d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 34–39

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

Apoptosis induced by the monomers HEMA and TEGDMAinvolves formation of ROS and differential activation of theMAP-kinases p38, JNK and ERK

Jan T. Samuelsena,∗, Jon E. Dahla, Stig Karlssona, Else Morisbaka, Rune Becherb

a NIOM–Nordic Institute of Dental Materials, P.O. Box 70, N-1305 Haslum, Norwayb Norwegian Institute of Public Health, Division of Environmental Medicine, Geitmyrsveien 75,P.O. Box 4404, Nydalen, N-0403 Oslo, Norway

a r t i c l e i n f o

Article history:

Received 25 June 2005

Received in revised form

24 November 2005

Accepted 28 November 2005

Keywords:

Dental monomers

Cell death

Signaling pathways

a b s t r a c t

Objectives. Cytotoxic methacrylate monomers have been identified in aqueous extracts of

freshly cured compomers. Some of these compounds, including HEMA and TEGDMA, induce

apoptosis and necrosis in vitro. The aim of the present study was to elucidate possible sig-

naling pathways involved in apoptosis following exposure to HEMA or TEGDMA in a salivary

gland cell line.

Methods. The cells were exposed to various concentrations of HEMA or TEGDMA. ROS for-

mation was determined by dichlorofluorescein assay. Phosphorylated MAP-kinases ERK1/2,

p38 and JNK, as well as specific caspases were identified by Western blotting. Apoptosis was

assayed by fluorescence microscopy.

Results. HEMA or TEGDMA exposure resulted in ROS formation and concentration-dependent

apoptosis as well as phosphorylation of ERK. Phosphorylation of JNK and p38 was induced

by HEMA. Selective inhibitors of ERK and JNK modified the apoptotic response after HEMA

and TEGDMA exposure, whereas p38 inhibition modified the apoptotic response only after

HEMA exposure. Vitamin C reduced HEMA-induced apoptosis.

Significance. ROS formation and differential MAP kinase activation appear to be involved in

the apoptotic response following exposure to HEMA and TEGDMA.

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. Introduction

-Hydroxyethyl methacrylate (HEMA) and triethyleneglycolimethacrylate (TEGDMA) are commonly identified leachablesf resin-based materials [1,2]. The cytotoxicity of methacrylateonomers including HEMA and TEGDMA has been described

n several studies [3–5]. Furthermore, HEMA and TEGDMAnduce apoptosis in vitro [6,7]. Apoptosis is a major formf cell death in biological systems as well as in chemically

nduced injury. A common denominator in the development

∗ Corresponding author. Tel.: +47 67 51 22 00; fax: +47 67 59 15 30.E-mail address: jts@niom.no (J.T. Samuelsen).

109-5641/$ – see front matter © 2005 Academy of Dental Materials. Puoi:10.1016/j.dental.2005.11.037

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

of apoptosis is activation of caspases [8]. Depending on the ini-tial apoptotic signal, specific initiator caspases are activated.These subsequently activate effector caspases responsible forthe cleavage of key cellular proteins. This leads to the typicalmorphological changes associated with apoptotic cells.

Leachables from resin-based materials are a likely cause

of cellular stress via the formation of reactive oxygen species(ROS). This statement is supported by the finding that suchleachables influence the intracellular level of glutathionewhich plays a dominant role in protecting cells against oxida-

blished by Elsevier Ltd. All rights reserved.

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ive damage [9]. ROS and mitochondria are important actorsn the induction of apoptosis. Caspase activation triggeredy cytochrome c release from mitochondria is mediatedy oxidative stress [10] created by imbalance between ROSormation and defense mechanisms [11]. Furthermore, ROS

ay interact with mitogen-activated protein (MAP)-kinases12]. MAP-kinases participate in the transmission of signalso the cell nucleus. In mammalian systems, three subfamiliesf MAP-kinases have been identified. These include theRK1/2, JNK and p38 MAP-kinase cascades. ERK is part ofhe ras/raf/MEK pathway associated with proliferation andurvival [13,14]. It has also been reported that induction ofpoptosis may be mediated via ERK [15]. The stress-activatedrotein kinases (SAPKs) JNK and p38 have been related mainlyo oxidative stress and apoptosis, as well as to inflammatoryesponses following exposure to chemical agents [13,16–18].owever, various isoforms of JNK and p38 may have different

unctions and localization in cells [19,20]. Published data alsondicate that the role of JNK and p38 in apoptosis differsetween cell types [21]. Although salivary gland cells used inhe present study have been shown to express p38 and JNK22], the expression and functions of different isoforms ofhese kinases have not been characterized in this cell line.

