electrochemically reduced water protects neural cells from...

Research ArticleElectrochemically Reduced Water Protects Neural Cells fromOxidative Damage

Taichi Kashiwagi1 Hanxu Yan2 Takeki Hamasaki2 Tomoya Kinjo2

Noboru Nakamichi1 Kiichiro Teruya12 Shigeru Kabayama3 and Sanetaka Shirahata12

1 Department of Bioscience and Biotechnology Faculty of Agriculture Kyushu University Fukuoka 812-8581 Japan2Division of Life Engineering Graduate School of Systems Life Sciences Kyushu University 6-10-1 Hakozaki Higashi-kuFukuoka 812-0053 Japan

3Nihon Trim Co LTD 1-8-34 Oyodonaka Kita-ku Osaka 531-0076 Japan

Correspondence should be addressed to Sanetaka Shirahata sirahatagrtkyushu-uacjp

Received 11 March 2014 Revised 19 August 2014 Accepted 2 September 2014 Published 14 October 2014

Academic Editor Giles E Hardingham

Copyright copy 2014 Taichi Kashiwagi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Aging-related neurodegenerative disorders are closely associated with mitochondrial dysfunction and oxidative stresses and theirincidence tends to increase with aging Brain is the most vulnerable to reactive species generated by a higher rate of oxygenconsumption and glucose utilization compared to other organs Electrochemically reduced water (ERW) was demonstrated toscavenge reactive oxygen species (ROS) in several cell types In the present study the protective effect of ERW against hydrogenperoxide (H

2O2) and nitric oxide (NO) was investigated in several rodent neuronal cell lines and primary cells ERW was

found to significantly suppress H2O2(50ndash200120583M) induced PC12 and SFME cell deaths ERW scavenged intracellular ROS and

exhibited a protective effect against neuronal network damage caused by 200120583M H2O2in N1E-115 cells ERW significantly

suppressed NO-induced cytotoxicity in PC12 cells despite the fact that it did not have the ability to scavenge intracellular NOERW significantly suppressed both glutamate induced Ca2+ influx and the resulting cytotoxicity in primary cells These resultscollectively demonstrated for the first time that ERW protects several types of neuronal cells by scavenging ROS because of thepresence of hydrogen and platinum nanoparticles dissolved in ERW

1 Introduction

Living organisms inevitably live with the process of agingwhich has been closely linked as a critical causative factorfor the development of neurodegenerative disorders (NDs)such as Alzheimerrsquos disease (AD) Parkinsonrsquos disease (PD)Huntingtonrsquos disease (HD) and Amyotrophic lateral scle-rosis (ALS) [1ndash3] Moreover the development of aging-related NDs has been shown to be closely associated withmitochondrial dysfunction and oxidative stresses [1 4 5]Oxidative stress is a state where the cell is overwhelmed withreactive oxygen species (ROS) and reactive nitrogen species(RNS) resulting from an imbalance between reactive speciesproduction and detoxification [3 6] Aerobic organismsabsolutely require oxygen for the maintenance of normalphysiological functions In particular the brain is responsible

for more than 20 of total body oxygen consumption and25 of total body glucose utilization although it representsless than 2 of the whole body weight [7] Such high oxygenconsumption in the brain consequently produces a largeamount of reactive species compared to other organs inthe body [6] ROS include a variety of diverse chemicalspecies such as superoxide anion (O

2

∙minus) hydroxyl radical(∙OH) and hydrogen peroxide (H

2O2) all of which are

capable of causing extensive oxidative damage to biologicalmacromolecules like DNA RNA proteins and lipids [8ndash10] In addition RNS such as nitric oxide (NO an unstablefree radical) is induced by glutamate a neurotransmitterabundant in the central nervous system (CNS) and essentialfor proper neurological processes However NO in excessleads to pathological complications such as NDs Accumu-lated data have shown that glutamate induces Ca2+ influx by

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 869121 18 pageshttpdxdoiorg1011552014869121

2 Oxidative Medicine and Cellular Longevity

way of the glutamate receptor channel N-methyl-D-aspartate(NMDA) The increased intracellular Ca2+ levels togetherwith endogenous calmodulin stimulate nitric oxide synthase(NOS) to produceNOwhich is converted to neurodestructiveNO radical (NO∙) depending on the redox conditions inthe neuronal cells NO∙ further reacts with O

2

∙minus generatinga potent oxidant ONOOminus Thereafter ONOOminus is rapidlyconverted to highly reactive ∙OH which is cytotoxic [2 10ndash12] Moreover the damage is exasperated by the fact that theCNS contains a high amount of polyunsaturated fatty acidswhich are readily peroxidized by toxic reactive species [10]and has relatively low levels of antioxidative enzymes [8 10]Therefore such inherent characteristics of the CNS and theROS and RNS individually or in combination contributeto the development of NDs Antioxidants metal chelatorsor other agents that improve endogenous enzymatic andnonenzymatic cellular defense systems have been suggestedas treatments for NDs [6] Control of ROS production bymitochondrially targeted antioxidant treatment is expectedto delay aging [1] Antioxidant therapies using plant extractsfrom seeds and leaves are reported to be beneficial [13]while some other prospective substances have been testedwith unsatisfactory results [2 14] For antioxidant therapiesto be effective they must cross the blood brain barrier(BBB) which protects the brain from toxins and reduces thebioavailability of exogenous antioxidants in the brain [2 6]Therefore it is highly desirable to develop alternative agentsthat are able to pass through the BBB and exert antioxidativeeffects in the brain

One promising candidate is electrochemically reducedwater (ERW) because it has the potential to cross the BBBERW produced near the cathode during electrolysis of 2mMNaOH model water has characteristics of high pH lowdissolved oxygen and an extremely negative redox poten-tial [15] ERW contains a high concentration of dissolvedhydrogen (04ndash09 ppm) and a small amount of platinumnanoparticles (Pt nps 01ndash25 ppb) both of which exhibitROS-scavenging ability [11 16 17] Neutralized ERW hasbeen shown to exhibit H

2O2-scavenging activity which is

correlated with protection of DNA [18 19] and carbontetrachloride induced liver damage [20] lifespan extensionof Caenorhabditis elegans [16 21] alloxan-induced type 1 dia-betes [15 19] hemodialysis-induced oxidative stress duringend-stage renal disease [22 23] and inhibitory effects ofHT1080 tumor cell invasion [24] ERW in combination withglutathione induced human leukemia HL-60 apoptotic celldeath whereas a cytotoxic effect was not observed in normalperipheral blood mononuclear cells [25] Despite the variousprotective functions exhibited by ERW its effect on neuronalcells has not been disclosed in the literature other than ina brief meeting abstract by Yan et al [26] who reported theprotective effect of ERW on H

2O2-induced cultured N1E-115

neuroblastoma cell deathCultures of nervous system tissues and cells are catego-

rized in the terms of their complexities whole-embryo wholebrain organotypic slices reaggregate cultures dissociatedprimary cell cultures and cell lines [27] The degree ofcomplexity of an in vitro model of dissociated primary cell

cultures is considered to more closely reflect the in vivo statethan that of the cell lines [27] In light of this view wealso usedmouse cerebral cortex neuronal primary (MCCNP)cells as a model to observe the effect of ERW in addition toimmortalized cell lines N1E-115 cells have been establishedas an adrenergic clone derived from mouse neuroblastomaC-300 [28] and are used as a model of CNS neurons[29ndash32] In addition in culture in the presence of severalfactors including DMSO these cells display morphologicalcharacteristics of neuritogenesis which we used as a markerfor changes upon treatment with ERW [33]The PC12 cell linewas established from a transplantable rat adrenal pheochro-mocytoma based on its response to nerve growth factor(NGF) PC12 cells possess the potential to be differentiatedinto either chromaffin cells or sympathetic neurons whenin the presence of NGF [34] This cell line has been usedas a model for studying the neuronal response to oxidativestress [35ndash37] Also the viability of PC12 cells is describedto be sensitive to NO stress thus this makes them useful fordetecting a subtle NO effect [38] Serum-free mouse embryo(SFME) cells were established from mouse embryo cells bymaintenance in the absence of serum [39] These cells showthe characteristics of an astrocyte a progenitor cell withoutsenescence which is the most abundant cell type in the CNS[39 40]

In the present study we utilized various cell types orig-inating from mouse and rat as a first step to explore theprotective effect of ERW on neurocytotoxicity caused byreactive species

2 Materials and Methods

21 Materials Dulbeccorsquos Modified Eaglersquos Medium(DMEM) and DMEMHamrsquos F12 Combined Medium(1 1) were purchased from Nissui Pharmaceutical Co LTD(Tokyo Japan) Insulin putrescine transferrin propidiumiodide (PI) Fluo-3AM pluronic F127 sodium glutamateand Ca2+ Mg2+-free Hankrsquos balanced salt solution (Ca2+Mg2+-free HBSS) were purchased from Sigma-Aldrich Japan(Tokyo Japan) 21015840 71015840-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Invitrogen Technologies (CarlsbadCA USA) Chemically defined lipid (CDL) and mouseepidermal growth factor (mEGF) were purchased fromLife Technologies Japan (Tokyo Japan) Cell counting kit-8(CCK-8) which uses WST-8 as a color indicator to measurelive cell numbers was purchased from Dojindo LaboratoriesCo (Tokyo Japan) and the kit is referred to as the WST-8kit hereafter Diaminorhodamine-4M acetoxymethyl ester(DAR-4M AM) was from Daiichi Pure Chemicals Co LTD(Tokyo Japan) N-Acetyl-L-cysteine (L-NAC) ascorbic acid(AsA) sodium nitroprusside (SNP) 4-[2-hydroxyethyl]-1-piperazineethane-sulfonic acid (HEPES) fetal bovineserum (FBS) bovine serum albumin (BSA) penicillinstreptomycin progesterone and all other chemicals wereobtained fromWako Pure Chemical Industries LTD (OsakaJapan) The gelatin sepharose 4B column was obtainedfrom GE Healthcare Japan (Tokyo Japan) Ultrapure water

Oxidative Medicine and Cellular Longevity 3

(MQ-water) was produced by a Millipore filtration system(Billerica MA USA)

22 Preparation of ERW ERWwas prepared by electrolysis ofMQ-water containing 2mMNaOH at 100V for 60min usinga TI-200 electrolysis device (Nihon Trim Co Osaka Japan)The device is a batch-type system composed of a 4-literelectrolysis vessel which is divided into two compartmentsby a semipermeable membrane Each compartment containsa platinum-coated titanium (Ti) electrode Further details ofthe device and the characteristics of ERW including pH dis-solved hydrogenoxygen and Pt nanoparticle concentrationsand redox potential are given in our previous reports [15 1641] ERW was neutralized with HEPES buffer or bicarbonatebuffer in medium before use NaOH solution was adjusted tothe same pH with that of freshly prepared ERW and used ascontrol water

23 Preparation of Cell Culture Medium 5x DMEMHamrsquosF12 medium (1 1 mixture of 5x DMEM and 5x Hamrsquos F12medium no FBS) was diluted by quadruple neutralized ERWor MQ-water as the control to make the control mediumNormal DMEMF12 medium supplemented with differentconcentrations of L-NAC or AsA was used as positivecontrols in this study

24 Cell Cultures Murine neuroblastoma N1E-115 cells werepurchased from ATCC (VA USA) and maintained in 90DMEMHamrsquos F12 medium supplemented with 10 FBS2mM glutamine 100UmL penicillin and 100 120583gmL strep-tomycin and incubated in a humidified atmosphere of 5CO2at 37∘C For differentiation a previously published

method was followed with modifications [31] N1E-115 cellswere cultured in 88 DMEMHamrsquos F12 medium containing10 FBS 2 dimethyl sulfoxide (DMSO) in poly-D-lysine(PDL)-coated dishes for 6 days and then the mediumwas replaced with serum-free DMEMF12 medium supple-mented with 5 120583gmL insulin 100 120583M putrescine 20 nMprogesterone and 5 120583gmL transferrinRat pheochromocy-toma PC12 cells were purchased from RIKEN Cell Bank(Tsukuba Japan) Cells were cultured in DMEM containing5horse serum (HS) 5 fetal bovine serum (FBS) 100UmLpenicillin and 100 120583gmL streptomycin and incubated in ahumidified atmosphere of 5 CO

