Cytotechnology 40: 139–149, 2002.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Protective mechanism of reduced water against alloxan-induced
pancreatic β-cell damage: Scavenging effect against reactive oxygen
Yuping Li1, Tomohiro Nishimura1, Kiichiro Teruya1, Tei Maki1, Takaaki Komatsu1,
Takeki Hamasaki1, TaichiKashiwagi1,
Yoshinori Katakura1,Kazuhiro Osada1,
Shinkatsu Morisawa2, Yoshitoki Ishii3, Zbigniew Gadek4& Sanetaka Shirahata1∗
1Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki,
Higashi-ku, Fukuoka, Japan;2Nihon Trim Co. Ltd., 1-8-34 Oyodonaka, Kita-ku, Osaka, Japan:3Hita Tenryosui
Co. Ltd., 647 Nakanoshima, Hita, Oita, Japan;4Center for Holistic Medicine and Naturopathy, Schmallenberg-
(∗Author for correspondence; E-mail: email@example.com;Fax: +81 92 642 3052)
Received 24 August 2002; accepted in revised form 10 October 2002
Key words: alloxan, apoptosis, diabetes, HIT-T15 cells, insulin, pancreatic beta cells, reactive oxygen species,
Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many dis-
eases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like
Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could
protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic
compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the pro-
duction of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels,
elevated cytosolic free Ca2+concentration, DNA fragmentation, decreased intracellular ATP levels and lowering
of cytosolic Ca2+concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin
release, and strongly blockedDNA fragmentation,partially suppressing the lowering of viability of alloxan-treated
cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and
2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The
protectiveactivityofRW was stableat 4◦C forovera month,butwas lost byautoclaving.Theseresults suggestthat
RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation.
RW may be useful in preventing alloxan-inducedtype 1-diabetes mellitus.
Abbreviations: BSA, bovine serum albumin; DCF, dichloroflurescein; DCFH-DA, 2?,7?-dichlorofluorescin-
diacetate; ELISA, enzyme-linked immunosorbent assay; ERW, electrolyzed-reduced water; HBSS, Hank’s
balanced salt solution; IDDM, insulin-dependent diabetes mellitus; IIDM, insulin-independent diabetes mellitus;
KRB, Krebs Ringer bicarbonate buffer; NRW, nature reduced water; ROS, reactive oxygen species; RW, reduced
Diabetes is mainly grouped into insulin-dependent
diabetes mellitus (IDDM) (Type 1-diabetes) and
insulin-independentdiabetes mellitus (IIDM)(Type2-
diabetes). Type 1-diabetes is caused by a deficiency
in insulin secretion from pancreatic β cells. Type 2-
diabetes mellitus is related to damage in the insulin
signaling pathway. Chemical compounds that select-
ively damage pancreatic β-cells constitute a class of
diabetogenic drugs. Alloxan, a cyclic urea derivative,
is a potent diabetogenic agent that has been widely
used for the induction of experimental type 1 dia-
betes (Rho et al., 2000). It has been reported that
alloxan rapidly and selectively accumulates in β-cells
in comparison with non-β cells (Gorus et al., 1982).
Although the precise diabetogenic mechanism of al-
loxan is not fully understood, evidence indicates that
pancreatic β-cell damage induced by alloxan is me-
diated through the generation of cytotoxic reactive
oxygen species (ROS) (Yamamoto et al., 1981; Mal-
aisse and Lea, 1982; Takasu et al., 1991). Okamoto
(1985) proposed that the primary target of ROS pro-
duced by alloxan is the DNA of the pancreatic β-cells
and causes DNA strand breaks. Increase of cytosolic
Ca2+also plays an important role in the diabetogen-
esis of alloxan, in relation to radical generation and
DNA fragmentation (Park et al., 1995).
