d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) 1532–1540

avai lab le at www.sc iencedi rec t .com

journa l homepage: www. int l .e lsev ierhea l th .com/ journa ls /dema

N-Acetyl cysteine (NAC) inhibits proliferation, collagen genetranscription, and redox stress in rat palatal mucosal cells

N. Satoa, T. Uenoa, K. Kuboa, T. Suzukia, N. Tsukimuraa, W. Atta, M. Yamadaa,N. Horia, H. Maedab, T. Ogawaa,∗

a Laboratory of Bone and Implant Sciences (LBIS), Weintraub Center for Reconstructive Biotechnology, Division of AdvancedProsthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Los Angeles, CA, USAb Department of Pathology, Aichi-Gakuin University, School of Dentistry, Nagoya, Japan

a r t i c l e i n f o

Article history:

Received 14 May 2009

Received in revised form 7 July 2009

Accepted 18 July 2009

Keywords:

Antioxidant

Oxidative stress

Redox balance

Glutathione

Oral mucosa

a b s t r a c t

Objectives. Control of hyperplastic and invasively growing gingival tissue is crucial for

maintaining normal oral function and for successful bone regenerative therapy. We tested

the hypothesis that materials containing N-acetyl cysteine (NAC), an antioxidant cysteine

derivative, can control proliferation and function of oral mucosal cells.

Methods. Oral mucosal cells derived from the rat palatal tissue were cultured with or with-

out NAC at different concentrations (2.5–10.0 mM). To simulate inflammatory conditions,

cultures were treated with hydrogen peroxide. NAC was also applied via collagen materi-

als in membrane and sponge forms to explore the clinical applicability. The redox balance

inside the cells was evaluated by measuring the concentration of intracellular glutathione

(GSH).

Results. Adding NAC into cultures of oral mucosal cells reduced their proliferation, transcrip-

tional expression, and collagen production in an NAC-concentration-dependent manner

without cytotoxic effects. Furthermore, NAC substantially reduced the hydrogen peroxide-

induced elevation of cellular proliferation and collagen production. The controlling effects

of NAC were also demonstrated in cells cultured on NAC-containing collagen materials and

were associated with an increase in intracellular glutathione (GSH) reserves and a decrease

in the oxidized form of glutathione (GSSG).

Significance. These results indicate that NAC may abrogate inflammation- or oxidative-stress-

induced hyperfunction of oral mucosal cells and that it can be delivered effectively via

biodegradable materials. This study provides a basis to explore NAC-containing biomaterials

ed to

emy

capacity of the area. Overgrowth of other mucosal tissues, e.g.,

that are functionaliz

© 2009 Acad

1. Introduction

Current dental treatment often involves management of softtissue. For instance, hypertrophic gingival tissue, often seenaround denture abutments and dental implants, deepens peri-

∗ Corresponding author at: Laboratory for Bone and Implant SciencesBiotechnology, Division of Advanced Prosthodontics, Biomaterials anAvenue (B3-81 CHS), Box 951668, Los Angeles, CA 90095-1668, USA. Tel.

E-mail address: togawa@dentistry.ucla.edu (T. Ogawa).0109-5641/$ – see front matter © 2009 Academy of Dental Materials. Pudoi:10.1016/j.dental.2009.07.006

control oral soft tissue growth and function without cytotoxicity.

of Dental Materials. Published by Elsevier Ltd. All rights reserved.

odontal or peri-implant pockets and impairs the cleansing

(LBIS), The Jane and Jerry Weintraub Center for Reconstructived Hospital Dentistry, UCLA School of Dentistry, 10833 Le Conte: +1 310 825 0727; fax: +1 310 825 6345.

epithelial hyperplasia in the palate growing over the majorconnectors of removable partial dentures, also creates func-tional problems. During wound healing of oral mucosa, tissue

blished by Elsevier Ltd. All rights reserved.

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caring occurs in the areas of incisions and sutures [1], andcontractive force from the tissue growing down into the

xtraction socket could cause postextraction alveolar boneoss [2]. Successful bone augmentation during guided boneegeneration depends on the prevention of existing or graftedoft tissue from invading the space using a barrier membrane3]. Moreover, severe fibroblastic hyperplasia induces alveo-ar bone resorption, compromises aesthetics, and interferes

ith oral functions such as tooth eruption, mastication, andpeech motion [4,5]. Deregulation of cell proliferation, ECMetabolism and cytokine production caused by several eti-

logical factors including inflammation, drugs, and geneticnheritance may be behind these pathological situations [6,7].