2at 37∘C Medium was

changed every 2 days Differentiation into neuronal cells wascarried out by culturing in PDL-coated dishes containingDMEM supplemented with 05 HS 05 FBS and 20 nMnerve growth factor (NGF) for 4 days in a humidified atmo-sphere of 5CO

2at 37∘C Serum-freemouse embryo (SFME)

cells were kindly provided by Loo et al [39] and cultured ina 1 1 mixture of DMEM Hamrsquos F12 medium containing 10nM sodium selenite 12 gL sodium bicarbonate 10 120583gmLtransferrin 1 of CDL and 50 ngmL mEGF in fibronectin-coated 25 cm2 T-flasks Fibronectin was purified from BSAusing a gelatin sepharose 4B column by the publishedmethod [42]

25 Preparation of Mouse Cerebral Cortex Neuronal Primary(MCCNP) Cells MCCNP cells were prepared from the cere-bral cortex of fetal mouse (15-16 days gestation) that has beenremoved aseptically from an anesthetized and decapitatedmouse (ddy strain Asekku-Yoshitomi Co Fukuoka Japan)The embryos were removed under sterile conditions andplaced into an ice-cold 1 1 mixture of phosphate-bufferedsaline (PBS) and DMEM Cerebral cortex excised from thebrain was minced and incubated for 15min in a mixture of005 trypsin and PBS Single cells dissociated from the cere-bral cortex were counted adjusted to 05ndash10 times 105 cellsmLwith DMEM supplemented with 5 HS 5 FBS 100UmLpenicillin and 100 120583gmL streptomycin and seeded on PDL-coated 24-well plastic plates Cells were incubated for 2days in a humidified atmosphere of 5 CO

2at 37∘C

After the incubation 10120583M cytosine 120573-D-arabinofuranosidehydrochloride (Ara-C) a deoxyribonucleic acid synthesisinhibitor was added and the cells were incubated for further24 h to eliminate rapidly growing nonneuronal cells [43 44]Ara-C containingmediumwas replaced with DMEM growthmedium as described above and the cells were incubated for7 to 10 days prior to subsequent experiments

26 AnimalHandling Micewere handledwith care followingthe guidelines for Animal Experiments provided by theFaculty of Agriculture and the Graduate School of KyushuUniversity and the Law (No 105) and Notification (No 6) ofthe Government

27 Cell Viability Assay A WST-8 kit was used to obtaina viable cell count (Dojindo Co Tokyo Japan) The assayprotocol provided by the manufacturer was closely followedBriefly N1E115 cells (104 cellswell) were seeded in a PDL-coated 24-well plate and pr-incubated with FBSDMEMmedium Following cell differentiation with DMSO cellswere treated with serum-free medium made of MQ-waterERW L-NAC AsA or 2mM NaOH supplemented with orwithout different concentrations of H

2O2for 24 h To each

well 1 120583L of WST-8 dye was added and incubation was con-tinued for further 4 h Viable cells were measured at 450 nmusing a microtiter plate reader (Lancraft Inc Norcross GAUSA) PC12 cells (104 cellswell) were seeded in PDL-coated24-well plates and preincubated in FBSHSDMEMmediumDifferentiation was accomplished by adding NGF followedby a 4 day incubation (detail is given in the cell culturesection) When cells were differentiated they were treatedwith serum-free medium containing ERW with or withoutdifferent concentrations of H

2O2or SNP for 24 h Viable cells

weremeasured as described above SFME cells (105 cellswell)were seeded in 24-well plates and incubated in the presenceof FBSHSDMEM medium for 1 day Cells were treatedwith serum-free medium containing ERW with or withoutdifferent concentrations of H

2O2for 24 h (detail is given

in the cell culture section) Viable cells were measured asdescribed above

28 Intracellular H2O2Detection Using a DCFH-DA Probe

21015840 71015840-Dichlorofluorescin diacetate (DCFH-DA) can diffuse


into cells where it is hydrolyzed by intracellular esterasesresulting in the formation of DCFHThen DCFH is oxidizedby intracellular oxidizing species including H

2O2to form the

highly fluorescent product 21015840 71015840-dichlorofluorescin (DCF)which can be detected by appropriate devices such as a flowcytometer andor confocal laser microscope DCFH-DA hasbeen shown to be useful to estimate intracellular H

2O2in rat

neuron cells [45]

29 Flow Cytometric Analysis of Intracellular H2O2 N1E-115

cells (75 times 104 cells) were seeded in an uncoated 10 cm dishand precultured for 1 day Then the medium was replacedwithHBSS (137mMNaCl 5mMKCl 1mMNa

2HPO4 5mM

glucose 1mM CaCl2 05mM MgCl

2 1 mgmL BSA and

10mM HEPES pH 74) buffer containing ERW L-NAC (0110mM) AsA (01 10mM) or control MQ-water and wasincubated for 10min After removing the supernatant 2mLof 5 120583M DCFH-DA in Ca2+ Mg2+-free HBSS adjusted pHto 74 with 10mM HEPES buffer was added to the dishand incubated for further 15min Cells were then harvestedin 5mL of PBS buffer and collected by centrifugation at500timesg for 3min and then resuspended in 1mL of PBSFluorescence intensities weremeasured immediately using anEPICS XL System II-JK flow cytometer (Beckman CoulterMiami USA) with excitation and emission wavelengths of495 and 525 nm respectively Histograms were analyzed byusing FlowJo software supplied with the cytometer

210 Confocal Laser Microscopic Analysis of IntracellularH2O2 The intracellular H

2O2was visualized using the

DCFH-DA probe One milliliter of N1E115 cells (2 times104 cellsmL) was precultured in a 35mmdish for 1 dayThenthe medium was replaced with HBSS made with ERW orcontrol MQ-water with or without 200120583M of H

2O2 and

incubated for 10min After removing the supernatant 2mLof 5 120583MDCFH-DA in Ca2+ Mg2+-free HBSS adjusted to pH74 with 10mM HEPES buffer was added and the dish wasincubated for further 10min Cells were then washed withHBSS to remove unreacted DCFH-DA and the cytoplasmicfluorescence intensities were measured using a confocal laserscanning microscope (Molecular Dynamics Sunnyvale CAUSA) with a FITC barrier filter at excitation and emissionwavelengths of 488 and 530 nm respectively

211 Assessment of Nitric Oxide (NO) Toxicity Using a CellViability Assay Differentiated PC12 cells were cultured ina medium made of ERW or MQ-water each containing 0200 400 or 600120583M sodium nitroprusside (SNP) for 24 hThe viability of the cells was measured using the WST-8 kitmethod

212 Detection of Intracellular NO Intracellular NO in thecells can be detected using a rhodamine chromophore theDAR-4M AM NO bioimaging probe DAR-4M AM diffusesinto cells and is hydrolyzed by intracellular esterases resultingin the formation of membrane impermeable DAR-4M DAR-4M then reacts with NO in the presence of oxygen forminga fluorescent triazol compound DAR-4M T which can

be visualized using a fluorescence microscope at 560 nmexcitation and 575 nm emission [46] For PC12 cells 2 times105 cells were seeded in a 35mm dish and incubated for1 day Cells were then treated with the medium made ofERW or MQ-water each containing 200 120583M SNP for 20minFollowing the incubation period cells were washed once withHBSS and 10 120583M DAR-4M AM in HBSS was added and thecells were incubated for 30min The cells were washed oncewith HBSS and intracellular fluorescence was observed usinga fluorescence microscope (IX70 Olympus Japan) and wasphotographed

213 Measurement of Intracellular Ca2+ Intracellular Ca2+was measured using Fluo-3 AM a membrane-permeablecalcium sensitive dye Internalized Fluo-3 AM is hydrolyzedby intracellular esterases liberating Flou-3 which can reactwith intracellular free Ca2+ ions to form a fluorescent com-plex After pretreatment with glutamate (0 01 or 10mM)in medium made of ERW or MQ-water for 10 min PMNcells were stained with 300 120583L of dye mixture composed of4 120583M Fluo-3 AM and 08mgmL pluronic F127 in HBSS pH74 for 20min and then 1mL of 1 FBS containing HBSSwas added followed by a further 40min incubation [47]Cells were washed once with HBSS and suspended in 1mLHEPES buffer A fluorescence microscope was used to detectintracellular fluorescence and the images were recorded by adigital camera Digital images were converted to a numericalreadout with the aid of a NIH image analysis program andthen were analyzed by Excel software

214 Measurement of Glutamate Neurotoxicity Glutamatetoxicity was measured using PMN cells After cells weretreated with 0 01 and 05mM glutamate in the mediummade of ERW or MQ-water for 24 h nonviable cells weredetected using trypan blue dye which does not enter livecells because of their intact cell membranes Treated cellswere stained with 01 trypan blue for 10min fixed with4 paraformaldehyde for 15min at 4∘C and rinsed oncewith PBS The nonviable cells stained blue by the dye werecounted in 8 randomly selected fields as one set and 4 setsfor each treatment were counted under themicroscope (CK2Olympus Japan)

215 Statistical Analysis Three to four independent exper-iments were performed Data are presented as the mean plusmnstandard deviation (SD) Statistical significance was deter-mined by repeatedmeasures analysis of variancewith post hoccomparisons using the Tukey test and a 119875 value of lt 005 wasconsidered significant

3 Results

31 ERW Protects Neural Cells From H2O2-Induced Cell

Death ERW has been reported to suppress H2O2-induced

oxidative stress in neuroblastoma N1E115 cells [26] Wefirst examined whether H

2O2-induced oxidative stress was

suppressed by ERW in two other neuronal cell lines PC-12and SFME Because PC12 and SFME cells showed different


sensitivity to H2O2in terms of cell viability different H

2O2

concentrations were tested in the present studies (data notshown) Differentiated PC12 cells were treated for 24 h with0 100 200 or 500 120583M H

2O2with or without ERW ERW

significantly suppressed cell death when treated with 100 or200120583M H

2O2(Figure 1(a) lowast119875 lt 005) but not with 500120583M

H2O2 SFME cells were treated for 24 h with 0 50 100

or 200120583M H2O2with or without ERW ERW significantly

suppressed cell death when treated with 50 or 100 120583M H2O2

(Figure 1(b) lowast119875 lt 005) but not with 200120583M H2O2 When

these cell lines were treated with ERW alone (0 120583M H2O2)

the number of viable cells did not increase compared to thatof the controls (Figures 1(a) and 1(b)) Therefore the resultsindicate that ERW suppresses neuronal cell death caused byH2O2-induced oxidative damage

32 ERWandAntioxidants Protect Neuronal Cells fromH2O2-

Induced Cell Death We decided to further confirm ERWefficacy by using the known antioxidants L-NAC and AsAas system controls The cell viability for the absolute controlwas set as 100 (Figure 2) The cell viability for the positivecontrol was reduced to 60 compared to the absolute control(Figure 2) When the N1E-115 cells were cultured with themedium made of ERW containing 200120583M H

2O2 the cell

viability was significantly increased compared to the positivecontrol (Figure 2 lowast119875 lt 005) in agreement with theprevious result [26] We also found that autoclaved ERWwas not protective against 200120583M H