The clinical effect of electrolyzed-reduced water
(ERW) was recognized by the Ministry of Health
and Welfare of Japan in 1965. Double blind clinical
tests demonstrated that ERW was safe and effective
for intestinal abnormal fermentation, acid indigestion,
chronic diarrhea, chronic constipation, dyspepsia, and
antacid (Tashiro, 1999). However, the mechanism of
the clinical effect of ERW has not been clarified. Sev-
eral natural mineral waters in the world are called mir-
acle waters because of their curative powers against
various diseases. Tracote water in Mexico was found
in 1991. Nordenau water in Germany was found in
in 1997.Ithas beenreportedbymanypeoplethat daily
consumption of these waters results in improvement
in various diseases including diabetes, cancer, arteri-
osclerosis and allergies. However, strict double-blind
clinical tests have not yet been performed.
ERW contains a lot of hydrogen, scavenges ROS,
and protects DNA from oxidative damage, suggesting
that stable active hydrogen in ERW might be a re-
ducing agent responsible for scavenging against ROS
(Shirahata et al., 1997). We found that Hita Tenryou-
sui water, Nordenau water, and Tracote water were
all antioxidative waters that could scavenge intracel-
lular ROS and called those waters natural reduced
waters (NRW) (Shirahata, 2002). Based on extensive
evidence, Shirahata (2000, 2002) proposed the active
hydrogen theory of reduced water as follows: (1) Wa-
ter is a good supplier of active hydrogen. (2) Active
hydrogen can be easily produced by weak electrolysis
and stabilized in the form of hydrogenated or reduced
metal colloids in reduced water (RW) like ERW and
NRW. (3) The reducing activity of RW is lost by heat-
ing or autoclaving due to dehydrogenation of metal
colloids or instability of metal colloids. (4) Active
hydrogen in RW may be an ideal scavenger against
ROS because it does not produce oxidized molecules
C, vitamin E, and polyphenols). Recently, Hana-
oka (2001) reported the scavenging activity of ERW
against ROS. RW stimulates glucose uptake in muscle
cells and adipocytes, suggesting that RW might be
effective for preventing type 2-diabetes (Oda et al.,
1999; Shirahata et al., 2001). However, the effect of
RW on pancreaticβ-cells in relation to type 1 diabetes
is not understood. Here we report that RW strongly
protects pancreatic β cells from damage induced by
the diabetogenic agent alloxan, suggesting that RW
may be effective in preventing alloxan-inducedtype-1
Materials and methods
Bovine serum albumin (BSA), an ATP Biolumines-
cence Assay Kit CLS II and a cellular DNA fragment-
ation ELISA Kit for the detection of apoptosis were
obtained from Roche Diagnostics GmbH (Mannheim,
Germany). Krebs Ringer bicarbonate (KRB) buffer
Sigma Chemical Co. (St. Louis, MO, U.S.A.). WST-
8 Assay Kit was from Promega Co. (Madison, WI,
U.S.A.). RPMI 1640 medium was from Nissui Phar-
maceutical Co. (Tokyo, Japan). A rat insulin enzyme
immunoassay(EIA) kit, D(+)-glucose,HEPES ((4-[2-
hydroxyethyl]-1-piperazineethanesulfonic acid) and
all other chemicals were obtained from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan).
Reduced water and preparation of medium
Electrolyzed-reduced water (ERW) was prepared
by the electrolysis of ultra pure water contain-
ing 0.002 N NaOH using an electrolyzing device
equipped with platinum-coated titanium electrodes
(Type TI-200s, Nihon Trim Co., Osaka, Japan).