Reactive oxygen species (ROS) such as superoxide, hydroxyladical and hydrogen peroxide are generated primarily by

itochondria through normal cellular metabolism, and theirroduction increases under inflammatory conditions. A lowoncentration of oxidants enhances cell proliferation and col-agen production in fibroblasts [8], indicating a close linketween ROS and fibrogenetic disorder. N-Acetyl cysteine

NAC) is an aminothiol cysteine derivative and functions asn antioxidant by scavenging ROS [9]. NAC is also easilyeacetylated into cysteine, an important precursor of cellu-

ar glutathione, and promotes the glutathione redox cycle,hich is one of the important ROS-removing systems. Besides

ts antioxidant effects, NAC attenuates interstitial fibrosis ineveral pathological states such as myocardial hypertrophy9] and suppresses fibroblastic proliferation in hepatic stel-ate cells [10], palmer skin fibroblasts [11], and murine embryoNIH3T3) fibroblasts [12]. However, the effect of NAC on cellunctions other than proliferation, such as gene transcriptionnd collagen synthesis, has not been fully examined. Moremportantly, the effects of NAC on oral soft tissue cells, as wells potential effective forms of NAC in vivo delivery, have rarelyeen investigated.

To understand the potential therapeutic value of NAC inontrolling unfavorable oral soft tissue growth, this studyested the hypothesis that NAC controls the function of oral

ucosal cells and that it can be delivered via biodegradableound healing materials. The objectives of this study were to

xamine the effect of NAC in various concentrations on theroliferation, gene transcription, and collagen production ofral mucosal cells and to determine whether these effects arebtained when NAC is delivered by collagen materials. Theffects of NAC as well as its role in controlling redox balanceere also examined under a challenging condition contain-

ng hydrogen peroxide (H2O2). Adding H2O2 into cell culturesimics the inflammatory status of the biological environment

nd is known to propel fibroblastic proliferation and function13–16]. Lastly, a safely aspect of NAC was addressed by exam-ning the viability of the cells when they are exposed to NAC.

. Materials and methods

.1. Cell culture

ral mucosal cells were obtained from the palatal tissue of 8-eek-old Sprague–Dawley rats. After sacrificing the animals,

he palatal tissue was aseptically removed and washed with

0 0 9 ) 1532–1540 1533

1% phosphate buffered saline (MP Biomedicals, Solon, OH,USA). The tissue was dissected into small pieces and digestedwith 0.25% collagenase for 12 h. The liberated cells were col-lected into Dulbecco’s Modified Eagle’s medium supplementedwith 10% fetal bovine serum and an antibiotic–antimycoticsolution in a humidified atmosphere of 95% air and 5%CO2 at 37 ◦C. At 80% confluency, the cells were inoculatedonto 12-well polystyrene culture dishes at a density of1.5 × 104 cells/cm2. Experimental cultures were prepared byadding NAC at a concentration of 2.5, 5, or 10 mM (per 1 mlculture medium). NAC was prepared as 1 mol stock solution inHEPES buffer whose pH was adjusted 7.2. To simulate inflam-matory conditions, some of the cultures were treated with10 �M H2O2. An addition of H2O2 to cell cultures and tissueshas been used as in vitro and in vivo inflammation models,respectively [13–15]. It has been known that H2O2 readily pen-etrates cells [17], and its low concentrations may be importantto produce positive proliferation signals in certain cell types[16]. We used 10 �M for H2O2 because this concentration sig-nificantly elevated the proliferation of oral mucosal cells inour preliminary study. Renewal of the medium, as well as H2O2

and NAC treatments, were performed every 3 days (continuousNAC treatment). To address the potential clinical applicationof NAC, the cells were seeded onto 5 mg of collagen mem-brane (Bio-Gide, Osteohealth, Shirley, NY) and 3 mg of collagensponge (CollaPlug, Integra, Plainsboro, NJ) that were coatedonto a 24-well polystyrene dish. The collagen materials inexperimental groups were pre-absorbed with NAC to give finalconcentrations of 2.5 and 5 mM NAC when they were filledwith 0.5 ml culture medium. A 0.1 mol NAC stock solution wasprepared in HEPES buffer to facilitate spreading NAC evenlyonto these collagen materials. It was ensured that the colla-gen materials, soaked in an appropriate amount of the NACstock solution (12.5 and 25 �l NAC stock solution for 2.5 and5 mM NAC final concentrations, respectively), absorbed all ofthe solution to give each of the final concentrations when thewells were filled with 0.5 ml of culture medium. The protocolfor this study was approved by the University of California atLos Angeles Animal Research Committee.