ROS-induced stress and signaling through caspase cascades well as the MAP kinase pathways are important in the devel-pment of apoptotic responses. The aim of this study was to

nvestigate mechanisms involved in the apoptotic responsefter exposure to HEMA and TEGDMA in vitro, by address-ng the involvement of ROS formation, MAP-kinase signalingathways and caspase activation.

. Materials and methods

.1. Monomers

riethyleneglycol dimethacrylate (TEGDMA) (CAS no. 109-16-), purity ≥95.0% and 2-hydroxyethyl methacrylate (HEMA)CAS no. 868-77-9), purity ≥97.0% (Fluka Chemie AG, Buchs,witzerland) were added to the cell culture medium up tomM (TEGDMA) or 15 mM (HEMA).

.2. Other chemicals

-Carboxy-2′,7′-dichlorofluorescin diacetate (DCFH-DA) wasurchased from Molecular Probes, Inc. (Eugene, OR, USA). Bio-ad DC protein assay from Bio-Rad Laboratories, Inc. (Her-ules, CA, USA). All other chemicals were purchased fromommercial sources at the highest purity available.

.3. Antibodies

abbit polyclonal antibodies to human phospho-ERK (CS-101), phospho-JNK (CS-9251) and phospho-p38 (CS–9211)ere obtained from Cell Signaling Technology, Inc. (Beverly,A 01915, USA). Goat polyclonal antibody to human caspase

(SC-6134) and rabbit polyclonal antibody to human caspase 9

SC-8355) were obtained from Santa Cruz Biotechnology (Santaruz, CA 95060, USA). All antibodies cross-reacted with theirorresponding rat proteins. HRP-conjugated goat anti-rabbit

( 2 0 0 7 ) 34–39 35

IgG (CS-7074, Cell Signaling Technology Cell, Inc.) and HRP-conjugated rabbit anti-goat IgG (P 0449, DakoCytomation DK-2600 Glostrup, Denmark) were used as secondary antibody.

2.4. MAP-kinase inhibitors

The specific inhibitors for ERK (PD98059) and p38 (SB202190)were obtained from Merck KGaA (Darmstadt, Germany). TheJNK specific inhibitor SP600125 was obtained from BiosourceInternational (CA, USA).

2.5. Cell culture

Rat submandibular salivary gland acinar cells, SM 10–12[23], were kindly provided by Dr. H.K. Galtung, University ofOslo, Norway. The cells were cultured in Dulbecco’s medium(DMEM), supplemented with 50 �g/ml gentamycin-sulphate(Sigma, MO, USA) and 2.5% fetal bovine serum (Sigma, MO,USA), at 37 ◦C, in air atmosphere containing 5% CO2 and 95%relative humidity. Prior to the experiments, the cells wereplated in cell culture dishes (3 × 104 cells/cm2).

2.6. Exposure conditions

The cells were incubated for 2, 4, 6, 8, 10 or 16 h in cul-ture medium either alone (control) or with monomer. Themedium was subsequently changed and the cells further incu-bated in medium without monomers for up to a total timeof 24 h.

2.7. ROS analyses

ROS measurements were performed by means of dichloroflu-orescein (DCF) assays [24]. After the fluorochrome DCFH-DAwas removed, the cells were incubated with a medium con-taining the specified concentrations of HEMA or TEGDMA. Toassess the role of ROS formation on apoptosis after HEMAor TEGDMA exposure, cells were pre-incubated for 30 minwith the radical-scavenging antioxidant Vitamin C (50 �g/ml).Exposure to H2O2 was used as positive control. Cells were ana-lyzed using Fluostar Optima fluorescence plate reader (BMG-Labtech, Offenburg, Germany). The excitation and emissionfilters were set at 430 and 520 nm, respectively.

