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lutathione Can Efficiently Prevent Direct Current–inducedytotoxicity

uko Nakamura, DDS, PhD,* Akiko Shimetani, DDS, PhD,* Hiroko Fujii, PhD,†

samu Amano, DDS, PhD,† Hiroshi Sakagami, PhD,‡ and Keiso Takahashi, DDS, PhD§

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bstracte have reported that direct current (DC) with anti-

acterial agents used in iontophoresis for root canalisinfection induced host cell necrotic cytotoxicity, andhis DC-induced cytotoxicity may be because of gener-ted free radicals and metal ions eluted from metallectrodes. Iontophoresis is still used in some cases,nd thus it is necessary to consider how we mayrevent DC-induced cytotoxicity of host cells of peri-pical lesions. Thus, we compared the protective effectsf various antioxidants on the DC-induced cytotoxicitygainst host cells. N-acetyl-L-cysteine and glutathioneGSH) efficiently prevented DC-induced cytotoxicitygainst human polymorphnuclear cells (PMNs) (p �.01). The DC-induced cytotoxicity against PMNs wasignificantly enhanced by buthionine sulfoximine (p �.05), an inhibitor of GSH synthesis, and its effect wasescued by adding the exogenous GSH (p � 0.01). Inddition, DC treatment reduced the intracellular GSH

evels in a time-dependent manner (p � 0.05). Trans-ission electron microscopy showed that the DC in-

uced the intense vacuolization and accumulation ofellular debris in autophagolysosomes, and these mor-hological changes were blocked by adding exogenousSH. These results suggest that GSH, a thiol antioxi-ant, effectively prevents the DC-induced cytotoxicity.J Endod 2008;34:693–697)

ey Wordsntioxidants, cytotoxicity, direct current, endodontics,lutathione, iontophoresis

From the *Divisions of Endodontics, †Anatomy, and ‡Phar-acology, Meikai University School of Dentistry, Saitama,

apan; and §Department of Conservative Dentistry, Division oferiodontology, Ohu University School of Dentistry, Fuku-hima, Japan.

Address requests for reprints to Dr Keiso Takahashi, Divi-ion of Periodontology, Department of Conservative Dentistry,hu University School of Dentistry, 31-1, Misumido, Tomita-achi, Koriyama, Fukushima 963-8611, Japan. E-mail address:

[email protected]/$0 - see front matter

Copyright © 2008 by the American Association ofndodontists.oi:10.1016/j.joen.2008.02.021

d

OE — Volume 34, Number 6, June 2008

he mechanical debridement and instrumentation of root canal system are crucial forthe reduction of intracanal infection. In addition, iontophoresis for canal disinfec-

ion are still used in some cases as an adjunct tool for nonsurgical endodontic therapy1) because of the difficulties eradicating microorganisms from dentinal tubes bynstrumentation alone (2). However, the clinical effectiveness of iontophoresis is stillontroversial because of possible side effects. In fact, many patients experience post-perative pain and prolonged inflammation after this treatment.

Iontophoresis is a drug delivery method that uses electric current to facilitate theocal delivery of medicaments. Recently, it has been reported that iontophoresis in-reased the transdentinal delivery of several proteins that may inhibit invasive cervicalesorption (3). We have previously shown that direct current (DC) with antibacterialgents used for iontophoresis-induced necrotic cell death (4) and the DC-inducedytotoxicity may be because of the generated free radicals and metal ions released frometal electrodes (5). These results may explain the reason of the side effects of ionto-

horesis. Therefore, it is important to consider how we may prevent DC-induced cyto-oxicity of host cells in the periapical regions to reduce potential harmful effects ofontophoresis.

We have also shown that different types of free radicals were generated from metallectrodes as follows, alkoxyl radical (St, Ni-Ti electrode), hydrogen radical (Ag and Aulectrode), and both carbon and alkoxyl radicals (Zn electrode), respectively (5). Bothree radicals and metal ions are a potential cause of oxidative stress on various cells (6).

etal ions generate intracellular oxidative stressors such as peroxides and superoxideshat can cause significant cellular damage including DNA damage (7). Therefore, it ismportant to investigate the underlying mechanism of DC-induced cytotoxicity so as toevelop safe iontophoretic nonsurgical endodontic treatment.