2.2. Proliferation assay

Cell proliferative activity was measured by 5-bromodeoxyuridine (BrdU) incorporation during DNAsynthesis. At days 2 and 4 of the culture, 100 �l of 100 mM BrdUsolution (Roche Applied Science, Mannheim, Germany) wasadded into each well and incubated for 10 h. After trypsinizingthe cells and denaturing the DNA, the cultures were incubatedwith anti-BrdU antibody conjugated with peroxidase (RocheApplied Science, Mannheim, Germany) for 90 min and reactedwith tetramethyl benzidine for color development. The colordevelopment was assessed by measuring the absorbanceat a wavelength of 370 nm using an ELISA plate reader. Thenumber of cells was also counted on days 2 and 5 afterseeding. The cells were gently rinsed twice with PBS andtreated with 0.1% collagenase in 300 �l of 0.25% trypsin–1 mM

EDTA–4Na for 15 min at 37 ◦C. A hematocytometer was usedto count the number of detached cells. Because the cultureswere treated with trypsin and collagenase for an extendedperiod of 15 min, we expected all of the cells to be separated

s 2 5

as measured by BrdU incorporation (t-test, p < 0.01; Fig. 3A).

1534 d e n t a l m a t e r i a l

from the substrates regardless of the culture conditions,allowing us to evaluate the density of the propagated cells.Scanning electron micrograms of selected cultures wereobtained to re-confirm the absence of any cell remnants onthe substrates, as performed routinely in our studies [18].

2.3. Cell viability assay

Cell viability was evaluated by flow cytometric detection ofapoptosis using an annexin V-FITC Kit (BD Bioscience, SanJose, CA). This method is based on the measurement of thebinding properties of annexin V to phosphatidylserine and onthe DNA-intercalating capabilities of propidium iodide. Thecells incubated in the culture media with or without 10 mMNAC for 24 h were tested.

2.4. Glutathione detection

Glutathione (GSH) plays an important role in the protectionof cells against ROS. During the process, GSH is convertedinto glutathione disulfide (GSSG), the oxidized form of GSH.Because GSSG is enzymatically reduced by glutathione reduc-tase, GSH is the dominant form in organisms. To quantify totalintracellular glutathione, a 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB)-based total glutathione quantification kit (DojindoMolecular Technologies, Inc., Gaithersburg, MD) was used.Twenty-four hours after seeding, the culture was incubatedwith DTNB and glutathione reductase for 10 min at 37 ◦C.Total GSH concentration was determined by measuring theabsorbance at 412 nm. In some experiments, GSSG was quan-tified by masking the GSH thiols with 2-vinylpyridine [19,20].Pure GSH was then calculated by subtracting GSSG from thetotal GSH.

2.5. Transcriptional analysis for collagen-related genes

The gene transcription was evaluated at days 7 and 14 usingreverse transcription polymerase chain reaction (RT-PCR) asdescribed previously [18,21]. Fibroblastic gene mRNAs such as�-I type I collagen (collagen I), �-I type III collagen (collagenIII), and prolyl-4-hydroxylase-alpha (P4H) were examined.

2.6. Collagen colorimetry

The Sirius red staining-based colorimetric assay was used toquantify total collagen deposition. This method specificallydetects collagen type I and III molecules without detectingother components of the ECM [22]. Cell cultures from days7 and 14 were examined as previously described [18,23]. Tovisualize collagen deposition, day-7 cultures were examinedusing a confocal laser scanning microscope (Carl Zeiss LSM310, Jena, Germany) following dual staining with Sirius redand a green fluorescent nucleic-acid stain (SYTO 13, MolecularProbe, Eugene, OR).

2.7. Statistical analysis

Three independent cultures were prepared for each of theuntreated control and experimental groups for each of theassays. One-way ANOVA, at a significance level of p < 0.05, were

( 2 0 0 9 ) 1532–1540

employed to examine the differences in the variables amongthe different experimental culture conditions with variousNAC concentrations. When necessary, the Bonferroni multiplecomparison test was used as a post hoc evaluation. To com-pare the differences between two groups, i.e., the cultures withand without H2O2, a t-test was used.

3. Results

3.1. NAC decreases cell proliferation of oral mucosalcells without cytotoxicity

Cell density at days 2 and 5 decreased in a concentration-dependent manner when NAC was added to the cultures(1-way ANOVA, p < 0.05; Fig. 1A). At day 5, the number ofcells in the culture supplemented with 2.5 mM NAC washalved compared to the number in the untreated culture,whereas the cell number in the culture with 10 mM NACwas less than 20% of that in the untreated culture (1-wayANOVA, p < 0.01). Consistent with the cell density results,BrdU incorporation decreased in an NAC-concentration-dependent manner at days 2 and 4 (1-way ANOVA, p < 0.01;Fig. 1B).

To determine whether the NAC-induced reduction of cellproliferation was due to its possible cytotoxicity, we examinedcell viability with and without NAC in the cultures. The per-centage of viable cells increased by approximately 10% after a24-h culture with 10 mM NAC (1-way ANOVA, p < 0.05; Fig. 1C).Accordingly, the percentages of necrotic and apoptotic cellswere reduced significantly in the NAC culture (p < 0.05).