The electrolyzing device used was a batch type
one and consisted of a vessel of 4 l (190 mm
length×210 mm wide×140 mm height)divided by a
semi-permeable membrane (190 mm wide×130 mm
height). Two platinum-coated titanium electrodes
(70 mm×110 mm square) were placed at a distance
of 55 mm from each side of the semi-permeablemem-
brane for electrolysis with a direct current of 100 V
for 60 min. The alkaline ERW was stored in a closed
glass bottle at 4◦C and neutralized with HEPES buf-
fer in medium before use. Nordenau Water was kindly
supplied by Mr. Theo Tommes from Nordenau in Ger-
many. Hita Tenryosui water was obtained from Hita
were purchased from a local market in Japan. In order
to inactivate its reducing activity, the RW was auto-
claved in an open bottle at 121◦C for 20 min. In order
to investigate the protective effect of RW on alloxan-
induced cytotoxicity in HIT-T15 cells, a medium was
prepared using RW instead of ultra pure water.
A hamster pancreatic β cell line, HIT-T15, was sup-
plied by Dainippon Pharmaceutical Co. (Osaka, Ja-
pan). The cells were cultured in a RPMI 1640 medium
containing 10% fetal bovine serum (FBS), 2 mM L-
glutamine, 25 mM HEPES, 100 IU ml−1penicillin-G
and 100 µg ml−1streptomycin at 37◦C in a hu-
midified atmosphere of 5% CO2. The medium was
exchanged every 2 days.
Measurement of cell viability
HIT-T15 cells (1×105cells ml−1) were seeded
onto 24 well plates and pre-incubated in a 10%
FBS/RPMI 1640 medium containing RW (sterilized
with a 0.22 µm filter or autoclaved at 121◦C for
20 min) for 18 h. Alloxan dissolved in 0.2 mM cit-
rate buffer (pH 4.0) was added to the cells that were
then incubated for 1 h. The medium was exchanged
for fresh medium containing RW and the cells in-
cubated for 24 h. Cell viability was determined by
the WST-8 Reduction Assay (Mossman et al., 1983).
The absorbance at 450 nm was measured with a
Measurement of intracellular ROS
(A) Con-focal laser microscopic analysis
The amount of intracellular ROS, especially the intra-
cellular H2O2 produced by alloxan was determined
by using a fluorescent dye, 2?,7?-dichlorofluorescin-
diacetate (DCFH-DA) (LeBel et al., 1992). HIT-T15
cells were pre-cultured for 24 h in RPMI 1640 with or
without RW. The cells were then incubated for 30 min
in a KRB buffer with or without RW. After the ad-
dition of 1mM alloxan, the cells were incubated for
a further 30 min. After removal of the supernatant,
5 µM DCFH-DA in a Ca2+, Mg2+-free HBSS buffer
was added and the cells incubated for 60 min. The
cytoplasmic fluorescence intensity was measured us-
ing a con-focal laser scanning microscope (Molecular
Dynamics, U.S.A.) with a FITC barrier filter.
(B) DCFH-DA flow cytometric analysis
Cells treated as described in (A) were harvested by
trypsinization, washed with PBS, resuspended in PBS
and analyzed immediately using a flow cytometer
(Coulter Elite FACSCAN) with excitation and emis-
sion wavelengths of 495 and 525 nm, respectively.
Gating was performed to remove apoptotic cells and
cellular debris before data were collected.
Measurement of cytosolic free Ca2+concentration
The change of intracellular free Ca2+concentration
was analyzed using Fluo 3-AM by flow cytomet-
ric analysis. HIT-T15 cells were cultured for 24 h
in a RPMI 1640 medium with or without RW, and
then incubated for 30 min in a KRB buffer with or
without RW. After the addition of 1 mM alloxan or
0.5 mM H2O2, the cells were incubated for 60 min.
The medium was then removed and an HBSS buf-
fer containing 4 µM Fluo 3-AM (Sigma) added. The
cells were incubated for 60 min and then washed
twice. The cytoplasmic fluorescence intensity of the
cells was monitored, according to the manufacturer’s
Measurement of DNA fragmentation
The cellular DNA fragmentation of HIT-T15 cells
was determined according to the manufacturer in-
structions using a cellular DNA fragmentation ELISA
Kit (Roche). Briefly, HIT-T15 cells (2×105cells
ml−1) were pre-incubated in a RPMI 1640 medium
containing RW (sterilized with a 0.22 µm filter or
autoclaved at 121◦C for 20 min in a open bottle)
for 24 h. The cells were then incubated with 10 mM
BrdU overnight at 37◦C, centrifuged at 250×g for
10 min, adjusted to 1×105cells ml−1in a KRB
buffer containing RW, plated in a 96-multiwell plate.