2O2induced cell death

(Figure 2) When N1E-115 cells were cultured with the posi-tive control medium supplemented with the known antiox-idants 1mM L-NAC or 1mM AsA the viability increasedsignificantly compared with the positive control respectively(Figure 2 lowast119875 lt 005 lowastlowast119875 lt 001) and indicated theassay system was working properly Cell viability was notaffectedwhen the cells were culturedwith the positive controlmedium supplemented with 01mM L-NAC or AsA or 2mMNaOH (Figure 2) From the results ERW was confirmed toexert protective effects against H


33 ERWMaintains Differentiated Morphology against H2O2-

Induced Oxidative Stress We next tested whether ERWaffects N1E-115 cell morphology Undifferentiated cells exhib-ited a typical round shaped morphology (Figure 3(a)) whiledifferentiated cells showed a neuronal network representingneurite outgrowth (Figures 3(b) and 3(d)) These cellularmorphologies are quite similar to those of published data [33]When the differentiated cells were cultured in the mediummade of MQ-water containing 200120583M H

2O2 the neuronal

network disappeared (Figure 3(c)) while the cells culturedwith the medium made of ERW containing 200 120583M H

2O2

maintained the original state of differentiated morphol-ogy displaying a neuronal network and neurite outgrowth(Figure 3(e))These results demonstrated that ERW is capableof maintaining the morphological integrity of differentiatedneuronal cells against H

2O2-induced oxidative stress

34 ERW Scavenges Intracellular ROS The results obtainedthus far demonstrated the substantial protective effects of

ERW on N1E-115 cells against H2O2-induced cell death We

examined whether ERW is protecting cell death by scav-enging intrinsic intracellular ROS The intracellular H

2O2

level of the N1E-115 cells treated for 10min with ERW (meanvalue = 560) was substantially decreased compared with theMQ-water control (mean value = 1050) (Figure 4(a)) Theintracellular H

2O2level of the cells treated for 24 h with

the ERW (mean value = 617) decreased to a lesser degreefrom theMQ-water control (mean value = 779) (Figure 4(b))When the N1E-115 cells were treated for 10 min with themedium containing 1mM L-NAC or AsA the leftward shiftindicative of the intracellular H

2O2scavenging was small

but clearly recognizable (Figures 4(c) and 4(e)) While theintracellular H

2O2level of 1mM L-NAC treated cells for 24

h was substantially reduced compared to MQ-water control(Figure 4(d)) the scavenging effect of 1mMAsA was evidentbut to a lesser extent (Figure 4(f)) Although the sameexperiment was performed with 01mM of L-NAC and AsAthis concentration did not show an appreciable scavengingeffect after either 10min or 24 h treatment (data not shown)From these results ERWwas shown to scavenge intracellularH2O2within a short time while the scavenging capabilities

of AsA in particular L-NAC are exerted for at least 24 hoursTo confirm the flow cytometric data we further tested the

scavenging ability of ERWagainst induced intracellularH2O2

by using a combination of a DCFH-DA probe and a confocalmicroscope Fluorescence intensities reflecting intracellularH2O2levels obtained from the cells treated for 10min with

MQ-water alone (control) were set to 100 (Figures 5(e) and5(a)) When cells were treated for 10min with ERW alonethe fluorescence intensity was decreased approximately 30compared with that of the MQ-water control (Figures 5(e)and 5(b) lowast119875 lt 005) When the cells were cultured withthe medium made of MQ-water containing 100 120583M H

2O2

the intracellular H2O2level was increased by approximately

20 compared with that of the MQ-water control (Figures5(e) and 5(c))When the cells were cultured with themediummade of ERW containing 100 120583M H

2O2 the intracellu-

lar H2O2level was suppressed approximately 20 com-

pared with that of the MQ-water containing 100 120583M H2O2

(Figures 5(e) and 5(d) lowast119875 lt 005) Representative images of3 independent experiments are shown in Figures 5(a)ndash5(d)These images were used to generate Figure 5(e) by measuringthe fluorescence intensities of each treatment The sameconclusion ERW scavenges physiologically intrinsic H

2O2

and exogenously induced H2O2 was reached from two

independent detection methods

35 Suppressive Effect of ERW on Nitric Oxide (NO) Toxic-ity in PC12 Cells In the MQ-water control medium SNPreduced PC12 cell viability in a manner approximatingdose-dependency (Figure 6(a)) However when cultured inthe medium made of ERW the cell viability was restoredsignificantly compared with the respective controls at 200and 400 120583M SNP (Figure 6(a) lowast119875 lt 005) Thus ERWcould restore cell viability against NO generated by up to400 120583M SNP However this finding does not prove that ERWis directly scavenging NO To clarify this point we assessed


5002001000

Cel

l via

bilit

y (

)PC12 cell

0

20

40

60

80

100

120

ControlERW

lowastlowast

lowast

H2O2 (120583M)

(a)

SFME cell

lowastlowast

lowast

200100500

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

ControlERW

H2O2 (120583M)

(b)

Figure 1 Suppressive effect of ERW against H2O2-induced cytotoxicity on PC12 and SFME cells Viable cell numbers were measured using a

WST-8 kit (a) Pheochromocytoma PC12 cells were cultured in FBSHSDMEMmedium with or without ERW and the number of survivingcellswere set as controlsH

2O2(100 200 and 500 120583M)was added to the controlmediumand itwas cultured for 24 hViable cellsweremeasured

at 450 nm using a microtiter plate reader (b) Serum-free mouse embryo (SFME) cells were cultured in a 1 1 mixture of DMEM Hamrsquos F12medium with or without ERW and the number of surviving cells were set as controls To the control medium H

2O2(50 100 and 200 120583M)

was added and it was cultured for 24 h Viable cells were measured at 450 nm using a microtiter plate reader Data are presented as mean plusmnstandard deviation (SD) for three independent experiments (119899 = 3 each set) lowast119875 lt 005 and lowastlowast119875 lt 001

the intracellular NO level directly using the NO-specificDAR-4M AM probe Differentiated PC12 cells were treatedwith themediummade of ERWorMQ-water with or without200120583M SNP After the SNP treatments NO-induced DAR-4M intracellular fluorescence was observed by a fluorescencemicroscope and photographed (Figure 6(b)) Intracellularfluorescence was barely visible in the cells cultured with themedium made of either MQ-water or ERW (Figure 6(b) leftside panels designated as MQ and ERW) Intracellular redfluorescence indicative of SNP induced NO was evident inthe cells cultured with the medium made of MQ-water andERW (Figure 6(b) right side panels designated as MQ +200120583M SNP and ERW + 200120583M SNP) Using two of theright side panels the fluorescence intensities were scored for50 cells randomly picked in each panel Analysis revealed nostatistically significant differences between MQ- and ERW-treated cells (Figure 6(c))Thus ERWwas found to lack directNO scavenging activity in PC12 cells

36 Suppressive Effect of ERW on Glutamate-Induced Ca2+Influx and Cell Death We used MCCNP cells to examinewhether ERW can affect Ca2+ influx As shown in Figure 7the MQ-water control (red line) treated cells showed a newCa2+ peak as indicated by a downward arrow in the 01 and1mM glutamate treated cells compared with the 0mM gluta-mate treated cells ERW (black line) in the presence of 01mMglutamate treated cells did not show such a peak indicativeof Ca2+ influx Even though the 1mM glutamate treated

cells cultured in the presence of ERW showed a peak thecalcium concentration [Ca2+]

119894is less than that of the control

The results indicate that ERW suppresses glutamate inducedCa2+ influx in the MCCNPcells which suggests a reduc-tion of cell death This possibility was further tested usingthe trypan blue exclusion assay As shown in Figure 8(b)dead cells treated with the medium made of the MQ-water control with 01 and 05mM glutamate increased in adose-dependent manner compared to those grown withoutglutamate Similarly a dose-dependent increase in deadcells was observed when the cells were treated with themediummade of ERWcontaining 0 01 or 05mMglutamateHowever the number of dead cells grown in themediumwithERW containing 01 or 05mM glutamate was significantlyreduced compared with those grown in the presence of therespective MQ-water controls (Figure 8(b) lowastlowast119875 lt 001)The representative photographs are shown in Figure 8(a)Theresults indicate that ERW suppresses glutamate-induced celldeath which correlated well with the suppression of Ca2+influx

4 Discussion

In the present study ERW was confirmed to suppress H2O2-

induced cell death in N1E-115 PC12 and SFME cell linesFurther studies with N1E-115 cells demonstrated that thesuppressive effect of ERW stemmed from its ROS scavengingabilityThis finding is in accordance with the previous reports


N1E115 cell

+ + + + + + + +

L-NAC AsA

Abs

cont

Pos

cont

ERW

Auto

ERW

Cel

l via

bilit

y (

)

0

20

40

60

80

120

100 lowastlowast

lowast

lowast

200120583M H2O2

01

mM

10

mM

NaO

H20

mM

01

mM

10

mM

minus

Figure 2 Protective effect of ERWagainst H2O2-induced cytotoxic-

ity onN1E115 cells N1E115 cells were preincubated with FBSDMEMmedium and were treated with serum-free medium made of MQ-water ERW L-NAC (01 1mM) AsA (01 1mM) or 2mM NaOHsupplemented with or without 200120583M H

2O2for 24 h A WST-

8 kit was used to measure viable cell numbers at 450 nm usinga microtiter plate reader Data are expressed as mean plusmn standarddeviation (SD) for three independent experiments lowast119875 lt 005 lowastlowast119875 lt001 Abs cont absolute control Pos cont positive control AutoERW autoclaved ERW

obtained from pancreatic 120573-cell HIT-T15 cells [15 19] humanlung carcinoma A549 cells [41] HT1080 tumor cells [24] anddiabetic dbdbmice [48]

The current study further revealed that ERW significantlysuppresses cell death caused by glutamate- or SNP-inducedstresses although it does not have direct NO scavengingactivity We pursued further experiments to unravel themechanism underlying this phenomenon Glutamate toxic-ity is mainly mediated by the glutamate receptor NMDAinvolving Ca2+ influx into cerebral cortex cells in the CNS[49] Internalized Ca2+ and endogenous calmodulin togetheractivate neuronal NO synthase for the production of NOImportantly neuroprotective NO is converted to neurode-structive NO radical (NO∙) depending on the redox condi-tion in the neuronal cells [11] Following NO∙ productionit reacts with O

2

∙minus to form ONOOminus which decomposes tonitrite and highly reactive HO∙ and the reactions proceed togenerate stable nitritenitrate species Such radicals liberatedduring the NO-cascade contribute to neurocytotoxicity [210ndash12 50] Based on the information for Ca2+ influx inducedby glutamate in cerebral cortex cells [49] and the NO-cascadeas discussed above we used MCCNP cells to assess whetherERW scavenges NO Firstly we found that ERW clearlysuppressed Ca2+ influx inMCCNP cells and ERW suppressedextracellular H

2O2induced Ca2+ influx in PC12 cells (data

not shown) Also a previous study demonstrated that ERWprevents alloxan-induced elevation of intracellular Ca2+ lev-els in HIT-T15 120573-cells [19] Moreover exposure of rat mixed

cortical neuron primary cells to H2O2resulted in increases in

intracellular Ca2+ levels which are suggested to be involved inH2O2-induced cell death [51] Subsequently H

2O2-induced

neuronal cell death was shown to be closely associated withNMDA mediated Ca2+ influx [52] The present finding ofthe suppressive effect of ERW is intriguing because increasedCa2+ influx triggers calpain (a protease) activation leading tothe cleavage of a neuron specific p35 activator protein to p25which binds to cyclin dependent kinase 5 (Cdk5) forming ahyperactive Cdk5p25 complex This deregulated Cdk5p25activity has been linked to be a causative factor of variousneurodegenerative diseases [6] Secondly we used the trypanblue exclusion assay to evaluate the protective function ofERWon the cytotoxic effect induced by glutamate inMCCNPcells ERW significantly suppressed glutamate induced celldeath which is closely correlated with reduced Ca2+ influxFinally we performed experiments using SNP and PC12 cellsin combination with the NO-specific DAR-4M AM probe todetermine whether ERW scavenges NO directly We utilizedPC12 cells because this cell line has been described to besensitive to NO [38] To our surprise ERW was not found toscavenge NO nevertheless the cell viability was significantlyincreased In addition in the presence of ERW we did notobserve the reduction of intracellular fluorescence intensitiesat SNP concentrations ranging from 50 to 500 120583M (data notshown) SNP has been used as a NO-donor [30 49 50]and its metabolites have been suggested to be involved inredox cyclingwith oxygen to formO