It has been reported that antioxidants inhibit or stimulate the cytotoxic effect ofree radicals, depending on the experimental conditions (8). There are many differentypes of antioxidants including enzymatic (superoxide dismutase, catalase, and coen-yme Q10), nonenzymatic (vitamin C), and thiol antioxidants (glutathione [GSH] and-acetyl-L-cysteine [NAC]) and metal chelator EDTA. A possible protective role ofntioxidants on the metal-induced cytotoxicity has been reported (9). Antioxidantsarticipate in significant cellular processes including the protection of cells from toxicompounds, oxidative damage, and radiation (10 –12). Therefore, we hypothesizedhat host cells can be protected from the DC-induced cytotoxicity by antioxidants.

To test this hypothesis, we have compared protective effects of various antioxidantsn the DC-induced cytotoxicity against human polymorphnuclear cells (PMNs). Weave also investigated the effect of GSH or NAC, which gave the highest protection againstC-induced cytotoxicity with different electrodes. Furthermore, we have morphologi-ally investigated the effect of GSH on PMNs survival from DC-induced cytotoxicity.

Materials and Methodseagents

The following chemicals and reagents were obtained from the indicated compa-ies: RPMI 1640 medium and fetal bovine serum (Gibco, Grand Island, NY); Mono-polyesolving medium (Dainippon Pharmaceutical Co Ltd, Osaka, Japan); NAC and catalaseTokyo Kasei Co Ltd, Tokyo, Japan); GSH, coenzyme-Q10 (CoQ10), and vitamin C (VC)Kyowa Hakko Kogyo Co Ltd, Tokyo, Japan); superoxide dismutase (SOD), EDTA, and
eferoxamine mesylate (Sigma Chem Co, St Louis, MO); L-buthionine sulfoximine
Glutathione Can Prevent DC-induced Cytotoxicity 693

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BSO) (Nakarai Kyoto, Japan); 5, 5-dimethyl-1-pyrroline-N-oxide andell counting kit (Total Glutathione Quantification Kit; Dojin, Kum-moto, Japan).

lectrodesThe following metal electrodes were obtained from the indicated

ompanies: Au (pure gold) (Toku Co, Tokyo, Japan), Ag (silver) and ZnShowa Pharmaceutical Ind, Co, Tokyo, Japan), and St (stainless steel)Dentsply-Sankin, Tokyo, Japan).

reparation of Human Peripheral Blood PMNsHuman peripheral blood PMNs were prepared according to our

revious report (4). This procedure yielded a PMN population of99% purity, as judged by Trypan blue dye exclusion and histochem-

cal staining, respectively.

he Treatment of DCThe PMNs were suspended at 2 � 106/mL in RPMI 1640 medium

upplemented with 10% fetal bovine serum in the 24-well culture plateFalcon 3047; Becton Dickinson, Franklin Lakes, NJ) and subjected toC (2 mA) for the indicated periods with occasional agitation at 25°C,sing KANTOP Jr (Showa Pharmaceutical Ind, Co) as described previ-usly (4). The cells were then incubated in the preconditioned mediumor the indicated periods of time before being tested for cell viability asescribed later.

ell ViabilityAfter incubation, 1 volume of trypan blue (0.4%, Sigma) was

dded to 1 volume of PMNs. After incubation at room temperature for 3inutes, the cells were counted in a hemocytometer. All counts were

erformed in triplicate.

ytotoxic AssayThe cytotoxic activity of the DC treatment against PMNs was as-

igure 1. The protective effects of NAC on the DC-induced cytotoxicity with fouourse of DC (2 mA)-induced cytotoxicity with four different electrodes: (A) A

essed, with the cell counting kit as described previously (4, 5). Briefly, A

94 Nakamura et al.

00 �L of the cell suspensions (5 � 105 cells/well) were transferred tohe 96-microwell culture plate (Falcon 3072, Becton Dickinson) andncubated for 4 hours with the cell counting kit. The absorbance washen measured at 540 nm by using microplate reader (Dainippon Phar-

aceutical Co).