After treatment with 1 mM alloxan for 2 h, DNA frag-
mentation was determined with the ELISA Kit using
a peroxidase-conjugated anti-BrdU antibody solution.
The absorbance was measured at 450 nm against a
substrate solution as a blank.
Measurement of intracellular ATP and insulin release
The concentration of intracellular ATP in HIT-T15
cells was determined by the luciferin-firefly luciferase
method (Ludin, 1978).Briefly, HIT-T15 cells (2×105
cells ml−1) were pre-incubated for 24 h in a RPMI
1640 medium with or without RW. The cells were
then incubated for 30 min with a KRB buffer with or
without 1mM alloxan. The alloxan-treated cells were
washed and incubated for 60 min with a KRB buffer
containing10mM glucoseThespentmediumwas col-
lected to examine the insulin release. The cells were
trypsinized and resuspended in PBS. The cell suspen-
sion (0.1 ml) was immediately mixed with 0.9 ml of
100 mM Tris-HCl buffer (pH = 7.8) containing 4 mM
EDTA. The relative amount of intracellular ATP was
determined according to the manufacturer’s instruc-
tions using an ATP assay Kit. For the measurement
of insulin release, the amount of hamster insulin in
the spent medium was determined by a competitive
ELISA technique using a rat insulin assay Kit because
of high homology between hamster and rat insulin.
Results and discussion
Effect of RW on cytotoxicity of alloxan in HIT-T15
Alloxan itself is non-toxic. It is reduced extracellu-
larly to dialuric acid in the presence of a reducing
agent (e.g. cysteine). Oxidation of dialuric acid in
the presence of oxygen results in the production of
both superoxide anion radicals and hydrogen perox-
ide, the latter of which can diffuse across the plasma
membrane and into the cell interior (Zhang et al.,
1995; Szkudelski,2001).Neitherof thesemoleculesis
overlyreactive, but they,in turn, leadto the production
of the highly reactive hydroxyl radical (Halliwell et
al., 1990), that is rapidly taken up by the pancreatic
β-cells with consequent possible damage (Tomita et
al., 1994). Alloxan is selectively toxic to pancreatic
β-cells. HIT-T15 cells were grown in the presence of
concentrations of alloxan ranging from 0.25 to 5 mM.
The addition of alloxan to the culture medium caused
a dose-dependent decrease in cell viability (data not
shown). At a concentration of 1 mM, alloxan was
able to kill about 48% of the cells compared with
the untreated control. Hence, all further studies were
conducted using this concentration of alloxan.
In order to evaluate the effect of RW on alloxan-
induced cytotoxicity, HIT-T15 cells were pre-cultured
for 18 h in media containing various waters and then
exposed to 1 mM alloxan for 1 h. The cells were
further cultured for 24 h. As shown in Figure 1a,
52.3% of the control cells treated with alloxan ex-
hibited viability. On the other hand, the cells treated
with Nordenau water, ERW and Hita Tenryosui wa-
ter showed significantly increased viability of 88.7,
82.2 and 66.7%, respectively, after alloxan treatment.
However, in contrast, two commercial natural min-
eral waters lowered the cell viability to 22.7 and
15.8% after alloxan treatment (Figure 1a). Since nat-
ural mineral water usually contains various kinds of
metal ions and organic compounds, the cytotoxicity
of alloxan might be potentiated by such substances
in the commercial natural mineral waters examined.
