lipoprotein-induced cell toxicity [50] Cells Medium
High glucose-induced oxidative stress [35] Cells Medium
control side, whereas hydrogen treatment improved the
reduction to 83.0%. We also started giving hydrogen-rich
water three days after surgery, and hemi-Parkinson’s sym-
ptoms were again suppressed, but not as much as those
observed in pretreated rats. The number of dopaminergic
neurons on the toxin-injected side was 76.3% of that on the
control side. Pretreated rats were also sacrificed 48 hrs
after toxin injection, and the tyrosine hydroxylase activity
at the striatum, where dopaminergic neurons terminate,
was decreased in both hydrogen and control groups. This
indicated that hydrogen did not directly detoxicate 6-OHDA
but exerted a delayed protective effect for dopaminergic cells.
Fujita and colleagues also demonstrated a similar prominent
effect of hydrogen-rich water on an MPTP-(1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine-) induced mouse model
of Parkinson’s disease [19]. MPTP is a neurotoxin that
blocks complex I of the mitochondrial electron transport
system and causes Parkinson’s disease in mice and humans.

Oxidative Medicine and Cellular Longevity 5
Table 2: Two disease models for which hydrogen has no effect.
Diseases Species Administration
Brain
Moderate to severe
neonatal brain hypoxia [9]
Rodent Gas
Muscle
Muscle disuse atrophy [13] Rodent Water
It is interesting to note that the concentration of hydrogen
that they used for the MPTP mice was only 0.08 ppm (5%
saturation), which is the second lowest among all the trials
published to date for rodents and humans. The lowest hydro-
gen concentration ever tested is 0.048 ppm in the dialysis
solution for patients receiving hemodialysis [20].
Alzheimer’s disease is the most common neurodegen-
erative disease and is characterized by abnormal aggrega-
tion of β-amyloid (Aβ) and tau, the large agg regates of
which are recognizable as senile plaques and neurofibrillary
tangles, respectively [21]. Effects of molecular hydrogen
on Alzheimer’s disease have been studied in three rodent
models. First, Nagata and colleagues made a mouse model
of dementia by restricting movement of mice for 10 hrs a
day [22]. They analyzed cognitive functions through passive
avoidance learning, object recognition tasks, and the Morris
water maze a nd demonstrated that ad libitum administration
of hydrogen-rich water efficiently ameliorated cognitive
impairment. They also showed that neural proliferation in
the dentate gyrus was restored by hydrogen. Second, Li
and colleagues made a rat model of Alzheimer’s disease
by intracerebroventricular injection of Aβ1-42 [23]. They
analyzed cognitive functions by the Morris water maze open
field tasks, and electrophysiological measurement of long-
term potentiation (LTP) and found that intraperitoneal
injection of hydrogen-rich saline for 14 days efficiently amel-
iorated cognitive decline and preserved LTP. The same team
later reported that the protective effects were mediated by
suppression of abnormal activation of IL1β, JNK, and NFκB
[24]. Third, Gu and colleagues used a senescence-accelerated
mouse strain (SAMP8) that exhibits early aging syndromes
including impairment in learning abilit y and memory [25].
Ad libitum administration of hydrogen-rich water for 30
days prevented cognitive decline, which was examined by
the Morris water maze. Additionally, ad libitum drinking of
hydr ogen water for 18 weeks showed efficient amelioration
of hippocampal neurodegeneration.
Cerebrovascular diseases are the most frequently re-
ported neurological diseases for which hydrogen has promi-
nent effects. As stated in Section 2, current hydrogen research
has broken out after Ohsawa reported a prominent effect of
2–4% hydrogen for a rat model of left cerebral artery
occlusion in 2007 [6].
In addition to neurodegenerative disorders of Parkin-
son’s disease and Alzheimer’s disease, effects of molecular
hydrogen have been reported in eight other brain diseases
listed under the categories of “brain” and “perinatal dis-
orders” in Tabl e 1. The brain consumes a large amount of
oxygen and is predisposed to be exposed to a large amount
of radical oxygen species especially under pathological con-
ditions. Molecular hydrogen is thus likely to exert a promi-
nent beneficial effect on brain diseases.