he Measurement of Cellular GSH Levels after DC TreatmentCellular glutathione was measured by using the Total Glutathione

uantification Kit as described later. PMNs (2 � 106) were centrifugedt 200g for 10 minutes at 4°C, and the supernatants were discarded.fter rinsing in phosphate-buffered saline (PBS), the cell pellets were

ysed with 80 �L of 10 mmol/L HCl, and then freezing and thawing wereepeated three times. Five percent sodium sulfate anhydrous (20 �L)Wako Co, Osaka, Japan) was added and centrifuged at 8,000g for 10inutes at 4°C. The supernatant was transferred to a new tube and used

or the total glutathione assay. Twenty microliters of enzyme workingolution, 140 �L of coenzyme working solutions, and 20 �L of eitherne of the GSH standard solution or sample solution were added to theach well in 96-well culture plate (Falcon 3072, Becton Dickinson).he plates were incubated at 37°C for 10 minutes and 20 �L of substrateorking solution was added and then incubated at room temperature

or 5 to 10 minutes. The absorbance of each plate was then measured at05 nm by using a microplate reader. The concentration of GSH in theample solution was determined by using a calibration curve.

ransmission Electron MicroscopyThe PMNs were exposed to DC treatment (2 mA, 5 minutes) with

he St electrode in the presence or absence of GSH (5 mmol/L). Samplesere rinsed twice in PBS and then fixed at room temperature for 40inutes in 2.5% glutaraldehyde in 0.1 mol/L of cacodylate buffer

pH7.4; Nissin EM Co Ltd, Tokyo, Japan). After rinsing in PBS, the cellsere fixed in the 1% osmium teroxide (Nissin EM Co). The samplesere dehydrated through a graded series of ethanol and embedded in

ent electrodes. PMNs were treated with DC for the indicated period. The timeAg, (C) St, and (D) Zn. �, with NAC; Œ, without NAC. *p � 0.01.

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raldite 502 (Nissin EM Co). Thin sections were cut with a diamond

JOE — Volume 34, Number 6, June 2008

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nife and doubly stained for 10 minutes in oxalic acid and ulanyl ace-ate. Specimens were observed by using a JEOL JEM-1210 electron

icroscope at an accelerating voltage of 100 kV (JEOL, Tokyo, Japan).

tatistical AnalysisIn the cytotoxic assay, each cell number represents mean � stan-

ard deviation from three independent wells. Most of the data are rep-esentative of three individual experiments with similar results. Differ-nces between the two groups were statistically treated by using annpaired Student t test.

Resultshe Protective Effects of NAC on DC-Induced Cytotoxicityith Metal Electrodes

The cytotoxic effects of DC (2 mA, 5 minutes) with 4 different metallectrodes and the protective effect of NAC (5 mmol/L) on the DC-nduced cytotoxicity, respectively, are shown in Figure 1. NAC protectedhe DC-induced cytotoxicity with all 4 electrodes (p � 0.01). The pro-ective effect of NAC on the DC-induced cytotoxicity with different metallectrodes was varied and was minimal with the Zn electrode among the

igure 2. (A) Comparison of protecting effects of antioxidants on the DC-induceith the St electrode for 5 minuets alone (�) or in the presence of GSH (5 mmo/mL), EDTA (1 mmol/L), and SOD (100 U/mL), respectively, or no DC treatmef DC-induced cytotoxicity in PMNs. : BSO alone, : BSO with GSH (5 mmoith DC (the St electrode) in the presence of increasing concentration of Bemocytometer after Trypan blue staining. (C) The decrease of intracellular Gndicated period with 2 mA � 5 minutes of DC, and [GSH]i was determined. *

electrodes. c


he Comparison of the Protective Effect of Antioxidants onC-Induced Cytotoxicity

The comparison of the protective effect of various antioxidants onhe DC-induced cytotoxicity with the St electrode against PMNs is shownn Figure 2A. Both GSH and NAC significantly protected the DC-inducedytotoxicity (p � 0.01), ascorbic acid and coenzyme Q10 moderatelyid, and the other antioxidants, SOD (100 U), and catalase (3,000/mL) did not. EDTA (1 mmol/L) showed little effect.

he Protective Effect of Glutathione on DC-Inducedytotoxicity

The DC-induced cytotoxic effect was significantly enhanced withSO (1, 0.1, and 0.01 mmol/L) (p � 0.05), and this effect was signif-

cantly neutralized with exogenous GSH (5 mmol/L) (p � 0.01, Fig.B). In addition, DC treatment (2 mA) with the ST electrode significantlyeduced the intracellular GSH levels in a time-dependent manner (p �.05, Fig. 2C).