2.8. Evaluation of apoptosis and necrosis

Cells were trypsinized, centrifuged and resuspended in 50 �lFBS containing 10 �g/ml Hoechst 33342 and 10 �g/ml propid-ium iodide (Sigma, MO, USA), and incubated for 0.5 h in darkat room temperature. A minimum of 300 cells were countedusing fluorescence microscopy (Leitz Orthoplan, Oberkochen,Germany) 100×, with an excitation filter 340–380 nm, andclassified as apoptotic, necrotic or viable. The percentage ofnecrotic and apoptotic cells was calculated.

2.9. Protein analyses

Cultured cells were scraped off the dish in PBS, centrifugedand the pellet dissolved in SDS-PAGE sample buffer. A totalof 10 �g protein from each sample was electrophoresed

l s 2 3 ( 2 0 0 7 ) 34–39

Fig. 1 – ROS formation in a submandibular acinar cell lineexposed to different concentrations of HEMA (a) or TEGDMA(b). Hundred micromoles of H2O2 was used as positivecontrol. All results are shown as mean ± S.D. of minimumof three separate experiments. All treatment groups weresignificantly different from control (p < 0.05). When the cellswere co-incubated with Vitamin C and either HEMA,TEGDMA or H2O2, no significant increases in ROS formationwere observed.

JNK were seen (Fig. 4b and c). In contrast to the results with

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

in SDS-polyacrylamide (SDS-PAGE) gels [25], blotted ontonitrocellulose filters and analyzed using specific antibod-ies [26]. The filters were blocked for 30 min with 3% BSAin TBS containing 0.1% Tween 20, and then incubatedwith antibodies diluted in TBS–Tween containing 1% BSA.Immunoreactive protein was detected using a chemilumines-cence system (West Dura, Pierce) after incubation with HRP-conjugated secondary antibody. Densitometry of the West-ern blots was done using QuantiScan software (BIOSOFT,Cambridge, UK).

2.10. Statistics

p-Values < 0.05 were considered significant. To assess theeffects of MAP-kinase inhibitors on apoptosis, either Stu-dent’s t-test or Mann–Whitney Rank sum test was used.Student–Newman–Keuls method for pairwise comparisonswas used to assess effects of Vitamin C on ROS formation.

3. Results

3.1. ROS analysis

The results show concentration-dependent increases in ROSformation after exposure to HEMA or TEGDMA for 1 h. Theincreases were significant for all concentrations tested. How-ever, TEGDMA appeared to be more potent than HEMA aslower TEGDMA concentrations were needed for significantincrease in ROS formation. H2O2 exposure caused a lin-ear, concentration-dependent increase in ROS formation with100 �M giving a similar response as the highest concentrationsof HEMA and TEGDMA tested (data shown for 100 �M H2O2

only). When the cells were co-incubated with Vitamin C andeither HEMA, TEGDMA or H2O2, no significant increases in ROSformation were observed (Fig. 1).

3.2. Evaluation of apoptosis with fluorescence stainedcells

Cells were exposed to HEMA or TEGDMA for 2–16 h. Subse-quent to the exposure, the cell culture medium was changedto monomer free medium. Cell death was evaluated 24 h afterstart of exposure. An apoptotic response was first detectedafter 8–10 h exposure. Using 10 h exposure, HEMA concentra-tions up to approximately 7.5 mM, resulted in an increasedpercentage of apoptotic cells. At higher concentrations, thepercentage of apoptotic cells leveled out whereas the numberof necrotic cells increased markedly (Fig. 2a). After 10 h expo-sure to TEGDMA, there was an increase in the percentage ofapoptotic and necrotic cells for concentrations up to approx-imately 2 mM. With increasing concentrations of TEGDMA,the percentage of necrotic cells continued to increasewhereas the percentage of apoptotic cells remained stable(Fig. 2b).