2

∙minus which then producesH2O2 which in turn produces HO∙ via the Fenton reaction

[30] Therefore the presently observed suppressive effect ofERW on SNP induced PC12 cell death is mostly caused by itsROS scavenging ability and it is less likely that it can scavengeNOdirectlyThe proposed points of action of ERWare shownin Figure 9

The question remains as to what factors are responsiblefor the presently observed scavenging activity of ERWagainstROS produced by glutamate SNP and H

2O2 ERW has

been shown to contain dissolved hydrogen (04ndash09 ppm) andPt nanoparticles (nps) (01ndash25 ppb) as potential ROS scav-enging substances [15ndash17] Recently we estimated the levelsof dissolved hydrogen to be 076 ppm in freshly preparedERW with a half-life of approximately 100min [24] Directinvolvement of hydrogen molecules as a ROS scavenger wasreported using rat myotube L6 cells [53] Also hydrogenmolecules were shown to enhance the expression of genes forantioxidant enzymes such as catalase glutathione peroxidaseand heme oxygenase [53] In another report ERWwas shownto restore the activities of superoxide dismutase catalase andglutathione peroxidase inmice supporting the idea that ERWacts as an antioxidant and an ROS scavenger [25] Similarlyan antioxidative role of ERW was reported in vitro and invivo [54ndash56] ERW was also reported to possess reducingactivities because of the presence of dissolved molecularhydrogen [57 58] In vivo 008 ppm of hydrogen moleculeswere reported to be sufficient to exert antineurodegenerativeeffects in PDmodelmice [59] Also hydrogenmoleculeswereshown to reduce ∙OH ONOOminus and oxidative stress in therat brain induced by focal ischemia and injury [60] Recently


N1E115 cell

(a)

N1E115 cell

(b)

(c) (d)

(e)

Figure 3 Protective effect of ERW on morphological changes against H2O2-induced oxidative stress on N1E115 cells N1E115 cells were

preincubated with FBSDMEMmedium and treated with serum-free medium made of MQ-water as the control (a) differentiated cells withDMSO (b) differentiated cells treated with 200120583MH

2O2(c) differentiated cells treated with ERW alone (d) and differentiated cells treated

with ERW and 200 120583MH2O2(e) for 24 h Morphological changes were observed microscopically and photographed

molecular hydrogen dissolved in ERWderived from tapwaterwas reported to reduce LPS-induced neuroinflammation inmice [61] Based on these studies it is highly probablethat hydrogen molecules in ERW can reach and deliver aneuroprotective effect in the brain

Another potential factor which plays a role in the ROSscavenging activity of ERW is the Pt nps derived fromthe Pt-coated Ti electrodes during electrolysis Comparativemeasurements using an inductively coupled plasma massspectrometer (ICP-Ms) have shown that Pt nps and relatedcompounds are detected only in the post-electrolyzed 2mMNaOH solution that is ERW and not in the preelectrolyzedNaOH solution [15 17] Furthermore Pt nps in several ERWpreparations were measured to range from 01 to 25 ppbusing ICP-MS [15ndash17 21] The variable amount of Pt npsin different ERW preparations was thought to result from

the gradual erosion of Pt coated Ti-electrodes from repeatedelectrolysis [24] It was also necessary to confirm if Pt npscan scavenge ROS In this respect it has been shown thatsynthetic Pt nps can directly scavenge O

2

∙minus HO∙ and H2O2

and are considered to be a multifaceted ROS scavenger [1762] In agreement with our results synthetic Pt nps havebeen reported to show ROS scavenging activity [63ndash65]Moreover it is noteworthy to mention that the liberated Ptnps generates active hydrogen from the hydrogen moleculessimultaneously produced during electrolysis In turn theactive hydrogen is adsorbedabsorbed to nearby Pt nps whichmay synergistically contribute to the scavenging effect of Ptnps [62 66] We tested whether autoclaving will affect theantioxidative ability of ERW which resulted in the loss of theprotective effect against H

2O2-induced N1E-115 cell death

Similar results were reported in other cell lines and protecting


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

MQ mean = 1050ERW mean = 560

Max

()

10 min treatment

(a)

ERW mean = 617MQ mean = 779

01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)

L-NAC mean = 791MQ mean = 1050

01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)

AsA mean = 746MQ mean = 1050

01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)

Figure 4 Flow cytometric analysis of the intracellular H2O2scavenging ability of ERW on N1E115 cells N1E-115 cells (75 times 104 cells) were

treated with ERW L-NAC (10mM) AsA (10mM) or control MQ-water and incubated for 10min ((a) (c) and (e)) or 24 h ((b) (d) and(f)) After each treatment cells were harvested and resuspended in 1 ml PBS Fluorescence intensities were measured immediately using anEPICS XL System II-JK flow cytometer with excitation and emission wavelengths of 495 and 525 nm respectively Histograms were analyzedby using FlowJo software provided with the cytometer


(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)

Figure 5 H2O2-induced ROS scavenging ability of ERW on N1E115 cells Differentiated N1E115 cells were treated for 10 min with ERW

with or without 200 120583M H2O2and intracellular H

2O2levels were detected with a DCFH-DA probe and measured using a confocal laser

microscope Representative photographs of three independent experiments are shown (a) MQ control (b) ERW (c) MQ + 100120583M H2O2

and (d) ERW + 100 120583MH2O2 (e) Photographs were used to calculate intracellular fluorescence intensities reflecting H

2O2levels in the cells

for each treatment Data are expressed as mean plusmn standard deviation (SD) for three independent experiments lowast119875 lt 005 and lowastlowast119875 lt 001

factors have been suggested to be heat unstable or volatilesubstances contained in the ERW [19] It has been reportedthat synthetic Pt nps will agglomerate after autoclavingleading to inefficient cellular uptake and dissolved hydrogenmolecules and active hydrogen adsorbedabsorbed in thePt nps are thought to be forced out [24 62] Thereforeautoclaving of ERW provides further support that it containshydrogenmolecules andPt nps as active substances For ERWto be useful for in vivo therapeutic applications Pt nps mustbe able to reach brain cells Following ingestion Pt nps arerequired to survive several obstacles including intestinal cellwall penetration blood flow and the BBB before reachingthe brain cells Pt nps were shown to be taken up by cellsvia mainly pinocytosis [62 67 68] Another critical obstaclewhich will limit the usefulness of Pt nps is their ability tocross the BBB as has been suggested for other antioxidants[2 6] Several diseases including AD leads to reduced BBBintegrity [69] suggesting that Pt nps has the opportunity topass through the diseased BBB more easily than the healthy

BBB to gain access to the brain cells A recent article identifiesthe hurdles associated with synthetic metal nps delivery tothe brain [69] Therefore further in vivo studies are requiredto investigate Pt nps containing ERW as suggested for othermetal nps [62 69ndash71]

ROS have been regarded as toxic by-products of phys-iological metabolism and increased ROS generation andreduced mitochondrial membrane potential (ΔΨm) areclosely associated with the oxidative stress in PC12 cells [72]In the present study ERW was found to protect 3 neuronalcell lines from direct oxidative damage induced by H

2O2

implying that ERW protects mitochondria in neuronal cellsHowever further investigation using N1E-115 cells treatedwith ERWH

2O2(200120583M) medium did not show significant

restoration of ΔΨm compared with the MQH2O2(200120583M)

medium treated control (data not shown)There are multiple pathways that induce cell apopto-

sis Among these ROS mediated pathways involving mito-chondria are relevant to the results obtained in this study


PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)

Figure 6 Suppressive effect of ERW on SNP-induced NO toxicity in PC12 cells Cells were cultured for 24 h with the medium containingERW orMQ-water each supplemented with 0 200 400 and 600120583M SNP (a) Cell viability was measured by aWST-8 kit Data are expressedas mean plusmn standard deviation (SD) for three independent experiments lowast119875 lt 005 (b) ERW was assessed for its intracellular NO scavengingability using theDAR-4MAMbioimaging probe Representative photographs of NOdependent fluorescence levels incubated withMQ ERWand 200 120583M SNP added-MQ and ERW are shown (c) Fluorescence intensities of 50 cells treated with MQ or ERW containing 200 120583M SNPwere measured and Tukeyrsquos multiple comparison method was used to compare the effect of MQ and ERW

ROS induced oxidative stress causes oxidative damage tovital cellular constituents which eventually affect cell via-bility ROS accumulation in cells mediates apoptosis by wayof mitochondria-dependent and mitochondria-independentpathways Elevated ROS levels in the cell will disturb thephysiological balance maintained by the proapoptotic (forexample Bax Bak Bok Bid and Bim) and antiapoptotic(for example Bcl-2 Bcl-X

119871 and Bcl-w) proteins all of which

belong to the Bcl-2 family of proteins and generally act toinduce apoptosis or survival of the cell Apoptosis is induced

when proapoptotic Bcl-2 family proteins (Bax Bak BokBid and Bim) are activated by death signals via deathreceptors (tissue necrosis factor receptor-1 (TNFR1) Fasand TRAIL-R1) Tissue necrosis factor-120572 (TNF120572) increasesthe ROS level which in turn activates the apoptosis signalregulating the kinase 1-c-Jun NH

2terminal kinase (ASK1-

JNK) apoptotic pathway Exogenously and endogenouslyderived ROS are considered the most effective activators ofASK1 which acts as a redox sensor because it contains theredox-sensitive regulatory protein thioredoxin (Trx) binding


Intr

acel

lula

r fluo

resc

ence

ERWControl

Glutamate MCCNP cell

[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl

Figure 7 Suppressive effect of ERW on glutamate-induced Ca2+ influx in MCCNP cells Intracellular Ca2+ was measured with Fluo-3 AM amembrane-permeable calcium sensitive dye Internalized Fluo-3 AM is hydrolyzed by intracellular esterases liberating Flou-3 which can reactwith intracellular free Ca2+ ions forming a fluorescent complex After pretreatment with glutamate (0 01 or 10mM) in the medium madewith ERW or MQ-water for 15min MCCNP cells were stained with Fluo-3 AM for 20min and post-incubated with FBSHBSS medium Afluorescence microscope was used to detect intracellular fluorescence and the images were recorded by a digital camera Digital images wereconverted to numerical data with the aid of the NIH image analysis program and then analyzed by Excel software Representative histogramsof triplicate independent experiments are shown

domain ROS inactivates Trx by oxidizing two cysteineresidues in the redox center of Trx causing it to dissociatefrom ASK1 This allows ASK1 to be bound by the tumornecrosis factor receptor-associated factor 2 (TRAF2) andTRAF6 to form an active ASK1 signalosome that induces theautophosphorylation of ASK1 During the ASK1 activationprocess under oxidative stress the 14-3-3 proteins bindingsite at Ser-966 of ASK1 is dephosphorylated resulting in thedissociation of 14-3-3 proteins which are facilitated by theROS activated mammalian sterile 20 (Mst) family memberSOK-1 The ROS activated ASK1 eventually activates theJNK cascade and sustained JNK activity leads to apoptosisThus oxidative stress activates ASK1 which phosphorylatesthe MAPKK-JNK and -p38 pathways Activated JNK directlyphosphorylates p53 rendering it to translocate to the nucleusallowing up-regulation of proapoptotic genes such as thep53 upregulated modulator of apoptosis (PUMA) and BCL2-associated X protein (Bax) Alternatively the phosphorylatedJNK relocates to the nucleus and phosphorylates c-jun toform activator protein 1 (AP-1) resulting in the up-regulationof proapoptotic genes such as TNF120572 Fas-L and BakMoreover activated JNK moves to the nearby mitochondriaand binds to the complexes of proapoptotic Bax and tBidproduced from proapoptotic Bid by active caspase-8 andthen tBid with BaxBax and BakBak oligomers initiate pore

formation in the outer mitochondrial membrane (OMM)causing the release of cytochrome c Additionally otherfactors including AIF EndoG and CAD which relocate tothe nucleus for DNA fragmentation and the proapoptoticprotein second mitochondrial activator of the caspasedirectinhibitor of the apoptosis protein binding protein withlow pI (SmacDIABLO) and mammalian serine protease(HtrA2Omi) induce apoptosis via indirect activation ofcaspase-8 apoptotic pathways Released cytochrome c formsan apoptosome with Apaf-1 and procaspase-9 resulting in theformation of active caspase-9 which activates the executionercaspase-3 Separately caspase-3 gets activated by the activecaspase-8 Activated caspase-3 executes cell destruction bycleaving various vital constituents in the cells Caspases areconstitutive residents of the cytosol as inactive zymogenmonomers and are activated for example by endogenouslyand exogenously generated H