ransmission Electron MicroscopyThe DC-treatment induced ultrastructural changes such as inten-

ive vacuolization and the accumulation of cellular debris in PMNs (Fig.A). Exogenous GSH inhibited the DC-induced cellular morphological

toxicity with the ST electrode. PMNs were treated with DC (2 mA � 5 minutes)AC (5 mmol/L ), Co-Q10 (250 �g/mL), vitamin C (5 mmol/L), catalase (3,000

a control. *p � 0.01. (B) The counteraction of GSH against BSO enhancementp � 0.01, **p � 0.05. PMNs were incubated for 1 hour without (control) orhout or with GSH (5 mmol/L), and the viable cell number was counted byncentration by DC treatment with the St electrode. PMNs were treated for the.01, **p � 0.05.

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ron-dense granules in the cytoplasm and mitochondria (Fig. 3B),hich showed similar morphology to untreated PMNs (Fig. 3C).

DiscussionAlthough iontophoresis for root canal disinfection is sometimes

sed in nonsurgical endodontic treatments, the clinical merit and theossible harmful effect are still controversial. This study indicates thathiol antioxidants such as GSH and NAC effectively protected the PMNsrom DC-induced cytotoxicity, and this effect may depend on intracel-ular GSH levels.

NAC reduced the DC-induced cytotoxicity generated from 4 differ-nt electrodes (Fig. 1). These results support our previous results (5)hat free radicals released from electrodes contribute to the DC-inducedytotoxicity. However, we did not know how much the free radicals arenvolved in the cytotoxic effects because metal ions are also eluted andhe metals could be toxic in some cases. It has been reported that metalons showed host cell cytotoxicity and their effect was also inhibited byntioxidants (7). Metal-mediated formation of free radicals may en-

igure 3. The ultrastructual changes in PMNs after treatment of DC with or withoB) of GSH (5 mmol/L), and the cells were processed for electron microscopym.

ance lipid peroxidation and changes in calcium and sulphydryl ho- D

96 Nakamura et al.

eostasis (7, 13). Chromium and nickel could be toxic at high doses.hese two metal ions were eluted when the St electrode (Fig. 1C) wassed, and thus both metal-induced free radicals and metal ions them-elves may be involved in the cytotoxicity. In contrast, the possible rolef zinc ion in reducing the oxidative stress as an antioxidant has beeneported (14). The DC-induced cytotoxicity with the Zn electrode (Fig.D) was the weakest among the 4 electrodes tested, in which NAChowed the lowest protective efficacy. We have already reported thatodine zinc iodide inhibited alkoxyl radical generation but not carbonadical (5), suggesting that the Zn ion could protect the host cells fromhe DC-induced cytotoxicity by reducing alkoxyl radical generation. TheC-induced cytotoxicity among the four metal electrodes was variedFig. 1); further study to investigate this phenomenon is feasible.

We compared the protective effects of various antioxidants andDTA on the DC-induced cytotoxicity with the St electrode (Fig. 2A).hiol antioxidants, GSH, and NAC showed the highest protective effectmong the antioxidants tested. These compounds contain a free SHroup, and the free SH might play a role in protecting the PMNs from

. PMNs were treated with DC (2 mA, 5 minutes) in the absence (A) or presenceis. Arrows indicates the mitochondria. (C) Negative control. EM scale bar, 500

ut GSHanalys

C-induced cytotoxicity. Glutathione is an intracellular molecule that

JOE — Volume 34, Number 6, June 2008

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ids in limiting cell damage by oxidative stress. Further study is requiredo elucidate the mechanism by which these antioxidants inhibit celleath. This study also showed that the DC-induced cytotoxicity was notffectively neutralized by catalase, SOD, or EDTA. EDTA, a metal ionhelator, showed little effect on the DC-induced cytotoxicity. In thistudy, both CoQ10 and ascorbate acted as cytoprotective antioxidantsFig. 2A). CoQ10 is recognized as a human dietary supplement thatarticipates in electron transport (15). It has been reported that ascor-ic acid (vitamin C) acts both as an antioxidant and as an oxidant,epending on the environment in which the molecule is present (8).