Pre-treatment and co-incubation of HEMA treated cells

with Vitamin C for 8 h reduced the apoptotic response signifi-cantly (Fig. 3). No statistically significant reduction in apop-tosis was seen following co-incubation of Vitamin C andTEGDMA. By using the ERK specific inhibitor PD98059 [27], a

statistically significant increase in the number of apoptoticcells was seen after both HEMA and TEGDMA exposure. Pre-incubation with the JNK specific inhibitor SP600125 [28] signifi-cantly reduced the number of apoptotic cells after exposure toboth HEMA and TEGDMA. The p38 specific inhibitor SB202190[29] was able to significantly reduce apoptosis only after HEMAexposure (Fig. 3).

3.3. Signal transduction proteins—Western blotting

After exposure to either 7.5 mM HEMA or 2 mM TEGDMA for6 h, a marked increase in phosphorylation of ERK1 and 2was observed (Fig. 4a). Following exposure to HEMA for 2and 6 h, increased levels of phosporylated (activated) p38 and

HEMA, exposure to TEGDMA did not result in large alterationsof phospho-p38 or phospho-JNK levels (Fig. 4b and c). Thelevels of total ERK, p38 and JNK remained stable (data notshown).

d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 34–39 37

Fig. 2 – Evaluation of apoptosis by fluorescence microscopyin a submandibular acinar cell line stained with Hoechstand propidium iodide following 8 h exposure to differentconcentrations of HEMA (a) or TEGDMA (b). All results arese

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Fig. 3 – Effects of MAP kinase inhibitors or Vitamin C onapoptosis induced by either HEMA (7.5 mM) or TEGDMA(2 mM) in a submandibular acinar cell line. The levels ofapoptosis for each treatment group were obtained asrelative increase compared to the respective treatmentcontrol. All results are shown as mean ± S.D. for aminimum of three separate experiments. Asterisk

hown as mean ± S.D. for a minimum of three separatexperiments.

.4. Caspase activation—Western blotting

xposure to HEMA (7.5 mM) for 6 h or TEGDMA (2 mM) for 2 hesulted in increased levels of caspase 9 p20 (Fig. 4d). Forhorter times, no induced caspase 9 activity was observed.o alteration in activity of caspase 8 was detected (data not

hown).

. Discussion

n agreement with previous studies, we showed that TEGDMAnd HEMA were able to induce apoptosis and necrosis in

concentration-dependent manner. To elucidate possibleechanisms involved in monomer-induced apoptosis, we

nvestigated the potential role of some key signaling pathwaysften involved in the toxicity of chemical compounds. High

evels of ROS may induce modifications that inhibit the activ-ty of cellular components or result in damage, repair and celleath. Previous results indicate that leachable composite com-

onents may exert some of their potential toxicity throughOS formation [9]. In support of this, we demonstrated thatxposure to HEMA and TEGDMA induced ROS formation in

salivary gland cell line. Vitamin C, a radical-scavenging

indicates treatment groups significantly different fromcontrol (p < 0.05).

antioxidant, inhibited this ROS formation and reduced HEMA-induced apoptosis. This indicates that initiation of apoptosisin vitro, at least by HEMA, could in part be mediated throughthe formation of ROS. Lack of correlation between inhibition ofROS formation and effect on TEGDMA-induced apoptosis, mayreflect that factors other than ROS are of greater importancefor the TEGDMA-induced apoptosis.

ROS and activation of MAP kinase cascades have beenshown to be associated in previous studies [30]. Our resultsindicate that exposure to HEMA or TEGDMA induces ERK1/2phosphorylation in a salivary gland cell line. Although someresults have challenged the view that ERK functions solely asa survival factor [15], the traditional view implicates ERK1/2as being activated in response to stimuli for proliferation andsurvival. In agreement with the latter, we found that inhibitionof ERK by the ERK inhibitor PD98059 resulted in a statisti-cally significant increased number of apoptotic cells followingexposure to HEMA or TEGDMA. This is in contrast to a pre-vious study [7] that failed to show ERK involvement in theapoptotic response of primary human pulp cells to TEGDMA.However, the sensitivity of ERK1/2 to oxidative signals is con-troversial and appears to vary among cell types [31]. Differ-ences in the sensitivity of ERK to oxidative signals in differentcell types could explain the observed differences in signal pat-tern responses in these two studies.