2O2through oxidation of the

cysteine catalytic sites Exposure to H2O2decreases the

mitochondrial transmembrane potential (ΔΨm) and oxidizesthe mitochondrial permeability proteins comprising volt-age dependent anion channel (VDAC) adenine nucleotidetranslocase (ANT) and cyclophilin D (CyD) leading tomito-chondrial Ca2+ influx Thus reduced ΔΨm elevated Ca2+levels and oxidative stress induce enlargement of the mito-chondrial permeability transition pore (MPTP) located at


Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400

0 01 05Glutamate (mM)

ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)

Figure 8 Protective effect of ERW against glutamate toxicity on MCCNP cells After MCCNP cells were treated with 0 01 and 05mMglutamate in the medium made of ERW or MQ-water for 24 h nonviable cells were stained with trypan blue dye Nonviable cells stainedblue were counted for 8 randomly selected fields as one set and 4 sets for each treatment were counted under the microscope Representativephotographs are presented in (a) from which stained cell counts were statistically analyzed as shown in (b) Data are expressed as mean plusmnstandard deviation (SD) for four independent experiments lowastlowast119875 lt 001


JNK

Nucleus

Cytochrome crelease

AP-1

Proapoptoticproteins

Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr

Figure 9 Schematic representation of possible points of action for the suppressive effects of ERW against stressors Glutamate induces Ca2+influx leading to increased neuronal cell death SNP increases neuronal cell death by inducing NO A reduction of neuronal cells eventuallyleads toNDs ERWsuppresses Ca2+ influx and scavenges ROS thereby protecting neuronal cell death Arrows indicate a single step ormultipleintermediary steps responsible for upregulating the proapoptotic genes triggered by ROS RNS and Ca2+ HO-1 and TrxRTrx which arecontrolled by vitagenes act as homeostasis keepers within a tolerable stress zone and are included in blue font indicates inactive state lowastindicates activated state (uarr) is increased intracellular Ca2+ and RNS levels Symbol (⊢) with red color represents prospective points of actionof ERW

the inner-outer membrane contact site to release cytochromec Elevated ROS will damage nuclear DNA directly andin particular H

2O2stimulates the nuclear accumulation

of Forkhead box O3 (FOXO3) transcription factor whichtransactivates the Bcl-2 interacting mediator of cell death(Bim) and other genes Activated Bim is then translocated tothe mitochondria causing a ROS burst via respiratory chainuncoupling At the intermitochondrial membrane (IMM)this and other events of proapoptotic protein activationinduce OMM perforation in a BaxBak dependent-manner[73ndash77] Therefore elevated ROS affects multiple sites thatdrive the cells toward apoptosis The present results can beused to elucidate the possible ERW action sites contribut-ing to the increased cell viability ERW contains hydrogenmolecules and Pt Nps as potential functional componentsas described above Dissolved hydrogen molecules diffusedin cells have been shown to reduce intracellular hydroxylradicals [78 79] Pt Nps have been shown to be taken up by

pinocytosis diffusion and other processes and act as novelROS scavengers in vitro and in vivo [80 81] Internalized func-tional components are capable of scavenging ROS producedby cytotoxic stimuli such as H

2O2 SNP and glutamate used

in this study Based on the pathways mentioned above ERWcould scavenge ROS to prevent thioredoxin (Trx) inactiva-tion and inhibit SOK1 activation thereby preventing activeASK1 signalosome formation An inactive ASK1 signalosomebecomes incapable of forwarding the ASK1-JNK pathwayand prevents subsequent apoptosis prone signals includingcytochrome c rerelease p53 activation and AP-1 formationH2O2scavenging prevents intracellular Ca2+ accumulation

Moreover scavenging H2O2is likely to prevent the nuclear

accumulation of FOXO3 that subsequently induces mito-chondrial ROS burst The present results of reduced ROSand [Ca2+]i levels cooperatively contribute to improve themitochondrial integrity leading to the increased cell viabilityAt present the mechanism by which ERW functioned to


reduce the [Ca2+]i level is not clear and further studies arerequired The proposed points of action of ERW on cellularstressors are shown in Figure 9

In the present study the cell viability was found to besignificantly improved because of the ROS scavenging abilityof ERW It is known that excessively produced ROS lead tooxidative stress and neurodegenerative diseases The presentstudy simulated excess ROSRNS levels by applying H

2O2

glutamate and SNP to the cells Recent reports emphasizethat cells that possess a group of genes termed vitagenes havea better ability tomaintain cellular homeostasis under variousstress conditions Based on the hormetic concept low dosesof oxidative stresses may induce cellular defense pathwaysto mediate a hormetic adaptive response conferred by theexpression of vitagenes coding for stress resistance proteinsthat maintain cellular homeostasis Vitagenes include thegenes coding for the heat shock proteins (HSP) such asHsp32(also called Heme oxygenase 1 HO-1) and Hsp70 sirtuinsand the thioredoxinthioredoxin reductase systems Underoxidative conditions the Hsps protects brain cells by degrad-ing the prooxidant heme to free iron carbon monoxideand biliverdin which in turn is reduced by biliverdin reduc-tase to bilirubin which possess endogenous NO and RNSscavenging abilities Conversely excess bilirubin productionbecomes neurotoxic by way of cell membrane disruptionmitochondrial transmembrane potential (ΔΨm) reductionand apoptotic pathway activation Thus HO-1 repression isimportant for cell survival as excess bilirubin is cytotoxicThe heme oxygenases function as oxidative stress sensors andmodulators of redox homeostasis SIRT1 (silent informationregulator 2 (Sir2) protein 1) is a sirtuin possessing histonedeacylase activity and it plays a role in the hormetic responseof cells to oxidative stressors Sirtuins in the presence ofNAD+ as a cofactor deacylate transcription factors such asNrf2 p53 FOXO (Forkhead box O) and nuclear factor-120581B(NF-120581B) H

2O2is known to upregulate SIRT2 expression and

it binds to FOXO3a enhancing its binding to target genessuch asMnSODThus increased SIRT1 and SIRT2 expressioncontributes to ROS reduction Also the FOXO subfamilyof transcription factors transactivates the Bcl-2 interactingmediator of cell death (Bim) and other genes Another vita-gene system is the thioredoxinthioredoxin reductase (Trxand Trx reductase) system Trx exists as a cytoplasmic protein(Trx-1) and a mitochondrial (Trx-2) protein essential for cellsurvival It regulates cellular redox balance by reversible oxi-dation of the two cysteine residues in the redox center of TrxTrx acts as an antioxidant or ROS scavenger as it eliminatessinglet oxygen (1O

2) hydroxyl radical (OH∙) and hydrogen

peroxide (H2O2) H2O2stimulates Trx-1 expression by the

transcription factor Nrf2 and numerous stimuli includingH2O2 hypoxia and UV irradiation will direct translocation

of Trx to the nucleus where it regulates the activities of criticaltranscription factors such as AP-1 members NF-kB p53 andJun which all are related to apoptosis pathways [82ndash87]Vitagenes could function in preserving cellular homeostasisduring stressful circumstances within a tolerable dose rangethe hormetic zone However beyond this hormetic zonehigher degrees of oxidative stresses are thought to disrupt

cellular redox homeostasis In the present study we mon-itored cell viabilities affected by various concentrations ofH2O2 glutamate and SNP The results demonstrated that

the cell viabilities were increased or recovered significantlyexcept samples treated with the lowest and the highestconcentrations of the stressors From the results it is possiblethat the reduced ROS induced by the presence of ERW inthe cell could have modulated the oxidative states such thatthey fell again within the range of the hormetic zone therebyallowing vitagenes to exert their activities

5 Conclusion

ERW was found to protect N1E-115 PC12 SFME andMCCNP cells from oxidative stresses caused by H

2O2 glu-

tamate and SNP treatments This neuronal cell protectionstemmed from the ROS specific scavenging ability of dis-solved hydrogen and Pt nps in the ERW The present com-munication provides encouraging data for the therapeuticapplicability of ERW against NDs

Abbreviations

AD Alzheimerrsquos diseaseALS Amyotrophic lateral sclerosisAra-C Cytosine 120573-D-arabinofuranoside

hydrochlorideAsA Ascorbic acidBBB Blood brain barrierCDL Chemically defined lipidCNS Central nervous systemDAR-4M AM Diaminorhodamine-4M acetoxymethyl

esterDCFH-DA 21015840 71015840-Dichlorofluorescin diacetateDMSO Dimethyl sulfoxideERW Electrolyzed reduced waterHD Huntingtonrsquos disease∙OH Hydroxyl radicalHEPES 4-[2-Hydroxyethyl]-1-

piperazineethane-sulfonic acidL-NAC N-Acetyl-L-cysteineNO Nitric oxidemEGF Mouse epidermal growth factorNOS Nitric oxide synthaseNO∙ Nitric oxide radicalNMDA N-Methyl-D-aspartateONOOminus PeroxynitritePD Parkinsonrsquos diseasePt PlatinumRNS Reactive nitrogen speciesROS Reactive oxygen speciesSNP Sodium nitroprussideO2

∙minus Superoxide radical

Conflict of Interests

The authors declare that there is no potential conflict ofinterests


References

[1] P H Reddy ldquoMitochondrial medicine for aging and neurode-generative diseasesrdquoNeuroMolecularMedicine vol 10 no 4 pp291ndash315 2008

[2] A Melo L Monteiro R M F Lima D M de OliveiraM D de Cerqueira and R S El-Bacha ldquoOxidative stressin neurodegenerative diseases mechanisms and therapeuticperspectivesrdquo Oxidative Medicine and Cellular Longevity vol2011 Article ID 467180 14 pages 2011

[3] S Gandhi andA Y Abramov ldquoMechanism of oxidative stress inneurodegenerationrdquoOxidativeMedicine and Cellular Longevityvol 2012 Article ID 428010 11 pages 2012

[4] M T Lin and M F Beal ldquoMitochondrial dysfunction andoxidative stress in neurodegenerative diseasesrdquoNature vol 443no 7113 pp 787ndash795 2006

[5] M Karbowski and A Neutzner ldquoNeurodegeneration as aconsequence of failed mitochondrial maintenancerdquo Acta Neu-ropathologica vol 123 no 2 pp 157ndash171 2012

[6] V Shukla S K Mishra and H C Pant ldquoOxidative stress inneurodegenerationrdquo Advances in Pharmacological Sciences vol2011 Article ID 572634 13 pages 2011