We have also investigated the effect of BSO to confirm the protec-ive role of GSH on the DC-induced cytotoxicity (Fig. 2B). BSO is annhibitor of �-glutamycysteine synthetase (16), which has been widelysed to inhibit GSH synthesis. BSO enhanced the DC-induced cytotox-city, which was reversed by exogenous GSH. The result indicated thatC-induced cytotoxicity has a relevance to intracellular GSH. We havelso shown that DC treatment reduces the intracellular GSH concentra-ion (Fig. 2C). These results suggest that the amounts of intracellularSH play a crucial role on the cell survival against DC-induced cytotox-

city. Furthermore, it is feasible to use GSH or NAC when undertakingontophoresis in endodontic therapy to reduce cytotoxicity against hostells.

Reactive oxygen species and metal ions can oxidize proteins, androtein conformation leads to upregulation of several signaling cas-ades and activates several redox-regulated transcription factors acti-ator protein-1, nuclear factor kappa B, nuclear factor of activated-cells (17). Therefore, further study is required to investigate the pos-ible signal transduction system that is involved in DC-induced cytotox-city involving metal ions and free radical.

Transmission electron microscopy showed that exogenous GSHnhibited the DC-induced cellular changes (Fig. 3). It is likely that ex-genous GSH was efficiently transported into PMNs and converted tontracellular GSH. These results suggest that DC-induced cytotoxicityppears to be mediated, at least in part, by the generation of reactivexygen species and the consequent depletion of intracellular GSH. Weould observe mitochondria in the DC-treated PMNs after the additionf GSH (arrowhead in Fig. 3B). Mitochondria are the major site of freeadical generation and are enriched with antioxidants including GSH,oQ10 and SOD, which are present on both sides of their membranesimed at minimizing oxidative stress in this organelle (18). The protec-
ive effects of GSH against DC-induced cytotoxicity may provide the basis

or a therapeutic strategy to protect host cells around periapical lesionsxposed to DC.

We showed the possibility that GSH may efficiently inhibit ionto-horesis-induced cytotoxic effect against host cells in the periapical

esions. GSH may inhibit posttreatment inflammatory reaction and thenduced pain. Further clinical study to investigate the usefulness ofxogenous GSH at iontophoresis will be required.

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4. Nakamura Y, Takahashi K, Shimetani A, Sakagami H, Nishikawa H. Cytotoxicity ofdirect current with antibacterial agents against host cells in vitro. J Endod2005;31:755– 8.

5. Nakamura Y, Takahashi K, Satoh K, Shimetani A, Sakagami H, Nishikawa H. Role offree radicals and metal ions in direct current-induced cytotoxicity. J Endod2006;32:442– 6.

6. Kasprzak KS. Possible role of oxidative damage in metal-induced carcinogenesis.Cancer Invest 1995;13:411–30.

7. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem2005;12:1161–208.

8. Sakagami H, Satoh K. Modulating factors of radical intensity and cytotoxic activity ofascorbate (review). Anticancer Res 1997;17:3513–20.

9. Satoh K, Sakagami H. Effect of cysteine, N-acetyl-L-cysteine and glutathione on cyto-toxic activity of antioxidants. Anticancer Res 1997;17:2175–9.

0. Meister A. Selective modification of glutathione metabolism. Science 1983;220:472–7.

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2. Kinoshita N, Yamamura T, Teranuma H, et al. Interaction between dental metals andantioxidants, assessed by cytotoxicity assay and ESR spectroscopy. Anticancer Res2002;22:4017–22.

3. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals andantioxidants in normal physiological functions and human disease. Int J Biochem CellBiol 2007;39:44 – 84.

4. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem2005;12:1161–208.

5. Sohal RS, Kamzalov S, Sumien N, et al. Effect of coenzyme Q10 intake on endogenouscoenzyme Q content, mitochondrial electron transport chain, antioxidative defenses,and life span of mice. Free Radic Biol Med 2006;40:480 –7.

6. Meister A. Glutathione metabolism and its selective modification. J Biol Chem1988;263:17205– 8.

7. Leonard SS, Harris GK, Shi X. Metal-induced oxidative stress and signal transduction.Free Radic Biol Med 2004;37:1921– 42.

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glutathione can efficiently prevent direct current–induced cytotoxicity

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