Furthermore, we found increased phosphorylation of bothp38 and JNK subsequent to HEMA but not to TEGDMA expo-sure. Treatment with the p38 specific inhibitor SB202190

resulted in a statistically significant reduction in the num-ber of apoptotic cells following exposure to HEMA only. Thisindicates that p38 is involved in the apoptotic response afterHEMA exposure. However, administration of the JNK inhibitor

38 d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 34–39

) andposu

Fig. 4 – Activation of MAP-kinases ERK1/2, p38 and JNK (a–cexposure to 7.5 mM HEMA or 2 mM TEGDMA for different ex

SB600125 significantly reduced the number of apoptotic cellsafter exposure to HEMA as well as TEGDMA. These resultsagree with the traditional view of p38 and JNK being impli-cated primarily in the induction of apoptosis and inflamma-tion after exposure to different agents [13,16–18]. Althoughno TEGDMA-induced JNK activation was observed by West-ern analysis the observation that inhibition of JNK appearedto reduce TEGDMA-induced apoptosis, may imply that JNK istransiently activated by TEGDMA at times not examined inour study. Thus, the results do not exclude a possible involve-ment of the JNK pathway also in TEGDMA-induced apoptosis.Overall, as an extended period of monomer exposure seemednecessary for the development of apoptosis, and since MAP-kinase activation appeared relatively rapidly after exposure,the results, at least regarding HEMA, could indicate that sus-tained activation of the MAP-kinase pathways is necessary.

Interestingly, the MAP-kinase signaling pattern subsequentto TEGDMA exposure has similarities to that described afterH2O2 exposure in HeLa cells [32]. The oxidant H2O2 wasreported to cause sustained activation of all three MAP-kinasesubfamilies. Furthermore, selective inhibition of ERK2 activa-tion by PD098059 markedly increased the apoptosis, whereasapoptosis decreased in transfected cells lacking the upstream

kinase responsible for activation of JNK. Inhibition of p38-kinase activity with p38-specific inhibitors had no effect oncell survival. Previously, various ROS have been shown to causedifferent cellular effects [33]. Our results could indicate that

caspase 9 (d) in a submandibular acinar cell line followingre periods.

different ROS or different localization of the ROS formation areinvolved in the apoptotic effects caused by HEMA compared tothose of TEGDMA exposure. TEGDMA-induced apoptosis mayinvolve H2O2 formation.

Two major pathways regulate induction of apoptosis inmammalian cells. In the extrinsic pathway, apoptosis isinduced through surface receptors and activation of caspase 8,whereas in the intrinsic pathway, apoptosis is induced withinthe cells, mainly through permeabilization of mitochondriaand activation of caspase 9. Enzymes important for H2O2 for-mation can be found in mitochondria. Results have demon-strated that methacrylates interfere with cellular cholesteroland phospholipids, which could alter membrane-associatedfunctions [34]. Furthermore, methyl methacrylate has beenshown to alter inner mitochondrial membrane structure [35].We showed that exposure to either HEMA or TEGDMA covari-ated with increased levels of active caspase 9, suggesting amitochondrial pathway for the induction of apoptosis. Ourresults indicate that some of the observed effects followingHEMA and TEGDMA exposure involve membrane alterationor damage of mitochondria.

Insight into mechanisms of toxicity of dental materials isimportant for understanding the potential of these materials

to cause adverse health effects in a clinical setting. In this con-text, we have shown that an apoptotic response subsequentto HEMA and, to some extent, TEGDMA exposure involves ROSformation as well as activation of the MAP kinase-signaling

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ascade. However, the results also indicate that involvementf other cellular events are important for the final determina-ion of the exposed cells’ fate.

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