These results indicate that RW can partially protect
HIT-T15 cells from alloxan-induced cytotoxicity. The
protective activity was stable in a closed glass bottle
at 4◦C for over a month. The activity was not lost
even after neutralization and repeated filtration with
a 0.22 µm filter. However, autoclaving of RW at
121◦C for 20 min resulted in the loss of the protective
activity (Figure 1b). No significant differencebetween
alloxan-treated HIT-T15 cells and alloxan/RW-treated
cells was found, suggesting that active agents in RWs
were lost by autoclaving. It has been reported that the
ROS-scavenging activity of ERW is stable at 4◦C for
more than one month and the activity is not lost by
neutralization and filtration, but lost by autoclaving
(Shirahata et al., 1997). These results support the act-
ive hydrogen theory that the reducing activity of RW
like ERW and NRW is lost due to dehydrogenation
of metal colloids or instability of metal colloids by
autoclaving (Shirahata, 2002).
Effect of RW on intracellular redox state
Free radicals are believed to be involved in the
cytotoxic action of alloxan on pancreatic β-cells.
Hence, we observed the intracellular redox state of
HIT-T15 cells treated with alloxan using an ROS-
sensitive probe DCFH-DA. The membrane-permeable
non-fluorescent fluorescein derivative, DCFH-DA (re-
duced 2?,7?-dichlorofluorescein diacetate) is cleaved
by intracellular esterases to membrane-impermeable
DCFH and oxidized by ROS like H2O2 in cells to
2?,7?-dichlorofluorescein(DCF), which is highlyfluor-
escent. We monitoredDCF fluorescenceby using both
a con-focal laser microscope and a flow cytometer.
As shown Figure 2a, the con-focal microscopic ana-
lysis revealed that RW scavenged intracellular ROS
Figure 1. Effects of pretreatment with various waters on alloxan-induced cytotoxicity of HIT-T15 cells. (a) Effect of RW filtrated with 0.22 µm
filter on alloxan-induced cytotoxicity. (b) Effect of RW treated with autoclaving at 121◦C for 20 min in an opened condition on alloxan-induced
cytotoxicity. After cells were pre-incubated with various waters for 18 h and treated with 1mM alloxan for 1 h, the medium was removed.
Incubation was continued in RPMI 1640 medium with or without various waters for 24 h. The WST-8 reduction assay was used for determining
viability. The alloxan-untreatment and alloxan-treatment were shown by – and +, respectively. Each value denotes the mean±S.D. of three
separate experiments. Statistical analysis was done using the Student’s t-test. * P≤0.05, ** P≤0.01, compared to alloxan untreated control.
a, ultrapure water (control); b, ERW; c, Hita Tenryosui water; d, Nordenau water; e, commercial natural mineral water A; f, commercial natural
mineral water B.
both in the absence and presence of alloxan. The
commercial natural mineral waters did not exhibit the
ROS-scavenging effect. Figure 2b shows the results
of the DCFH-DA flow cytometric analysis. The RWs
decreased the ROS level by about 35%. Alloxan el-
evated the ROS level of HIT-T15 cells by about 30%.
RW-treated cells sustained 10–30% lower intracellu-
lar ROS level than that of non-treated cells, even
after alloxan-treatment.Whereas, the commercial nat-
ural mineral waters did not suppress the intracellular
ROS level and the cells treated with the natural min-
eral waters exhibited 40% higher ROS levels than
non-treatedcells afteralloxan-treatment.These results
clearly show that RW is anti-oxidative water that can
scavenge intracellular ROS and protect β-cells from
alloxan-induced ROS stress.