4. Molecular Hydrogen Is Effective for
Six Human Diseases
As in other therapeutic modalities, effects of molecular
hydrogen have been tested mostly on rodents but have also
been studied in six human diseases. The reported human
diseases include diabetes mellitus type II [26], metabolic
syndrome [27], hemodialysis [20, 28], inflammator y and
mitochondrial myopathies [29], brain stem infarction [30],
and radiation-induced adverse effectsforlivertumor[31].
These studies are reviewed in detail here. In addition, a ther-
apeutic trial for Parkinson’s disease is currently in progress
and exhibits favorable responses as far as we know, but the
details are not yet disclosed.
First, Kajiyama and colleagues performed a randomized,
double-blind, placebo-controlled, crossover study in 30
patients with diabetes mellitus type II and 6 patients with
impaired glucose tolerance [26]. The patients consumed
either 900 mL of hydrogen-rich water or placebo water for
8 weeks, with a 12-week washout period. They measured 13
biomarkers to estimate lipid and glucose metabolisms at
baseline and at 8 weeks after hydrogen treatment. All the bio-
markers were favorably changed with hydrogen, but stati-
stical significance was observed only in improvement of elec-
tronegative charge-modified low-density lipoprotein-(LDL-)
cholesterol, small dense LDL, and urinary 8-isoprostanes.
In four of six patients with impaired glucose tolerance,
hydrogen normalized the oral glucose tolerance test. Lack of
statistical significance in their studies was likely due to the
small number of patients and the short observation period.
Lack of statistical significance, however, may also suggest a
less prominent effect in human diabetes mellitus compared
to rodent models [32, 33].
Second, Nakao and colleagues performed an open-label
trial in 20 subjects with potential metabolic syndrome [27].
Hydrogen-rich water was produced by placing a metallic
magnesium stick in water, which yielded 0.55–0.65 mM
hydrogen water (70–80% saturation). The participants con-
sumed 1.5–2.0 liters of hydrogen water p er day for 8 weeks
and showed a 39% increase in urinary superoxide dismutase
(SOD), an enzyme that catalyzes superoxide anion ( O
2
−
);
a 43% decrease in urinary thiobarbituric acid reactive
substances (TBARS), a marker of lipid peroxidation; an 8%
increase in high-density-lipoprotein-(HDL-) cholesterol; a
13% decrease in total cholesterol/HDL-cholesterol. The
aspartate aminotransferase (AST) and alanine transaminase
(ALT) levels remained unchanged, whereas the gamma glu-
tamyl transferase (GGT) level was increased by 24% but
was still within a normal range. Although the study was
not double blinded and placebo controlled, improvements in
biomarkers were much more than those in other hydrogen
studies in humans. As this study used a large amount of
hydrogen water, the amount of hydrogen might have been a

6 Oxidative Me dicine and Cellular Longevity
critical determinant. Alternatively, excessive hydration might
have prevented the participants from excessive food intake.
Third, Nakayama and colleagues performed an open-
label placebo-controlled crossover trial of 12 sessions of
hemodialysis in eight patients [28] and an open-label trial
of 78 sessions of hemodialysis in 21 patients [20]. In both
studies, continuous sessions of hemodialysis with hydrogen-
rich dialysis solution decreased systolic blood pressure before
and a fter dialysis. In the short-term study, plasma methyl-
guanidine was significantly decreased. In the long-term
study, plasma monocyte chemoattractant protein 1 and
myeloperoxidase were significantly decreased.