3.2. NAC decreases fibroblastic gene transcription andcollagen production in oral mucosal cells

The transcription of the collagen-related genes tested wasdownregulated and total collagen deposition decreased inan NAC-concentration-dependent manner (p < 0.05 and <0.01,respectively, 1-way ANOVA; Fig. 2A, left histogram in Fig. 2B).For instance, the addition of 10 mM NAC reduced collagendeposition to approximately 30% of the baseline level onculture days 7 and 14. The collagen deposition standard-ized relative to the number of the cells also decreased inan NAC-concentration-dependent manner (1-way ANOVA,p < 0.05; right histogram in Fig. 2B). Representative fluorescentimages of the cells after Sirius red staining confirmed thatNAC reduced extracellular deposition of collagen, as well asthe intracellular production of collagen molecules (top imagesin Fig. 2B)

3.3. NAC abrogates H2O2-induced hyperproliferationof oral mucosal cells

Treatment with 10 �M H2O2 increased the proliferative activ-ity of the cells by 40% and 35% at days 2 and 4, respectively,

However, there was no such promotive effect, when H2O2 andNAC were added to the cultures. The addition of NAC reducedthe rate of cell proliferation dose-dependently (1-way ANOVA,p < 0.01), regardless of H2O2.

d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) 1532–1540 1535

Fig. 1 – Proliferative activity and viability of rat oral mucosal cells treated with N-acetyl cysteine (NAC). (A) Cell densitymeasured by a hematocytometer in untreated control cultures and cultures supplemented with NAC at variousconcentrations. (B) Proliferation rate measured by BrdU incorporation during mitosis. (C) Results of oral mucosal cell viability24 h after seeding with and without 10 mM NAC. Representative flow cytometric images as well as the percentages ofapoptotic cells (quadrant 4 in the images), necrotic cells (quadrant 2) and viable cells (quadrant 3) are shown. The data ares indie

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hown as mean ± SD (n = 3) for all panels. *p < 0.05, **p < 0.01,xperimental groups (1-way ANOVA).

.4. NAC abrogates H2O2-induced hyperproduction ofollagen

reatment with 10 �M H2O2 significantly increased the deposi-ion of collagen by 10% on day 7 (t-test, p < 0.05; Fig. 3B). Whenhe cultures were co-treated with NAC, there was a consistentAC-concentration-dependent reduction of collagen deposi-

ion (p < 0.01, ANOVA), but there were no differences betweenhe cultures with and without H2O2.

.5. NAC-mediated improvement of glutathione redoxalance

dding NAC increased the amount of the reduced formf glutathione (GSH) in a concentration-dependent man-er (1-way ANOVA, p < 0.01; Fig. 3C). Adding H2O2 further

ncreased the GSH level (p < 0.05), except in the culture with0 mM NAC. Treatment of the cells with H2O2 alone substan-ially increased the level of the oxidized form of glutathioneGSSG) (p < 0.01, t-test); however, when the cells were treated

ith NAC, no GSSG was detected, regardless of the H2O2

reatment (Fig. 3C). The percentage of GSSG relative to the

otal glutathione (GSH + GSSG) increased from approximately0% to 60% by the addition of H2O2 (Fig. 3D). However,t remained at the baseline level of 0 after co-treatment

ith NAC.

cating a statistically significant difference among the

3.6. Effective suppression of fibrogenetic phenotypesby collagen vehicle-delivered NAC

The cells cultured on the collagen membrane that contained2.5 or 5 mM NAC proliferated at a significantly lower rate thanthe cells cultured on the untreated collagen membrane atdays 2 and 4 (1-way ANOVA, p < 0.05; Fig. 4A). Likewise, NACabsorbed in the collagen sponge was effective in reducing therate of proliferation in a concentration-dependent manner (1-way ANOVA, p < 0.05; Fig. 4B). The transcriptional expressionof the collagen-related genes tested was significantly down-regulated on the 5 mM NAC-containing collagen substrates(p < 0.05), except for collagen III expression on the collagensponge (Fig. 4C). The total glutathione level increased in asso-ciation with an increase in the NAC absorbed into the collagenvehicles (p < 0.05, 1-way ANOVA; Fig. 4D).

4. Discussion

The present study demonstrated that NAC treatment of oralmucosal cells resulted in a dose-dependent decrease in cell

proliferation and function, as represented by the downregu-lated gene transcription and reduced collagen production anddeposition. Moreover, these suppressive effects were found tobe effective in an inflammatory condition simulated by H2O2