[7] D G Smith R Cappai and K J Barnham ldquoThe redox chem-istry of the Alzheimerrsquos disease amyloid 120573 peptiderdquo Biochimicaet Biophysica Acta vol 1768 no 8 pp 1976ndash1990 2007

[8] A Nunomura K Honda A Takeda et al ldquoOxidative damageto RNA in neurodegenerative diseasesrdquo Journal of Biomedicineand Biotechnology vol 2006 Article ID 82323 6 pages 2006

[9] M Tanaka P B Chock and E R Stadtman ldquoOxidized messen-ger RNA induces translation errorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 104 no1 pp 66ndash71 2007

[10] Z Radak Z Zhao S Goto and E Koltai ldquoAge-associatedneurodegeneration and oxidative damage to lipids proteins andDNArdquoMolecular Aspects of Medicine vol 32 no 4ndash6 pp 305ndash315 2011

[11] S A Lipton Y-B Choi Z-H Pan et al ldquoA redox-based mech-anism for the neuroprotective and neurodestructive effects ofnitric oxide and related nitroso-compoundsrdquo Nature vol 364no 6438 pp 626ndash632 1993

[12] T Kume H Katsuki and A Akaike ldquoEndogenous factorsregulating neuronal death induced by radical stressrdquo Biologicaland Pharmaceutical Bulletin vol 27 no 7 pp 964ndash967 2004

[13] N Ghosh R Ghosh and S CMandal ldquoAntioxidant protectiona promising therapeutic intervention in neurodegenerativediseaserdquo Free Radical Research vol 45 no 8 pp 888ndash905 2011

[14] B Halliwell ldquoFree radicals and antioxidants updating a per-sonal viewrdquo Nutrition Reviews vol 70 no 5 pp 257ndash265 2012

[15] Y Li T Hamasaki N Nakamichi et al ldquoSuppressive effects ofelectrolyzed reduced water on alloxan-induced apoptosis andtype 1 diabetes mellitusrdquo Cytotechnology vol 63 no 2 pp 119ndash131 2011

[16] H Yan H Tian T Kinjo et al ldquoExtension of the lifespan ofcaenorhabditis elegans by the use of electrolyzed reducedwaterrdquoBioscience Biotechnology and Biochemistry vol 74 no 10 pp2011ndash2015 2010

[17] S Shirahata T Hamasaki and K Teruya ldquoAdvanced researchon the health benefit of reduced waterrdquo Trends in Food Scienceamp Technology vol 23 no 2 pp 124ndash131 2012

[18] S Shirahata S Kabayama M Nakano et al ldquoElectrolyzed-reduced water scavenges active oxygen species and protects

DNA from oxidative damagerdquo Biochemical and BiophysicalResearch Communications vol 234 no 1 pp 269ndash274 1997

[19] Y Li T Nishimura K Teruya et al ldquoProtective mechanismof reduced water against alloxan-induced pancreatic 120573-celldamage scavenging effect against reactive oxygen speciesrdquoCytotechnology vol 40 no 1ndash3 pp 139ndash149 2002

[20] C-F Tsai Y-WHsuW-K Chen et al ldquoHepatoprotective effectof electrolyzed reduced water against carbon tetrachloride-induced liver damage in micerdquo Food and Chemical Toxicologyvol 47 no 8 pp 2031ndash2036 2009

[21] H Yan T Kinjo H Tian et al ldquoMechanism of the lifespanextension of Caenorhabditis elegans by electrolyzed reducedwatermdashparticipation of PT nanoparticlesrdquo Bioscience Biotech-nology and Biochemistry vol 75 no 7 pp 1295ndash1299 2011

[22] K-C Huang C-C Yang K-T Lee and C-T Chien ldquoReducedhemodialysis-induced oxidative stress in end-stage renal dis-ease patients by electrolyzed reduced waterrdquo Kidney Interna-tional vol 64 no 2 pp 704ndash714 2003

[23] K-C Huang S-P Hsu C-C Yang et al ldquoElectrolysed-reducedwater dialysate improves T-cell damage in end-stage renal dis-ease patients with chronic haemodialysisrdquo Nephrology DialysisTransplantation vol 25 no 8 pp 2730ndash2737 2010

[24] T Kinjo J Ye H Yan et al ldquoSuppressive effects of electrochem-ically reduced water on matrix metalloproteinase-2 activitiesand in vitro invasion of human fibrosarcoma HT1080 cellsrdquoCytotechnology vol 64 no 3 pp 357ndash371 2012

[25] C-F Tsai Y-W Hsu W-K Chen Y-C Ho and F-J LuldquoEnhanced induction of mitochondrial damage and apoptosisin human leukemia HL-60 cells due to electrolyzed-reducedwater and glutathionerdquo Bioscience Biotechnology and Biochem-istry vol 73 no 2 pp 280ndash287 2009

[26] H Yan T Kashiwaki T Hamasaki et al ldquoThe neuroprotectiveeffects of electrolyzed reduced water and its model watercontaining molecular hydrogen and Pt nanoparticlesrdquo BMCProceedings vol 5 article P69 supplement 8 2011

[27] G J HarryM Billingsley A Bruinink et al ldquoIn vitro techniquesfor the assessment of neurotoxicityrdquo Environmental HealthPerspectives vol 106 supplement 1 pp 131ndash158 1998

[28] T Amano E Richelson and M Nirenberg ldquoNeurotransmittersynthesis by neuroblastoma clonesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 69 no1 pp 258ndash263 1972

[29] H Li H-C Chen and F L Huang ldquoIdentification of arapidly dephosphorylating 95-kDa protein as elongation factor2 during 8-Br-cAMP treatment of N1E115 neuroblastoma cellsrdquoBiochemical and Biophysical Research Communications vol 217no 1 pp 131ndash137 1995

[30] MYamadaKMomose E Richelson andMYamada ldquoSodiumnitroprusside-induced apoptotic cellular death via productionof hydrogen peroxide in murine neuroblastoma N1E-115 cellsrdquoJournal of Pharmacological and Toxicological Methods vol 35no 1 pp 11ndash17 1996

[31] J-E Oh K R Karlmark J-H Shin et al ldquoDifferentiationof neuroblastoma cell line N1E-115 involves several signalingcascadesrdquo Neurochemical Research vol 30 no 3 pp 333ndash3482005

[32] NMatuszak LHamtiaux B Baldeyroux et al ldquoDual inhibitionof MAGL and type II topoisomerase by N-phenylmaleimidesas a potential strategy to reduce neuroblastoma cell growthrdquoEuropean Journal of Pharmaceutical Sciences vol 45 no 3 pp263ndash271 2012


[33] J-E Oh A Freilinger E Gelpi A Pollak M Hengstschlagerand G Lubec ldquoProteins involved in neuronal differentiation ofneuroblastoma cell line N1E-115rdquo Electrophoresis vol 28 no 12pp 2009ndash2017 2007

[34] L A Greene and A S Tischler ldquoEstablishment of a noradren-ergic clonal line of rat adrenal pheochromocytoma cells whichrespond to nerve growth factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 73 no7 pp 2424ndash2428 1976

[35] B Liu P Yang Y Ye et al ldquoRole of ROS in the protective effectof silibinin on sodium nitroprusside-induced apoptosis in ratpheochromocytoma PC12 cellsrdquo Free Radical Research vol 45no 7 pp 835ndash847 2011

[36] Z Kawakami H Kanno Y Ikarashi and Y Kase ldquoYokukansana kampo medicine protects against glutamate cytotoxicity dueto oxidative stress in PC12 cellsrdquo Journal of Ethnopharmacologyvol 134 no 1 pp 74ndash81 2011

[37] SMaH LiuH Jiao et al ldquoNeuroprotective effect of ginkgolideK on glutamate-induced cytotoxicity in PC 12 cells via inhibi-tion of ROS generation and Ca2+ influxrdquo NeuroToxicology vol33 no 1 pp 59ndash69 2012

[38] J Y Jung C R Han Y J Jeong et al ldquoEpigallocatechingallate inhibits nitric oxide-induced apoptosis in rat PC12 cellsrdquoNeuroscience Letters vol 411 no 3 pp 222ndash227 2007

[39] D T Loo J I Fuquay C L Rawson and D W BarnesldquoExtended culture of mouse embryo cells without senescenceinhibition by serumrdquo Science vol 236 no 4798 pp 200ndash2021987

[40] H Yamaguchi J Zhu T Yu et al ldquoSerum-free mouse embryocells generate a self-sustaining feedback loop for an astrocytemarker protein and respond to cytokines and bisphenol A inaccordance with the subtle difference in their differentiationstaterdquoCell Biology International vol 31 no 6 pp 638ndash644 2007

[41] J Ye Y Li T Hamasaki et al ldquoInhibitory effect of electrolyzedreduced water on tumor angiogenesisrdquo Biological and Pharma-ceutical Bulletin vol 31 no 1 pp 19ndash26 2008

[42] E Ruoslahti E G Hayman M Pierschbacher and E EngvallldquoFibronectin purification immunochemical properties andbiological activitiesrdquoMethods in Enzymology vol 82 part A pp803ndash831 1982

[43] R A Tobey ldquoEffects of cytosine arabinoside daunomycinmithramycin azacytidine adriamycin and camptothecin onmammalian cell cycle traverserdquo Cancer Research vol 32 no 12pp 2720ndash2725 1972

[44] N K Blank F J Seil and R M Herndon ldquoAn ultrastructuralstudy of cortical remodeling in cytosine arabinoside inducedgranuloprival cerebellum in tissue culturerdquoNeuroscience vol 7no 6 pp 1509ndash1531 1982

[45] Y Oyama A Hayashi T Ueha and K Maekawa ldquoCharacter-ization of 2101584071015840-dichlorofluorescin fluorescence in dissociatedmammalian brain neurons estimation on intracellular contentof hydrogen peroxiderdquo Brain Research vol 635 no 1-2 pp 113ndash117 1994

[46] H Kojima M Hirotani N Nakatsubo et al ldquoBioimaging ofnitric oxide with fluorescent indicators based on the rhodaminechromophorerdquo Analytical Chemistry vol 73 no 9 pp 1967ndash1973 2001

[47] P A Vandenberghe and J L Ceuppens ldquoFlow cytometricmeasurement of cytoplasmic free calcium in human peripheralblood T lymphocytes with fluo-3 a new fluorescent calciumindicatorrdquo Journal of Immunological Methods vol 127 no 2 pp197ndash205 1990

[48] M-J Kim K H Jung Y K Uhm K-H Leem and H KKim ldquoPreservative effect of electrolyzed reduced water onpancreatic 120573-cell mass in diabetic dbdb micerdquo Biological andPharmaceutical Bulletin vol 30 no 2 pp 234ndash236 2007

[49] AAkaikeH Katsuki T Kume andTMaeda ldquoReactive oxygenspecies in NMDA receptor-mediated glutamate neurotoxicityrdquoParkinsonism amp Related Disorders vol 5 no 4 pp 203ndash2071999

[50] C Ghosh and D K Lahiri ldquoIncreased vulnerability of neuronalcell lines to sodium nitroprusside-mediated toxicity is causedby the decreased level of nitric oxide metabolitesrdquo Journal ofMolecular Neuroscience vol 13 no 1-2 pp 77ndash92 1999

[51] E R Whittemore D T Loo J A Watt and C W CotmansldquoA detailed analysis of hydrogen peroxide-induced cell death inprimary neuronal culturerdquo Neuroscience vol 67 no 4 pp 921ndash932 1995

[52] F Mailly P Marin M Israel J Glowinski and J PremontldquoIncrease in external glutamate and NMDA receptor activationcontribute to H

2O2-induced neuronal apoptosisrdquo Journal of

Neurochemistry vol 73 no 3 pp 1181ndash1188 1999[53] S Shirahata T Hamasaki K Haramaki et al ldquoAnti-diabetes

effect of water containing hydrogen molecule and Pt nanopar-ticlesrdquo BMC Proceedings vol 5 supplement 8 pp 18ndash20 2011