Fourth, we performed an open-label tr ial of 1.0 liter of
hydrogen water per day for 12 weeks in 14 patients with mus-
cular diseases including muscular dystrophies, polymyosi-
tis/dermatomyositis, and mitochondrial myopathies, as well
as a randomized, double-blind, placebo-controlled, cross-
over trial of 0.5 liter of hydrogen water or dehydrogenized
water per day for 8 weeks in 22 patients with dermatomyositis
and mitochondrial myopathies [29]. In the open-label
trial, significant improvements were observed in lactate-
to-pyruvate ratio, fasting blood glucose, serum mat rix
metalloproteinase-3 (MMP3), and triglycerides. Especially,
the lactate-to-pyruvate ratio, which is a sensitive biomarker
for the compromised mitochondrial electron transport sys-
tem, was decreased by 28% in mitochondrial myopathies.
In addition, MMP3, which represents the activit y of inflam-
mation, was decreased by 27% in dermatomyositis. In the
double-blind trial, a statistically significant improvement was
observed only in serum lactate in mitochondrial myopathies,
but lactate-to-pyruvate ratio in mitochondrial myopathies
and MMP3 in dermatomyositis were also decreased. Lack of
statistical significance with the double-blind study was likely
due to the shorter observation period and the lower amount
of hydrogen compared to those of the open-label trial.
Fifth, Kang and colleagues perfor med a randomized
placebo-controlled study of 1.5–2.0 liters of 0.55–0.65 mM
hydrogen water per day for 6 weeks in 49 patients receiving
radiation therapy for malignant liver tumors. Hydrogen sup-
pressed the elevation of total hydroperoxide levels, main-
tained serum antioxidant capacity, and improved the qualit y
of life (QOL) scores. In particular, hydrogen efficiently pre-
vented loss of appetite. Although the patients were randomly
assigned to the hydrogen and placebo groups, the study could
not be completely blinded because hydrogen was produced
with a metallic magnesium stick, which generated hydrogen
bubbles.
Sixth, Ono and colleagues intravenously administered
hydrogen along with Edaravone, a clinically approved radical
scavenger, in 8 patients with acute brain stem infarction and
compared MRI indices of 26 patients who received Edar-
avone alone [30]. The relative diffusion-weighted images
(rDWIs), reg ional apparent diffusion coefficients (rADCs),
and pseudonormalization time of rDWI and rADC were
all improved with the combined infusion of Edaravone and
hydrogen.
No adverse effect of hydrogen has been documented
in the six human diseases described above. Among the six
diseases, the most prominent effect was obser ved in subjects
with metabolic syndrome, who consumed 1.5–2.0 liters of
hydrogen water per day [27]. The amount of hydrogen water
may be a critical parameter that determines clinical out-
come. It is also interesting to note that lipid and glucose
metabolisms were analyzed in three studies and all showed
favorable responses to hydrogen [26, 27, 29].
5. Molecular Bases of Hydrogen Effects
Effects of hydrogen on various diseases have been attributed
to four major molecular mechanisms: a specific scaveng-
ing activity of hydroxyl radical, a scavenging activity of
peroxynitrite, alterations of gene expressions, and signal-
modulating activities. The four mechanisms are not mutually
exclusive and some of them may be causally associated with
other mechanisms.
The first molecular mechanism identified for hydrogen
was its specific scavenging activity of hydroxyl radical [6].
Indeed, oxidative stress markers like 8-OHdG, 4-hydroxyl-
2-nonenal (4-HNE), malondialdehyde (MDA), and thiobar-
bituric acid reactive substances (TBARSs) are decreased in
all the examined patients and rodents. As hydrogen can
easily dissipate in exhalation, hydrogen in drinking water is
able to stay in human and rodent bodies in less than
10 min (unpublished data). Hydrogen, however, can bind to
glycogen, and the dwell time of hydrogen is prolonged in rat
liver after food intake [33]. A question still remains if mice
and humans can take a sufficient amount of hydrogen that
efficiently scavenges hydroxyl radicals that a re continuously
generated in normal and disease states.