1536 d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) 1532–1540

Fig. 2 – Functional phenotypes of oral mucosal cells treated with N-acetyl cysteine (NAC). (A) Expression of collagen I,collagen III and prolyl-4 hydroxylase (P4H) genes analyzed by reverse transcription polymerase chain reaction (RT-PCR).Upper panel shows representative electrophoresis images visualized with ethidium bromide staining. Lower panel showsthe intensity of the target gene bands normalized to the GAPDH expression level. Data are mean ± SD (n = 3). (B) Totalcollagen deposition, as well as the collagen deposition standardized relative to the cell number, evaluated by Sirius redstaining-based colorimetry (histograms). Data are mean ± SD (n = 3). Representative confocal laser scanning microscopicimages of day 7 cultures following dual staining with Sirius red and a green fluorescent nucleic-acid stain (SYTO 13) are alsopresented. *p < 0.05, **p < 0.01, indicating a statistically significant difference among the experimental groups (1-wayANOVA).

d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) 1532–1540 1537

Fig. 3 – Oral mucosal cell function and redox status with N-acetyl cysteine (NAC) and H2O2 co-treatment. (A) Proliferationrate measured by BrdU incorporation during mitosis. The cultures with and without 10 �M H2O2 were treated with variousconcentrations of NAC. (B) Total collagen deposition evaluated by Sirius red staining-based colorimetry. (C) Levels ofintracellular GSH (the reduced form of glutathione) and GSSG (the oxidized form of glutathione) standardized to cell number,evaluated by DTNB (5,5′-dithiobis(2-nitrobenzoic acid))-based enzymatic reaction 24 h after seeding. (D) A GSSG ratio to totalglutathione (GSH + GSSG) varying with and without H2O2 at various NAC concentrations. Data are mean ± SD (n = 3) for allp t dif(

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anels. *p < 0.05, **p < 0.01, indicating a statistically significant-test).

reatment; NAC abrogated H2O2-induced hyperproliferationnd collagen deposition. Such suppressive effect was not dueo cytotoxicity of NAC. On the contrary, the viability of theral mucosal cells was even higher with NAC in the cultures,hich is consistent with the other cytoprotective effects ofAC against various exotic stimuli such as stress injuries [24]nd oxidative stress from medications and foreign materials9,25–28].

The reduced proliferation of oral mucosal cells may beelated to the reported inhibition of the mitogen-activated pro-ein (MAP) kinase pathway [12] and TGF-beta signaling [11]y NAC, which results in reduced proliferation of fibroblas-ic cells. Studies have also shown that treatment of fibroblastsith NAC decreases the expression of cyclin D1 protein, which

s responsible for an arrest of G(1) phase of cell proliferation29]. The G(1) arrest is linked with NAC-blocked activationf MAP kinases p42MAPK and p44MAPK [12]. Further, an

xidative stress–antioxidant interaction regulates activationnd phosphorylation of extracellular signal-regulated proteininase (pERK), which plays an important role in fibroblasticroliferation [30]. Nuclear factor E2-related factor-2 (Nrf2) is a

ference between the cultures with and without 10 �M H2O2

transcription factor orchestrating antioxidant and cytoprotec-tive responses against oxidative stress [31]. Nrf2 binds to theantioxidant response element (ARE) in the regulatory regionsof multiple antioxidant genes and activates the expressionof these genes [31]. A critical role of Nrf2 in maintaining theGSH redox state was demonstrated in Nrf2-deficient mice [32].Proven antiinflammatory effects of Nrf2 in many cell types,such as vascular cells and pulmonary cells, imply that Nrf2could be a potential therapeutic target for pathophysiology inwhich inflammation and oxidative stress play a role [33–35].Nrfs may inhibit cell proliferation via a mechanism indepen-dent of the nuclear factor kappa B (NF-kappa B) inhibition [36].In this study, NAC inhibited the cell proliferation in associa-tion with the remarkable modulation of the redox status, i.e.,the GSH/GSSG ratio. To identify the mechanism underlying theNAC-induced inhibition of cellular proliferation, future stud-ies should pay a particular attention to the effect of NAC on the

expression and activation of these transcription factors underconditions with or without oxidative stress.

Little information is available on the mechanism concern-ing the suppressive effect of NAC on fibroblastic function,

1538 d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) 1532–1540

Fig. 4 – The effects of NAC delivered via biomaterials and at lower concentrations. (A–D) Effects of collagen vehicle-carriedNAC on oral mucosal cell function. Proliferation rate measured by BrdU incorporation in oral mucosal cells cultured on acollagen membrane (A) and collagen sponge (B) with or without the pre-absorption of NAC. (C) Expression of collagen I,collagen III and prolyl-4 hydroxylase (P4H) genes analyzed by reverse transcription polymerase chain reaction (RT-PCR).Upper panel shows representative electrophoresis images visualized with ethidium bromide staining. Lower panel showsthe intensity of the target gene bands normalized to the GAPDH expression level. (D) Intracellular glutathione concentration

ll pa

standardized to cell number. Data are mean ± SD (n = 3) for adifference among the experimental groups (1-way ANOVA).