[54] K Miyashita M Yasuda T Ota and T Suzuki ldquoAntioxidativeactivity of a cathodic solution produced by the electrolysis ofa dilute NaCl solutionrdquo Bioscience Biotechnology and Biochem-istry vol 63 no 2 pp 421ndash423 1999

[55] K Hanaoka ldquoAntioxidant effects of reduced water producedby electrolysis of sodium chloride solutionsrdquo Journal of AppliedElectrochemistry vol 31 no 12 pp 1307ndash1313 2001

[56] T Yanagihara K Arai K Miyamae et al ldquoElectrolyzedhydrogen-saturated water for drinking use elicits an antioxida-tive effect a feeding test with ratsrdquoBioscience Biotechnology andBiochemistry vol 69 no 10 pp 1985ndash1987 2005

[57] A Hiraoka M Takemoto T Suzuki et al ldquoStudies on theproperties and real existence of aqueous solution systems thatare assumed to have antioxidant activities by the action ofldquoactive hydrogenrdquordquo Journal of Health Science vol 50 no 5 pp456ndash465 2004

[58] A Hiraoka S Sasaki T Yamada A Shinohara andM ChibabldquoEffects of drinking a water product with anti-oxidant activitiesin vitro on the blood levels of biomarker substances for theoxidative stressrdquo Journal of Health Science vol 52 no 6 pp 817ndash820 2006

[59] K Fujita T Seike N Yutsudo et al ldquoHydrogen in drinkingwater reduces dopaminergic neuronal loss in the 1-methyl-4-phenyl-1236-tetrahydropyridine mouse model of Parkinsonrsquosdiseaserdquo PLoS ONE vol 4 no 9 Article ID e7247 2009

[60] I Ohsawa M Ishikawa K Takahashi et al ldquoHydrogen actsas a therapeutic antioxidant by selectively reducing cytotoxicoxygen radicalsrdquo Nature Medicine vol 13 no 6 pp 688ndash6942007

[61] S Spulber K Edoff L Hong S Morisawa S Shirahata and SCeccatelli ldquoMolecular hydrogen reduces lps-induced neuroin-flammation and promotes recovery from sickness behaviour inmicerdquo PLoS ONE vol 7 no 7 Article ID e42078 12 pages 2012

[62] T Hamasaki T Kashiwagi T Imada et al ldquoKinetic analysisof superoxide anion radical-scavenging and hydroxyl radical-scavenging activities of platinum nanoparticlesrdquo Langmuir vol24 no 14 pp 7354ndash7364 2008

[63] M Kajita K Hikosaka M Iitsuka A Kanayama N Toshimaand Y Miyamoto ldquoPlatinum nanoparticle is a useful scavenger


of superoxide anion and hydrogen peroxiderdquo Free RadicalResearch vol 41 no 6 pp 615ndash626 2007

[64] J Kim M Takahashi T Shimizu et al ldquoEffects of apotent antioxidant platinum nanoparticle on the lifespan ofCaenorhabditis elegansrdquo Mechanisms of Ageing and Develop-ment vol 129 no 6 pp 322ndash331 2008

[65] Y Yoshihisa Q-L Zhao M A Hassan et al ldquoSODcatalasemimetic platinum nanoparticles inhibit heat-induced apoptosisin human lymphomaU937 andHHcellsrdquo Free Radical Researchvol 45 no 3 pp 326ndash335 2011

[66] Y Isobe M Yamauchi R Ikeda and H Kitagawa ldquoA study onhydrogen adsorption of polymer protected Pt nanoparticlesrdquoSynthetic Metals vol 135-136 pp 757ndash758 2003

[67] J Pelka H Gehrke M Esselen et al ldquoCellular uptake ofplatinum nanoparticles in human colon carcinoma cells andtheir impact on cellular redox systems and DNA integrityrdquoChemical Research in Toxicology vol 22 no 4 pp 649ndash6592009

[68] H Gehrke J Pelka C G Hartinger et al ldquoPlatinum nanopar-ticles and their cellular uptake and DNA platination at non-cytotoxic concentrationsrdquo Archives of Toxicology vol 85 no 7pp 799ndash812 2011

[69] S Krol ldquoChallenges in drug delivery to the brain nature isagainst usrdquo Journal of Controlled Release vol 164 no 2 pp 145ndash155 2012

[70] S Wohlfart S Gelperina and J Kreuter ldquoTransport of drugsacross the blood-brain barrier by nanoparticlesrdquo Journal ofControlled Release vol 161 no 2 pp 264ndash273 2012

[71] R R Arvizo S Bhattacharyya R A Kudgus K Giri R Bhat-tacharya and P Mukherjee ldquoIntrinsic therapeutic applicationsof noblemetal nanoparticles past present and futurerdquoChemicalSociety Reviews vol 41 no 7 pp 2943ndash2970 2012

[72] T Satoh Y Enokido H Aoshima et al ldquoChanges in mito-chondrial membrane potential during oxidative stress-inducedapoptosis in PC12 cellsrdquo Journal of Neuroscience Research vol50 pp 413ndash420 1997

[73] Y Zhang and B R Bhavnani ldquoGlutamate-induced apoptosisin neuronal cells is mediated via caspase-dependent and inde-pendent mechanisms involving calpain and caspase-3 proteasesas well as apoptosis inducing factor (AIF) and this process isinhibited by equine estrogensrdquo BMCNeuroscience vol 7 article49 2006

[74] M L Circu and T Y Aw ldquoReactive oxygen species cellu-lar redox systems and apoptosisrdquo Free Radical Biology andMedicine vol 48 no 6 pp 749ndash762 2010

[75] M S Ola M Nawaz and H Ahsan ldquoRole of Bcl-2 familyproteins and caspases in the regulation of apoptosisrdquoMolecularand Cellular Biochemistry vol 351 no 1-2 pp 41ndash58 2011

[76] K Sinha J Das P B Pal and P C Sil ldquoOxidative stressthe mitochondria-dependent and mitochondria-independentpathways of apoptosisrdquo Archives of Toxicology vol 87 no 7 pp1157ndash1180 2013

[77] R J Mailloux X Jin and W G Willmore ldquoRedox regulationof mitochondrial function with emphasis on cysteine oxidationreactionsrdquo Redox Biology vol 2 no 1 pp 123ndash139 2014

[78] Y Yang B Li C Liu et al ldquoHydrogen-rich saline protectsimmunocytes from radiation-induced apoptosisrdquo Medical Sci-ence Monitor vol 18 no 4 pp BR144ndashBR148 2012

[79] S Kato Y Saitoh K Iwai and N Miwa ldquoHydrogen-rich elec-trolyzed warm water represses wrinkle formation against UVAray together with type-I collagen production and oxidative-stress diminishment in fibroblasts and cell-injury prevention in

keratinocytesrdquo Journal of Photochemistry and Photobiology BBiology vol 106 no 1 pp 24ndash33 2012

[80] M Takamiya Y Miyamoto T Yamashita K Deguchi Y Ohtaand K Abe ldquoStrong neuroprotection with a novel platinumnanoparticle against ischemic stroke- and tissue plasminogenactivator-related brain damages in micerdquoNeuroscience vol 221pp 47ndash55 2012

[81] H Nakanishi T Hamasaki T Kinjo et al ldquoLow concentrationplatinum nanoparticles effectively scavenge reactive oxygenspecies in rat skeletal L6 cellsrdquoNano Biomedicine and Engineer-ing vol 5 no 2 pp 76ndash85 2013

[82] V Calabrese E GuaglianoM Sapienza et al ldquoRedox regulationof cellular stress response in aging and neurodegenerativedisorders role of vitagenesrdquoNeurochemical Research vol 32 no4-5 pp 757ndash773 2007

[83] V Calabrese C Cornelius S Cuzzocrea I Iavicoli E Rizzarelliand E J Calabrese ldquoHormesis cellular stress response andvitagenes as critical determinants in aging and longevityrdquoMolecular Aspects of Medicine vol 32 no 4ndash6 pp 279ndash3042011

[84] V Calabrese C Cornelius A T Dinkova-Kostova et al ldquoCel-lular stress responses hormetic phytochemicals and vitagenesin aging and longevityrdquo Biochimica et Biophysica Acta vol 1822no 5 pp 753ndash783 2012

[85] C Cornelius R Perrotta A Graziano E J Calabrese and VCalabrese ldquoStress responses vitagenes and hormesis as criticaldeterminants in aging and longevity mitochondria as a lsquochirsquordquoImmunity and Ageing vol 10 no 1 article 15 2013

[86] C Cornelius A Trovato Salinaro M Scuto et al ldquoCellularstress response sirtuins andUCPproteins inAlzheimer diseaserole of vitagenesrdquo Immunity and Ageing vol 10 no 1 article 412013

[87] A T Salinaro C Cornelius and G Koverech ldquoCellular stressresponse redoxstatus and vitagenes in glaucoma a systemicoxidant disorder linked to Alzheimers diseaserdquo ExperimentalPharmacology and Drug Discovery vol 5 article 129 pp 1ndash82014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


way of the glutamate receptor channel N-methyl-D-aspartate(NMDA) The increased intracellular Ca2+ levels togetherwith endogenous calmodulin stimulate nitric oxide synthase(NOS) to produceNOwhich is converted to neurodestructiveNO radical (NO∙) depending on the redox conditions inthe neuronal cells NO∙ further reacts with O

2

∙minus generatinga potent oxidant ONOOminus Thereafter ONOOminus is rapidlyconverted to highly reactive ∙OH which is cytotoxic [2 10ndash12] Moreover the damage is exasperated by the fact that theCNS contains a high amount of polyunsaturated fatty acidswhich are readily peroxidized by toxic reactive species [10]and has relatively low levels of antioxidative enzymes [8 10]Therefore such inherent characteristics of the CNS and theROS and RNS individually or in combination contributeto the development of NDs Antioxidants metal chelatorsor other agents that improve endogenous enzymatic andnonenzymatic cellular defense systems have been suggestedas treatments for NDs [6] Control of ROS production bymitochondrially targeted antioxidant treatment is expectedto delay aging [1] Antioxidant therapies using plant extractsfrom seeds and leaves are reported to be beneficial [13]while some other prospective substances have been testedwith unsatisfactory results [2 14] For antioxidant therapiesto be effective they must cross the blood brain barrier(BBB) which protects the brain from toxins and reduces thebioavailability of exogenous antioxidants in the brain [2 6]Therefore it is highly desirable to develop alternative agentsthat are able to pass through the BBB and exert antioxidativeeffects in the brain

One promising candidate is electrochemically reducedwater (ERW) because it has the potential to cross the BBBERW produced near the cathode during electrolysis of 2mMNaOH model water has characteristics of high pH lowdissolved oxygen and an extremely negative redox poten-tial [15] ERW contains a high concentration of dissolvedhydrogen (04ndash09 ppm) and a small amount of platinumnanoparticles (Pt nps 01ndash25 ppb) both of which exhibitROS-scavenging ability [11 16 17] Neutralized ERW hasbeen shown to exhibit H

2O2-scavenging activity which is

correlated with protection of DNA [18 19] and carbontetrachloride induced liver damage [20] lifespan extensionof Caenorhabditis elegans [16 21] alloxan-induced type 1 dia-betes [15 19] hemodialysis-induced oxidative stress duringend-stage renal disease [22 23] and inhibitory effects ofHT1080 tumor cell invasion [24] ERW in combination withglutathione induced human leukemia HL-60 apoptotic celldeath whereas a cytotoxic effect was not observed in normalperipheral blood mononuclear cells [25] Despite the variousprotective functions exhibited by ERW its effect on neuronalcells has not been disclosed in the literature other than ina brief meeting abstract by Yan et al [26] who reported theprotective effect of ERW on H