Another molecular mechanism of hydrogen effect is its
peroxynitrite-(ONOO
−
-) scavenging activity [6]. Although
hydrogen cannot eliminate peroxynitrite as efficiently as
hydroxyl radical in vitro [6], hydrogen can efficiently reduce
nitric-oxide-(NO-) induced production of nitrotyrosine in
rodents [34–38]. NO is a gaseous molecule that also exerts
therapeutic effects including relaxation of blood vessels and
inhibition of platelet aggregation [39].NO,however,isalso
toxic at higher concentrations because NO leads to ONOO
−
-
mediated production of nitrotyrosine, which compromises
protein functions. A part of hydrogen effects may thus be
attributed to the reduced production of nitrotyrosine.
Expression profiling of rat liver demonstrated that hydro-
gen has a minimal effect on expression levels of individual
genesinnormalrats[40]. Gene ontology analysis, however,
revealed that oxidoreduction-related genes were upregulated.
In disease models of rodents, expression of individual genes
and proteins is analyzed. In many disease models, hydrogen
downregulated proinflammatory cytokines including tumor
necrosis-factor-(TNF-) α, interleukin-(IL-) 1β,IL-6,IL-
12, interferon-(IFN-) γ, and high mobility group box 1
(HMGB1) [4, 23, 24, 36, 41–59]. Hydrogen also downregu-
lated nuclear factors including nuclear factor kappa B
(NFκB), JNK, and proliferation cell nuclear antigen (PCNA)
[24, 44, 50, 55, 60–63]. Caspases were also downregulated
[10, 55–57, 62, 64, 65]. Other interesting molecules studied
to date include vascular endothelial growth factor (VEGF)

Oxidative Medicine and Cellular Longevity 7
[15]; MMP2 and MMP9 [34]; brain natriuretic peptide [48];
interce llular-adhesion-molecule-1 (ICAM-1) and myeloper-
oxidase [36]; B-cell lymphoma 2 (Bcl2) and Bcl2-associated
Xprotein(Bax)[60]; MMP3 and MMP13 [38]; cyclooxyge-
nase 2 (COX-2), neuronal nitric oxide synthase (nNOS), and
connexins 30 and 43 [66]; ionized calcium binding adaptor
molecule 1 (Iba1) [52]; fibroblast growth factor 21 (FGF21)
[33]. Most molecules, however, are probably passengers that
are secondarily changed by hydrogen administration, and
some are potentially direct t argets of hydrogen effects, which
need to be identified in the future.
Using rat RBL-2H3 mast cells, we demonstrated that
hydrogen attenuates phosphorylation of FcεRI-associated
Lyn and its downstream signaling molecules [64]. As phos-
phorylation of Lyn is ag ain regulated by the downstream
signaling molecules and makes a loop of signal transduction
pathways, we could not identify the exact target of hydrogen.
Our study also demonstrated that hydrogen ameliorates an
immediate-type allergic reaction not by radical-scavenging
activity but by direct modulation of signaling pathway(s).
In addition, using murine RAW264 macrophage cells, we
demonstrated that hydrogen reduces LPS/IFNγ-induced NO
production [67]. We found that hydrogen inhibits phospho-
rylation of ASK1 and its downstream signaling molecules,
p38 MAP kinase, JNK, and IκBα without affecting ROS
production by NADPH oxidase. Both studies point to a
notion that hydrogen is a gaseous signal modulator. More
animal and cells models are expected to be explored to
confirm that hydrogen exerts its beneficial effect as a signal
modulator.
6. Enigmas of Hydrogen Effects
Two enigmas remain to be solved for hydrogen effects. First,
no dose-response effect of hydrogen has been observed.
Hydrogen has been administered to animals and humans in
the forms of hydrogen gas, hydrogen-rich water , hydrogen-
rich saline, instillation, and dialysis solution (Tabl e 1).
Supposing that a 60-kg person drinks 1000 mL of satu-
rated hydrogen-rich water (1.6 ppm or 0.8 mM) per day,
0.8 mmoles of hydrogen is consumed by the body each day,
which is predicted to give rise to a hydrogen concentration of
0.8 mmoles/(60 kg
× 60%) = 0.022 mM (2.8% saturation =
0.022 mM/0.8 mM). As hydrogen mostly disappears in
10 min by dissipation in exhalation (unpublished data), an
individual is exposed to 2.8% hydrogen only for 10 min.