Effect of RW on cytosolic free Ca2+concentration
It has become apparent that Ca2+is important in both
physiological and toxicological processes (Kim et al.,
1994).Theassociation ofcytotoxicitywith a sustained
Figure 2. Effect of pretreatment with various waters on intracellular redox state of HIT-T15 cells treated with alloxan. (a) Con-focal laser
microscopic analysis. After cells were pre-incubated with various waters for 24 h and treated with or without 1 mM alloxan for 30 min, the
medium was removed. Incubation was continued with 5 µM DCFH-DA in HBSS buffer for 60 min. The change of cytoplasmic fluorescence
intensity was measured by a con-focal laser microscope (a) and flow cytometer (b). Each value in (b) denotes the mean±S.D. of three separate
experiments. The alloxan-non-treatment and alloxan-treatment were shown by – and +, respectively. Statistical analysis was done using the
T-test. * P≤0.05; ** P < 0.01, compared to non-treated control. # P≤0.05; ## P≤0.01, compared to alloxan-treated control. a, ultrapure water
(control); b, ERW; c, Hita Tenryosui water; d, Nordenau water; e, natural mineral water A; f, natural mineral water B.
increase of cytosolic Ca2+levels has been reported
in many different cell types (Orrenius et al., 1989).
The oxidative stress is accompanied by an increase in
cytosolic free Ca2+(Masumoto et al., 1990; Kim et
al., 1994; Janciauskiene and Ahren, 1998). Alloxan-
derived ROS may disturb intracellular Ca2+homeo-
stasis. This results in secondary reactions ultimately
leading to DNA strand breaks and cytotoxicity of pan-
creatic β-cells (Kim et al., 1994; Szkudelski, 2001),
suggesting the importance of the Ca2+-dependent
Figure 3. Effects of pretreatment with various waters on cytosolic free Ca2+in alloxan-treated HIT-T15 cells. After cells were pre-incubated
with various waters for 24 h and treated with 1 mM alloxan for 60 min, the medium was removed, and treated with 4 µM Fluo 3-AM (Sigma)
in HBSS buffer at 37◦C for 60 min and then washed twice. The change of cytoplasmic fluorescence intensity of selected cells was monitored
by flow cytometry. The alloxan-non-treatment and alloxan-treatment were shown by – and +, respectively. Each value denotes the mean±S.D.
of three separate experiments. a, ultrapure water (control); b, ERW; c, Hita Tenryosui water; d, Nordenau water; e, natural mineral water A;
f, natural mineral water B.
pathway in cell damage. To verify the effect of RW on
Ca2+-influx, the change of cytosolic free Ca2+con-
centration was traced using Fluo 3-AM in HIT-T15
cells. As shown in Figure 3, alloxan-treated cells ex-
Ca2+than non-treated cells. The three kinds of RWs
completely inhibited the alloxan-induced increase of
cytosolic free Ca2+. While, the two commercial nat-
ural mineral waters could not strongly suppress the
alloxan-induced increase of cytosolic free Ca2+. Sim-
ilar results were obtained by using 0.5 mM H2O2
instead of alloxan (data not shown). These results sug-
gest that the protective action of RW against alloxan-
induced pancreatic β-cell damage is exerted primarily
by inhibiting ROS generation before the change of
Effect of RW on DNA fragmentation
It is known that alloxan can cause accumulation
of enough ROS to induce an increase of Ca2+in-
flux, which results in secondary reactions ultimately
leading to the fragmentation of DNA of β-cells
(Yamamoto et al., 1981). Okamoto (1985) has pro-
posed that the primary target of ROS produced from
alloxan is the DNA of pancreatic β-cells, and that
causes alloxan DNA strand breaks. The DNA frag-
mentation by alloxan is a critical step in the induction
of alloxan-diabetes. In order to investigate the effect
of RW on DNA strand break, the DNA fragmentation
was measured using the ELISA method. As shown
in Figure 4a, alloxan increased approximately 8-fold
the extent of DNA fragmentation when HIT-T15 cells
were treated with 1 mM alloxan for 2 h. However,
the DNA fragmentation induced by alloxan was al-
most perfectly inhibited in HIT-T15 cells by RW. The
two commercial natural mineral waters did not inhibit
this DNA fragmentation. Autoclaving of RW resul-
ted in almost complete loss of the protective activity
of RW against the alloxan-induced DNA fragmenta-
tion (Figure 4b), because no significant differencewas
found between the values of alloxan-treated control
and alloxan/RW-treated cells. These results suggest
that the protective activity of RW against the alloxan-
induced DNA fragmentation is lost by heating.