as represented by the downregulated gene expression andimpaired collagen production. A high-throughput cDNAanalysis of NAC-treated keratinocytes revealed the downregu-lation of a large number of genes involved in cell proliferationand regulation of the cell cycle, and an upregulation of genesinvolved in cytoskeletal reorganization [37]. However, therewas no significant alteration of transcription pattern to implya regulatory mechanism for collagen-related genes. The GSHredox status (e.g., GSH/GSSG ratio) has been reported to play animportant role in the differentiation and phenotypic expres-sion of some cell types [38–42]. Moreover, it is hypothesizedthat NAC affects or inhibits activation of redox-sensitive tran-scription factors, such as activator protein-1 (AP-1), c-JunN-terminal kinase, and nuclear factor kappa B (NF-�B), pre-

sumably by altering intracellular redox status [9,17,43,44].These transcriptional factors are suggested to play a key rolein mediating cell differentiation [45–47]. The present studyrevealed that adding NAC dramatically changed the GSH/GSSG

nels. *p < 0.05, **p < 0.01, indicating a statistically significant

ratio regardless of H2O2. Future studies should explore themechanism underlying the NAC-induced suppressive pheno-type of oral mucosal cells.

The addition of 10 �M H2O2 significantly increased theproliferation of oral mucosal cells as we expected. The sig-nificance of our findings is that the H2O2-induced increasein cell proliferation and collagen production was reducedto a normal level or even lower by NAC, indicating a com-plete abrogation of H2O2-indued inflammatory effects. Theseresults were associated with the findings that the GSSG levelsubstantially increased by H2O2 was reduced to the baselinelevel of 0 by NAC co-treatment and that, in contrast, the GSHlevel increased by NAC. These imply a potential role for NACas an immediate antioxidant scavenger for H2O2 and a cys-

teine provider for the antioxidant redox system, which couldprovide a pharmacodynamic basis for a therapeutic strategyagainst fibroproliferative disorders in oral soft tissues. We alsofound that adding H2O2 increased intracellular GSH (reduced

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orm of glutathione). Oxidative stress, unless in over-dose,nhances the uptake of cysteine in human fibroblasts [48],hich results in the increased synthesis of intracellular GSH

49]. It seems that an ample supplement of cysteine couldllow for more effective H2O2 stress-induced GSH synthesis.

Surgical wounds are subjected to various oxidativetressors under inflammatory conditions. The exploratoryxperiments in this study showed the successful inhibitionf proliferation and collagen production in oral mucosal cellsy NAC when it was delivered via collagen carriers. This mayrovide an opportunity to explore the possible use of NAC inonjunction with biomaterials in in vivo to control unfavor-ble soft tissue reaction. Simultaneously, the effect of NACn osteoblasts and other cell types needs to be addressed.n conclusion, the addition of NAC in the culture of ratalatal mucosal cells inhibited their cell proliferation, andene transcription and production of collagens without cyto-oxicity. Similar effects were confirmed when the cells wereultured on biodegradable collagen materials containing NAC.oreover, NAC was effective in ameliorating H2O2-induced

yperproliferation and hyperproduction of collagen in theells.

cknowledgement

his work was supported by DRP Co., Ltd.

e f e r e n c e s

[1] Velvart P, Peters CI. Soft tissue management in endodonticsurgery. J Endod 2005;31:4.

[2] Sukotjo C, Lin A, Song K, Ogawa T, Wu B, Nishimura I. Oralfibroblast expression of wound-inducible transcript 3.0(wit3.0) accelerates the collagen gel contraction in vitro. JBiol Chem 2003;278:51527.

[3] Schwartzmann M. Use of collagen membranes for guidedbone regeneration: a review. Implant Dent 2000;9:63.

[4] Kavvadia K, Pepelassi E, Alexandridis C, Arkadopoulou A,Polyzois G, Tossios K. Gingival fibromatosis and significanttooth eruption delay in an 11-year-old male: a 30-monthfollow-up. Int J Paediatr Dent 2005;15:294.

[5] Baptista IP. Hereditary gingival fibromatosis: a case report. JClin Periodontol 2002;29:871.

[6] Trackman PC, Kantarci A. Connective tissue metabolism andgingival overgrowth. Crit Rev Oral Biol Med 2004;15:165.

[7] Nakao K, Yoneda K, Osaki T. Enhanced cytokine productionand collagen synthesis of gingival fibroblasts from patientswith denture fibromatosis. J Dent Res 1995;74:1072.

[8] Liu RM, Liu Y, Forman HJ, Olman M, Tarpey MM. Glutathioneregulates transforming growth factor-beta-stimulatedcollagen production in fibroblasts. Am J Physiol Lung CellMol Physiol 2004;286:L121.

[9] Zafarullah M, Li WQ, Sylvester J, Ahmad M. Molecularmechanisms of N-acetylcysteine actions. Cell Mol Life Sci2003;60:6.