2O2-induced cultured N1E-115

neuroblastoma cell deathCultures of nervous system tissues and cells are catego-

rized in the terms of their complexities whole-embryo wholebrain organotypic slices reaggregate cultures dissociatedprimary cell cultures and cell lines [27] The degree ofcomplexity of an in vitro model of dissociated primary cell

cultures is considered to more closely reflect the in vivo statethan that of the cell lines [27] In light of this view wealso usedmouse cerebral cortex neuronal primary (MCCNP)cells as a model to observe the effect of ERW in addition toimmortalized cell lines N1E-115 cells have been establishedas an adrenergic clone derived from mouse neuroblastomaC-300 [28] and are used as a model of CNS neurons[29ndash32] In addition in culture in the presence of severalfactors including DMSO these cells display morphologicalcharacteristics of neuritogenesis which we used as a markerfor changes upon treatment with ERW [33]The PC12 cell linewas established from a transplantable rat adrenal pheochro-mocytoma based on its response to nerve growth factor(NGF) PC12 cells possess the potential to be differentiatedinto either chromaffin cells or sympathetic neurons whenin the presence of NGF [34] This cell line has been usedas a model for studying the neuronal response to oxidativestress [35ndash37] Also the viability of PC12 cells is describedto be sensitive to NO stress thus this makes them useful fordetecting a subtle NO effect [38] Serum-free mouse embryo(SFME) cells were established from mouse embryo cells bymaintenance in the absence of serum [39] These cells showthe characteristics of an astrocyte a progenitor cell withoutsenescence which is the most abundant cell type in the CNS[39 40]

In the present study we utilized various cell types orig-inating from mouse and rat as a first step to explore theprotective effect of ERW on neurocytotoxicity caused byreactive species

2 Materials and Methods

21 Materials Dulbeccorsquos Modified Eaglersquos Medium(DMEM) and DMEMHamrsquos F12 Combined Medium(1 1) were purchased from Nissui Pharmaceutical Co LTD(Tokyo Japan) Insulin putrescine transferrin propidiumiodide (PI) Fluo-3AM pluronic F127 sodium glutamateand Ca2+ Mg2+-free Hankrsquos balanced salt solution (Ca2+Mg2+-free HBSS) were purchased from Sigma-Aldrich Japan(Tokyo Japan) 21015840 71015840-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Invitrogen Technologies (CarlsbadCA USA) Chemically defined lipid (CDL) and mouseepidermal growth factor (mEGF) were purchased fromLife Technologies Japan (Tokyo Japan) Cell counting kit-8(CCK-8) which uses WST-8 as a color indicator to measurelive cell numbers was purchased from Dojindo LaboratoriesCo (Tokyo Japan) and the kit is referred to as the WST-8kit hereafter Diaminorhodamine-4M acetoxymethyl ester(DAR-4M AM) was from Daiichi Pure Chemicals Co LTD(Tokyo Japan) N-Acetyl-L-cysteine (L-NAC) ascorbic acid(AsA) sodium nitroprusside (SNP) 4-[2-hydroxyethyl]-1-piperazineethane-sulfonic acid (HEPES) fetal bovineserum (FBS) bovine serum albumin (BSA) penicillinstreptomycin progesterone and all other chemicals wereobtained fromWako Pure Chemical Industries LTD (OsakaJapan) The gelatin sepharose 4B column was obtainedfrom GE Healthcare Japan (Tokyo Japan) Ultrapure water












2at 37∘C




2O2for 24 h To each










2O2to form the


2O2in rat

neuron cells [45]



2HPO4 5mM


2 1 mgmL BSA and





2O2 and








3 Results








2O2













2O2 the cell







2O2 the neuronal


2O2






2O2












2O2



2O2 the intracellu-




2O2





5002001000

Cel

l via

bilit

y (

)PC12 cell

0

20

40

60

80

100

120

ControlERW

lowastlowast

lowast

H2O2 (120583M)

(a)

SFME cell

lowastlowast

lowast

200100500

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

ControlERW

H2O2 (120583M)

(b)





2O2(50 100 and 200 120583M)







4 Discussion




N1E115 cell

+ + + + + + + +

L-NAC AsA

Abs

cont

Pos

cont

ERW

Auto

ERW

Cel

l via

bilit

y (

)

0

20

40

60

80

120

100 lowastlowast

lowast

lowast

200120583M H2O2

01

mM

10

mM

NaO

H20

mM

01

mM

10

mM

minus



2O2for 24 h A WST-




2






2O2-induced


2




2O2 ERW has



N1E115 cell

(a)

N1E115 cell

(b)

(c) (d)

(e)








2






01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100


Max

()

10 min treatment

(a)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)




(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























2at 37∘C




2O2for 24 h To each










2O2to form the


2O2in rat

neuron cells [45]



2HPO4 5mM


2 1 mgmL BSA and





2O2 and








3 Results








2O2













2O2 the cell







2O2 the neuronal


2O2






2O2












2O2



2O2 the intracellu-




2O2





5002001000

Cel

l via

bilit

y (

)PC12 cell

0

20

40

60

80

100

120

ControlERW

lowastlowast

lowast

H2O2 (120583M)

(a)

SFME cell

lowastlowast

lowast

200100500

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

ControlERW

H2O2 (120583M)

(b)





2O2(50 100 and 200 120583M)







4 Discussion




N1E115 cell

+ + + + + + + +

L-NAC AsA

Abs

cont

Pos

cont

ERW

Auto

ERW

Cel

l via

bilit

y (

)

0

20

40

60

80

120

100 lowastlowast

lowast

lowast

200120583M H2O2

01

mM

10

mM

NaO

H20

mM

01

mM

10

mM

minus



2O2for 24 h A WST-




2






2O2-induced


2




2O2 ERW has



N1E115 cell

(a)

N1E115 cell

(b)

(c) (d)

(e)








2






01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100


Max

()

10 min treatment

(a)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)




(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2O2to form the


2O2in rat

neuron cells [45]



2HPO4 5mM


2 1 mgmL BSA and





2O2 and








3 Results








2O2













2O2 the cell







2O2 the neuronal


2O2






2O2












2O2



2O2 the intracellu-




2O2





5002001000

Cel

l via

bilit

y (

)PC12 cell

0

20

40

60

80

100

120

ControlERW

lowastlowast

lowast

H2O2 (120583M)

(a)

SFME cell

lowastlowast

lowast

200100500

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

ControlERW

H2O2 (120583M)

(b)





2O2(50 100 and 200 120583M)







4 Discussion




N1E115 cell

+ + + + + + + +

L-NAC AsA

Abs

cont

Pos

cont

ERW

Auto

ERW

Cel

l via

bilit

y (

)

0

20

40

60

80

120

100 lowastlowast

lowast

lowast

200120583M H2O2

01

mM

10

mM

NaO

H20

mM

01

mM

10

mM

minus



2O2for 24 h A WST-




2






2O2-induced


2




2O2 ERW has



N1E115 cell

(a)

N1E115 cell

(b)

(c) (d)

(e)








2






01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100


Max

()

10 min treatment

(a)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)




(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2O2













2O2 the cell







2O2 the neuronal


2O2






2O2












2O2



2O2 the intracellu-




2O2





5002001000

Cel

l via

bilit

y (

)PC12 cell

0

20

40

60

80

100

120

ControlERW

lowastlowast

lowast

H2O2 (120583M)

(a)

SFME cell

lowastlowast

lowast

200100500

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

ControlERW

H2O2 (120583M)

(b)





2O2(50 100 and 200 120583M)







4 Discussion




N1E115 cell

+ + + + + + + +

L-NAC AsA

Abs

cont

Pos

cont

ERW

Auto

ERW

Cel

l via

bilit

y (

)

0

20

40

60

80

120

100 lowastlowast

lowast

lowast

200120583M H2O2

01

mM

10

mM

NaO

H20

mM

01

mM

10

mM

minus



2O2for 24 h A WST-




2






2O2-induced


2




2O2 ERW has



N1E115 cell

(a)

N1E115 cell

(b)

(c) (d)

(e)








2






01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100


Max

()

10 min treatment

(a)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)




(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5002001000

Cel

l via

bilit

y (

)PC12 cell

0

20

40

60

80

100

120

ControlERW

lowastlowast

lowast

H2O2 (120583M)

(a)

SFME cell

lowastlowast

lowast

200100500

Cel

l via

bilit

y (

)

0

20

40

60

80

100

120

ControlERW

H2O2 (120583M)

(b)





2O2(50 100 and 200 120583M)







4 Discussion




N1E115 cell

+ + + + + + + +

L-NAC AsA

Abs

cont

Pos

cont

ERW

Auto

ERW

Cel

l via

bilit

y (

)

0

20

40

60

80

120

100 lowastlowast

lowast

lowast

200120583M H2O2

01

mM

10

mM

NaO

H20

mM

01

mM

10

mM

minus



2O2for 24 h A WST-




2






2O2-induced


2




2O2 ERW has



N1E115 cell

(a)

N1E115 cell

(b)

(c) (d)

(e)








2






01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100


Max

()

10 min treatment

(a)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)




(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















N1E115 cell

+ + + + + + + +

L-NAC AsA

Abs

cont

Pos

cont

ERW

Auto

ERW

Cel

l via

bilit

y (

)

0

20

40

60

80

120

100 lowastlowast

lowast

lowast

200120583M H2O2

01

mM

10

mM

NaO

H20

mM

01

mM

10

mM

minus



2O2for 24 h A WST-




2






2O2-induced


2




2O2 ERW has



N1E115 cell

(a)

N1E115 cell

(b)

(c) (d)

(e)








2






01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100


Max

()

10 min treatment

(a)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)




(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















N1E115 cell

(a)

N1E115 cell

(b)

(c) (d)

(e)








2






01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100


Max

()

10 min treatment

(a)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)




(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100


Max

()

10 min treatment

(a)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

24 h treatment

(b)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(c)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100M

ax (

)

(d)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(e)


01 1 10 100 1000FL1 LOG FL1 LOG

0

20

40

60

80

100

Max

()

(f)




(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c) (d)

0

N1E115 cell

DCF

fluo

resc

ence

()

100

0

20

40

60

80

100

120

140

ControlERW

H2O2 (120583M)

lowastlowast

lowast

(e)











2O2







PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PC12 cell

Viab

ility

()

200 60040000

20

40

60

80

100

120

ControlERW

SNP (120583M)

lowast

lowast

(a)

MQ

ERW

MQ + 200120583M SNP

ERW + 200120583M SNP

(b)

0

40

80

120

160

200

MQ ERW

Fluo

resc

ent i

nten

sity

(rel

ativ

e rat

e)

(c)









Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Intr

acel

lula

r fluo

resc

ence

ERWControl


[Ca2+]i

[Ca2+]i

[Ca2+]i

01mM0mM 1mM

ERWControl

ERWControl








Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control ERW

05mM05mM

0mM0mM

01mM01mM

(a)

0

100

200

300

400


ControlERW

Num

ber o

f dea

d ce

lls

lowastlowast

lowastlowast

(b)



JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















JNK

Nucleus

Cytochrome crelease

AP-1


Mitochondria

Trx14-3-3

Trx SOK114-3-3

p53

Glutamate SNP

c-Jun

Apoptosis

DNA

OMM

IMM

Cytosol


AD PD HD ALS CVD

ROS

ERW

KEAP1Nrf2

KEAP1

HO-1TrxRTrx

NOSROS

ERW

Ca2+ H2O2

[RNS]i uarr

lowastNrf2lowastp53

lowastASK1

ASK1-NO∙

(H2 Pt NPs)

(H2 Pt NPs)

[Ca2+]iuarr

[Ca2+]iuarr

[Ca2+] uarr













2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2O2










5 Conclusion


2O2 glu-


Abbreviations









References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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