The DNA fragmentation induced by alloxan may
be mediated by Ca2+-dependent endonuclease, the
activity of which is affected by ATP. This initiates the
repair process involving the activation of poly (ADP-
ribose) synthetase and the associated NAD utilization.
Alloxan causes the depletion of cellular ATP and this
is believed to be a result of a lack of NAD+. It is
believed that NAD depletion is so precipitous that it
Figure 4. Effects of pretreatment with various waters on alloxan-induced DNA fragmentation in HIT-T15 cells. (a) Effect of RW on al-
loxan-induced DNA fragmentation. (b) Effect of autoclaved RW on alloxan-induced DNA fragmentation. Cells were pre-incubated with various
waters for 24 h and treated with 1mM alloxan for 2 h. The amount of DNA fragmentation in 1×105cells ml−1was measured by ELISA.
Each value denotes the mean±S.D. of three separate experiments. a, ultrapure water (control); b, Control + alloxan; c, ERW + alloxan; d, Hita
Tenryosui water + alloxan; e, Nordenau water + alloxan; f, natural mineral water A + alloxan; g, natural mineral water B + alloxan.
becomes irreversible and results in a virtual cessation
of NAD-dependent metabolism leading to cell death.
This is supported by the fact that nicotinamide sup-
plementation and free radical quenchers can prevent
alloxan-induced diabetes (Suresh et al., 2001).
Effect of RW on intracellular ATP and insulin release
and the depletion of cellular ATP is a known hall-
mark for the deterioration of cell metabolism. Alloxan
has a direct effect on islet cell permeability and acts
at the site of hexose transport. It also interferes with
the generationof glucose-derivedenergyby inhibiting
glycolytic flux and pyruvate oxidation, and decreases
ATP production (Borg et al., 1979). Alloxan deple-
tion of the cellular ATP, leads to the opening of K+
channels and to cell membrane hyper-polarization.
The latter event closes voltage-dependent Ca2+chan-
nels, decreases [Ca2+]I and suppresses Ca2+oscilla-
tions, eventually leading to the inhibition of insulin
secretion. Thus, we investigated the effect of RW
on glucose-stimulated cellular ATP levels (Figure 5)
Figure 5. Effects of pretreatment with various waters on glucose-stimulated intracellular ATP level in HIT-T15 cells treated with alloxan. After
cells were pre-incubated with various waters for 24 h and treated with 1 mM alloxan for 30 min, the medium was removed. After washing, the
cells were incubated with KRB buffer containing 10 mM glucose for 60 min. The relative amount of intracellular ATP was determined using
an ATP assay Kit by chemiluminescence method. The alloxan-non-treatment and alloxan-treatment were shown by – and +, respectively. Each
value denotes the mean±S.D. of three separate experiments. a, ultrapure water (control); b, ERW; c, Hita Tenryosui water; d, Nordenau water;
e, natural mineral water A; f, natural mineral water B.
Figure 6. Effects of pretreatment with various waters on the glucose-stimulated insulin release in HIT-T15 cells treated with alloxan. Cells
were pre-incubated with various waters for 24 h and treated with 1mM alloxan for 30 min. After washing, the cells were incubated with KRB
buffer containing 10 mM glucose for 60 min. The released insulin in the spent medium was determined by radioimmunoassay with rat insulin
as standard. The secreted insulin concentration of 100% was 2.38 ng ml−1. The alloxan-non-treatment and alloxan-treatment were shown by –
and +, respectively. Each value denotes the mean±S.D. of three separate experiments. a, ultrapure water (control); b, ERW; c, Hita Tenryosui
water; d, Nordenau water; e, natural mineral water A; f, natural mineral water B.
and the glucose-stimulated increase of insulin release
(Figure 6). ERW and Hita Tenryosui water increased
glucose-stimulated ATP levels to 270% and Nordenau
water 150%. Alloxan decreased glucose-stimulated
cellular ATP levels to 47% of the level of non-treated
HIT-T15 cells. Even after alloxan treatment, ERW and
Hita Tenryosui-treated cells sustained the high ATP
level of 220%and Nordenauwater-treatedcells 100%.