[10] Meurer SK, Lahme B, Tihaa L, Weiskirchen R, Gressner AM.N-Acetyl-l-cysteine suppresses TGF-beta signaling at

distinct molecular steps: the biochemical and biologicalefficacy of a multifunctional, antifibrotic drug. BiochemPharmacol 2005;70:1026.

[11] Kopp J, Seyhan H, Muller B, Lanczak J, Pausch E, GressnerAM, et al. N-Acetyl-l-cysteine abrogates fibrogenic

0 0 9 ) 1532–1540 1539

properties of fibroblasts isolated from Dupuytren’s diseaseby blunting TGF-beta signalling. J Cell Mol Med 2006;10:157.

[12] Sekharam M, Trotti A, Cunnick JM, Wu J. Suppression offibroblast cell cycle progression in G1 phase byN-acetylcysteine. Toxicol Appl Pharmacol 1998;149:210.

[13] Mapp PI, Walsh DA, Garrett NE, Kidd BL, Cruwys SC, PolakJM, et al. Effect of three animal models of inflammation onnerve fibres in the synovium. Ann Rheum Dis 1994;53:240.

[14] Griffiths HR, Dowling EJ, Sahinoglu T, Blake DR, Parnham M,Lunec J. The selective protection afforded by ebselen againstlipid peroxidation in an ROS-dependent model ofinflammation. Agents Actions 1992;36:107.

[15] Spillert CR, Pelosi Jr MA, Parmer LP, Lazaro EJ. Aperoxide-induced inflammation model for drug testing.Agents Actions 1987;21:297.

[16] Burdon RH. Superoxide and hydrogen peroxide in relation tomammalian cell proliferation. Free Radic Biol Med1995;18:775.

[17] Kim KY, Rhim T, Choi I, Kim SS. N-Acetylcysteine inducescell cycle arrest in hepatic stellate cells through its reducingactivity. J Biol Chem 2001;276:40591.

[18] Saruwatari L, Aita H, Butz F, Nakamura HK, Ouyang J, Yang Y,et al. Osteoblasts generate harder, stiffer, and moredelamination-resistant mineralized tissue on titanium thanon polystyrene, associated with distinct tissue micro- andultrastructure. J Bone Miner Res 2005;20:2002.

[19] Griffith OW. Determination of glutathione and glutathionedisulfide using glutathione reductase and 2-vinylpyridine.Anal Biochem 1980;106:207.

[20] Teare JP, Punchard NA, Powell JJ, Lumb PJ, Mitchell WD,Thompson RP. Automated spectrophotometric method fordetermining oxidized and reduced glutathione in liver. ClinChem 1993;39:686.

[21] Ogawa T, Nishimura I. Different bone integration profiles ofturned and acid-etched implants associated with modulatedexpression of extracellular matrix genes. Int J OralMaxillofac Implants 2003;18:200.

[22] Tullberg-Reinert H, Jundt G. In situ measurement of collagensynthesis by human bone cells with a sirius red-basedcolorimetric microassay: effects of transforming growthfactor beta2 and ascorbic acid 2-phosphate. Histochem CellBiol 1999;112:271.

[23] Takeuchi K, Saruwatari L, Nakamura HK, Yang JM, Ogawa T.Enhanced intrinsic biomechanical properties of osteoblasticmineralized tissue on roughened titanium surface. J BiomedMater Res A 2005;72A:296.

[24] Neal R, Cooper K, Gurer H, Ercal N. Effects ofN-acetylcysteine and 2,3-dimercaptosuccinic acid on leadinduced oxidative stress in rat lenses. Toxicology1998;130:167.

[25] Kojima N, Yamada M, Paranjpe A, Tsukimura N, Kubo K,Jewett A, et al. Restored viability and function of dental pulpcells on poly-methylmethacrylate (PMMA)-based dentalresin supplemented with N-acetyl cysteine (NAC). DentMater 2008;24:1686.

[26] Schweikl H, Hartmann A, Hiller KA, Spagnuolo G, Bolay C,Brockhoff G, et al. Inhibition of TEGDMA and HEMA-inducedgenotoxicity and cell cycle arrest by N-acetylcysteine. DentMater 2007;23:688.

[27] Att W, Yamada M, Kojima N, Ogawa T. N-Acetyl cysteineprevents suppression of oral fibroblast function onpoly(methylmethacrylate) resin. Acta Biomater2009;5:391.

[28] Yamada M, Kojima N, Paranjpe A, Att W, Aita H, Jewett A, et

al. N-Acetyl cysteine (NAC)-assisted detoxification of PMMAresin. J Dent Res 2008;87:372.

[29] Menon SG, Sarsour EH, Kalen AL, Venkataraman S, HitchlerMJ, Domann FE, et al. Superoxide signaling mediates

s 2 5

transport activity in human fibroblasts by oxygen. J Biol

1540 d e n t a l m a t e r i a l

N-acetyl-l-cysteine-induced G1 arrest: regulatory role ofcyclin D1 and manganese superoxide dismutase. Cancer Res2007;67:6392.