On the other hand, when a person is placed in a 2%
hydrogen environment for 24 hrs, body water is predicted
to become 2% saturation (0.016 mM). Even if we suppose
that the hydrogen concentration after drinking hydrogen
water remains the same for 10 min, areas under the curves of
hydrogen water and 2% hydrogen gas are 0.022 mM
× 1/6 hrs
and 0.016 mM
× 24 hrs, respectively. Thus, the amount of
hydrogen given by 2% hydrogen gas should be 104 or more
times higher than that given by drinking hydrogen water. In
addition, animals and patients are usually not able to drink
100%-saturated hydrogen water. If the hydrogen concentra-
tion is 72% of the saturation level, the peak concentrations
achieved by drinking hydrogen water and 2% hydrogen
gas should be identical (0.022 mM
× 72% = 0.016 mM). Nev-
ertheless, hydrogen water is as effective as, or sometimes
more effec tive than, hydrogen gas. In addition, orally taken
hydrogen can be readily distributed in the stomach, intestine,
liver, heart, and lung but is mostly lost in exhalation. Thus,
hydrogen concentrations in the arteries are predicted to be
very low. Nevertheless, marked hydrogen effects are observed
in the brain, spinal cord, kidney, pancreas muscle, and
cartilage, where hydrogen is carried via arteries.
The second enigma is intestinal production of hydrogen
gas in rodents and humans. Although no mammalian cells
can produce hydrogen endogenously, hydrogen is produced
by intestinal bacteria carrying hydrogenase in both rodents
and humans. We humans are able to make a maximum of
12 liters of hydrogen in our intestines [68, 69]. Specific-
pathogen-free (SPF) animals are different from aseptic
animals and carry intestinal bacteria that produce hydrogen.
The amount of hydrogen taken by water or gas is much
less than that produced by intestinal bacteria, but the
exogenously administered hydrogen demonstrates a promi-
nent effect. In a mouse model of Concanavalin A-induced
hepatitis, Kajiya and colleagues killed intestinal bacteria by
prescribing a cocktail of antibiotics [43]. Elimination of
intestinal hydrogen worsened hepatitis. Restitution of a
hydrogenase-negative str a in of E. coli had no effects, whereas
that of a hydrogenase-positive strain of E. coli ameliorated
hepatitis. This is the only report that addressed a beneficial
effect of intestinal bacteria, and no human study has been
reported to date. Kajiya and colleagues also demonstrated
that drinking hydrogen-rich water was more effective than
the restitution of hydrogenase-positive bacteria. If intestinal
hydrogen is as effective as the other hydrogen administration
methods, we can easily increase hydrogen concentrations
in our bodies by an α-glucosidase inhibitor, acarbose [70],
an ingredient of curry, turmeric [71], or a nonabsorbable
synthetic disaccharide, lactulose [68, 72, 73]. The enigma of
intestinal bacteria thus needs to be solved in the future.
7. Summary and Conclusions
Effects of hydrogen have been reported in 63 disease models
and human diseases (Ta ble 1). Only two diseases of cerebral
infarction and metabolic syndrome have been analyzed in
both rodents a nd humans. Lack of any adverse effects
of hydrogen enabled clinical studies even in the absence
of animal studies. Some other human studies including
Parkinson’s disease are currently in progress, and promising
effects of hydrogen are expected to emerge for many other
human diseases. We also have to elucidate molecular bases of
hydrogen effects in detail.
8. Added Note in Proof
We recently reported a line of evidence that molecular
hydrogen has no dose-response effect in a rat model of
Parkinson’s disease [74].

8 Oxidative Me dicine and Cellular Longevity
Acknowledgments
Works performed in the authors’ laboratories were sup-
ported by Grants-in-Aid from the MEXT and MHLW of
Japan and from the Priority Research Project of Aichi.
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