The commercial natural mineral waters did not ex-
hibit such an enhancing effect on glucose-stimulated
damage. It is suggested that RW increased glucose-
stimulated ATP production, leading to the closing of
The latter event may open voltage-dependent Ca2+
channels, increase [Ca2+]Iand eventually lead to the
increase of insulin secretion.
As shown in Figure 6, Hita Tenryousui wa-
ter, Nordeanu water and ERW enhanced glucose-
stimulated insulin release to 480, 310, and 280%
compared to that of non-treated cells. On the other
hand, 1 mM alloxan decreased the glucose-stimulated
insulin release of HIT-T15 cells to 36% of that of
non-treated cells. Even after alloxan treatment, Hita
Tenryosui water, ERW, and Nordenau water enhanced
glucose-stimulated insulin release to 220, 210 and
130%. The commercial natural mineral water did not
exhibit an enhancing effect on the glucose-stimulated
insulin release and exhibited low insulin release level
of 48and 33% of that of non-treatedcells after alloxan
treatment. Because RW neither increased cellular ATP
levels nor enhanced insulin release without glucose
stimulation (data not shown), it is suggested that RW
might activate the function of β-cells by increasing
the glucose sensitivity and response of pancreatic β-
cells against glucose. These results suggest that RW is
effective for prevention of alloxan-induced IDDM.
Environmental agents modulate the incidence of
IDDM, possibly by inducing the initial β-cell lesions.
Numerous immunological and genetic studies includ-
ing those with non-obese diabetic mice (Wogensen et
al., 1994) have established that cellular and humoral
autoimmunity against pancreatic β-cells is import-
ant in the pathogenesis of IDDM (Eisenbarth et al.,
1986), presumably subsequent to β-cell injury by vir-
uses and/or chemotoxins (Krowlewski et al., 1987).
Cellular responses to such factors often involve the
generation of ROS. Oxidative stress such as the pro-
duction of ROS, especially the generation of H2O2
and hydroxyl radicals in cells have been implicated in
ATPchannels and to cell membrane depolarization.
numerous reports as a mechanism of cell death in a
number of disease states.
This is the first paper reporting the protective ef-
fect of RW against pancreatic β-cells. We confirmed
that RW could scavenge intracellular ROS and pre-
vent alloxan-induced β-cell damage by ROS. Anti-
oxidative water like RW would be beneficial in as-
sisting the treatment of diabetes mellitus as well as
prevention of diabetes, because water can rapidly per-
meate all the body, it has no calorie and the uptake
of large quantities of water is possible. In clinical
tests of Nordenau water on 139 diabetes patients, a
downward trend of average blood sugar, HbA1c, cho-
lesterol, triglyceride and LDL values was exhibited
(Shirahata et al., 2001). Daily comsumption of RW
may prevent not only environmental agent-induced
diseases causedby ROS includingcancer, arterioscler-
osis, neural disease and allergies. The detailed action
mechanism of the reducing agents in RW respons-
ible for the ROS scavenging activity will be reported
(1)RWs like ERW, Hita Tenryosui water and Norde-
nau water were all anti-oxidative waters that could
scavenge intracellular ROS in hamster pancreatic
(2)Alloxan induced lowering of viability, increase of
cytosolic free Ca2+, increase of DNA fragmenta-
tion, decrease of intracellular ATP, and decrease
of glucose-stimulated insulin release. RW could
inhibit all those cytotoxic effects of alloxan, in-
creasing markedly the glucose-stimulated increase
of ATP levels and insulin release.
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