[30] Pat B, Yang T, Kong C, Watters D, Johnson DW, Gobe G.Activation of ERK in renal fibrosis after unilateral ureteralobstruction: modulation by antioxidants. Kidney Int2005;67:931.

[31] Chen XL, Kunsch C. Induction of cytoprotective genesthrough Nrf2/antioxidant response element pathway: a newtherapeutic approach for the treatment of inflammatorydiseases. Curr Pharm Des 2004;10:879.

[32] Harvey CJ, Thimmulappa RK, Singh A, Blake DJ, Ling G,Wakabayashi N, et al. Nrf2-regulated glutathione recyclingindependent of biosynthesis is critical for cell survivalduring oxidative stress. Free Radic Biol Med 2009;46:443.

[33] Cho HY, Reddy SP, Yamamoto M, Kleeberger SR. Thetranscription factor NRF2 protects against pulmonaryfibrosis. FASEB J 2004;18:1258.

[34] Levonen AL, Inkala M, Heikura T, Jauhiainen S, JyrkkanenHK, Kansanen E, et al. Nrf2 gene transfer inducesantioxidant enzymes and suppresses smooth muscle cellgrowth in vitro and reduces oxidative stress in rabbit aortain vivo. Arterioscler Thromb Vasc Biol 2007;27:741.

[35] Siow RC, Ishii T, Mann GE. Modulation of antioxidant geneexpression by 4-hydroxynonenal: atheroprotective role ofthe Nrf2/ARE transcription pathway. Redox Rep 2007;12:11.

[36] Kim JY, Cho HJ, Sir JJ, Kim BK, Hur J, Youn SW, et al.Sulfasalazine induces haem oxygenase-1 viaROS-dependent Nrf2 signalling, leading to control ofneointimal hyperplasia. Cardiovasc Res 2009;82:550.

[37] Edlundh-Rose E, Kupershmidt I, Gustafsson AC, Parasassi T,Serafino A, Bracci-Laudiero L, et al. Gene expression analysisof human epidermal keratinocytes after N-acetyl l-cysteine

treatment demonstrates cell cycle arrest and increaseddifferentiation. Pathobiology 2005;72:203.

[38] Therond P, Abella A, Laurent D, Couturier M, Chalas J,Legrand A, et al. In vitro study of the cytotoxicity of isolatedoxidized lipid low-density lipoproteins fractions in human

( 2 0 0 9 ) 1532–1540

endothelial cells: relationship with the glutathione statusand cell morphology. Free Radic Biol Med 2000;28:585.

[39] Kim JM, Kim H, Kwon SB, Lee SY, Chung SC, Jeong DW, et al.Intracellular glutathione status regulates mouse bonemarrow monocyte-derived macrophage differentiation andphagocytic activity. Biochem Biophys Res Commun2004;325:101.

[40] Mallery SR, Lantry LE, Laufman HB, Stephens RE, Brierley GP.Modulation of human microvascular endothelial cellbioenergetic status and glutathione levels duringproliferative and differentiated growth. J Cell Biochem1993;53:360.

[41] Slim R, Toborek M, Robertson LW, Lehmler HJ, Hennig B.Cellular glutathione status modulates polychlorinatedbiphenyl-induced stress response and apoptosis in vascularendothelial cells. Toxicol Appl Pharmacol 2000;166:36.

[42] Navarro J, Obrador E, Carretero J, Petschen I, Avino J, Perez P,et al. Changes in glutathione status and the antioxidantsystem in blood and in cancer cells associate with tumourgrowth in vivo. Free Radic Biol Med 1999;26:410.

[43] Oka S, Kamata H, Kamata K, Yagisawa H, Hirata H.N-Acetylcysteine suppresses TNF-induced NF-kappaBactivation through inhibition of IkappaB kinases. FEBS Lett2000;472:196.

[44] Schweikl H, Spagnuolo G, Schmalz G. Genetic and cellulartoxicology of dental resin monomers. J Dent Res 2006;85:870.

[45] Haddad JJ. Oxygen sensing and oxidant/redox-relatedpathways. Biochem Biophys Res Commun 2004;316:969.

[46] Wagner EF. Functions of AP1 (Fos/Jun) in bone development.Ann Rheum Dis 2002;61(Suppl. 2):ii40.

[47] Karin M, Lin A. NF-kappaB at the crossroads of life anddeath. Nat Immunol 2002;3:221.

[48] Bannai S, Sato H, Ishii T, Sugita Y. Induction of cystine

Chem 1989;264:18480.[49] Griffith OW. Biologic and pharmacologic regulation of

mammalian glutathione synthesis. Free Radic Biol Med1999;27:922.