Effect of N-acetyl-l -cysteine on ROS production and cell death caused by HEMA in human primary gingival fibroblasts

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Biomaterials 27 (2006) 1803–1809 www.elsevier.com/locate/biomaterials

Effect of N-acetyl-L-cysteine on ROS production and cell death caused by HEMA in human primary gingival fibroblasts Gianrico Spagnuoloa,, Vincenzo D’Anto`a, Claudia Cosentinob, Gottfried Schmalzc, Helmut Schweiklc, Sandro Rengoa a Department of Oral and Maxillofacial Sciences, University of Naples ‘‘Federico II’’ via S. Pansini 5, 80131-Naples, Italy Department of Cellular and Molecular Biology and Pathology, University of Naples ‘‘Federico II’’, via S. Pansini 5, 80131-Naples, Italy c Department of Operative Dentistry and Periodontology, University of Regensburg, D-93042 Regensburg, Germany

b

Received 16 July 2005; accepted 29 October 2005 Available online 14 November 2005

Abstract Previous investigations have shown that 2-hydroxyethyl methacrylate (HEMA) causes reactive oxygen species (ROS) production, which in turn affects cell survival and cell death. The purpose of this study was to evaluate the effects of the antioxidant N-acetyl-Lcysteine (NAC) on HEMA-induced toxicity in human primary gingival fibroblasts (HGF). HGF were treated with various concentrations of HEMA (0–12 mM) in the absence and presence of NAC (1, 5, and 10 mM). The 3-(4,5 dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the mitochondrial dehydrogenase activity after HEMA exposure. Viability and cell death were determined by flow cytometry using Annexin V and PI staining. ROS production was detected by the increasing fluorescence of the oxidation-sensitive dye 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) after HEMA treatment. After a 24 h incubation period, HEMA concentrations higher then 10 mM caused a decrease of cell viability, mitochondrial activity, and an increase of cell death. HEMA concentrations of 4–12 mM markedly increased ROS levels in a dose-dependent manner. High NAC concentrations (5 and 10 mM) significantly reduced cell death, and restored the mitochondrial activity after a 24 h co-treatment, but 1 mM NAC increased HEMA toxicity (po0.05). All NAC concentrations significantly reduced ROS levels induced by HEMA after a 2 h exposure (po0.05), but no such reduction was observed after a 4 h treatment. Furthermore, treatment with 10 mM HEMA and 1 mM NAC for 6 h caused an increase in ROS levels compared to 10 mM HEMA alone (po0.05). In conclusion, our results suggest that high NAC concentrations protect HGF against HEMA cytotoxicity by reducing the induced ROS levels. r 2005 Elsevier Ltd. All rights reserved. Keywords: Dental resin; HEMA; Human gingival fibroblasts; NAC; Reactive oxygen species

1. Introduction Several studies have shown that various monomers and other components, such as initiators or activators of dental resin materials, can be released into the oral environment even after polymerization [1]. It has been shown that resinbased materials release un-reacted monomers like triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) might diffuse through dentin to the pulp causing adverse phenomena [2,3].

Corresponding author. Tel./fax: +39 081 7462385.

E-mail address: [email protected] (G. Spagnuolo). 0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.10.022

Cytotoxicity of the monomers has been widely investigated and experimental parameters, cell lines or assay procedures influenced the evaluation of their cytotoxicity [4,5]. Recent investigations focused on the mechanisms of monomers which lead to cell death via apoptosis or initiate survival cellular responses [6,7]. It is well established that HEMA caused cell death by activating apoptosis processes in primary fibroblasts [6]. In addition, HEMA can suppress the growth of several kinds of cells [8,9] and, in particular, it can induce cell cycle delay in human pulp fibroblasts [10]. It has also been shown that HEMA increased the production of reactive oxygen species (ROS), which can cause a delay in cell cycle progression [10]. We showed that HEMA-induced ROS production was very important to

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modulate the activation of nuclear factor kappa B (NF-kB) which played a protective role to counteract HEMA cytotoxicity [6]. Furthermore, other researches have shown that the increase of ROS induced by dental materials and monomers [6,10–13] might be involved in their cytotoxicity. In fact, reports have shown the ability of antioxidants such as N-acetyl-L-cysteine (NAC) to reduce cell damage induced by TEGDMA or dental composite [11,12]. A recent study demonstrated that high NAC concentrations protected DNA from oxidative damage induced by VLirradiated camphorquinone (CQ)/dimethyl-p-toluidine (DMT), a resin dental materials initiator, in a cell-free environment [14]. In addition, Pagoria et al. [15] suggested that 10 mM NAC may reduce or revert the oxidative stress in murine cementoblast cell line (OCCM.30) and immortalized murine fibroblast cell line, 3T3-Swiss albino (3T3), exposed to VL-irradiated CQ/DMT. Therefore, in this study we tested the hypothesis that NAC may protect human primary gingival fibroblasts (HGF) against HEMA cytotoxicity and cell death by decreasing the induced-ROS production. We assessed the HEMA effect on ROS levels, cell death, viability and mitochondrial activity in the presence or absence of NAC. 2. Materials and methods 2.1. Reagents

2.4. Measurement of reactive oxygen species levels (ROS) Production of ROS in cultured cells was quantified using the cellpermeant fluorescence probe DCFH-DA, as previously described [17]. HGF (1
105 cells) were incubated with different concentrations of HEMA (0–12 mM) in the presence or absence of NAC for 2, 4 or 6 h. In order to measure ROS production induced by HEMA, cells were stained with 10 mM of DCFH-DA for 30 min at 37 1C, detached with trypsin/ EDTA, washed, re-suspended in PBS, and then immediately analyzed by flow cytometry. We used a FACScan flow cytometer to measure ROS generation by the fluorescence intensity (FL-1, 530 nm) of 20,000 cells. Mean fluorescence intensity was obtained by histogram statistics using WinMDI 2.8.

2.5. Cell death detection Cells were exposed to HEMA (0–12 mM) and NAC for different periods of time at 37 1C. Morphological changes were observed and documented by phase contrast microscopy. Flow cytometry was used to detect cell death. After treatments, floating and adherent cells were collected and harvested by centrifugation, then washed once with PBS. Next, the cells were suspended in 500 mL of binding buffer. Untreated and treated cells were stained with annexin VFITC and PI (MBL Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), and incubated at room temperature for 15 min before being analyzed by flow cytometry (FACScan, Becton-Dickinson, San Jose, CA, USA). Viable cells (no staining), apoptotic cells (annexin V+) and necrotic cells (both PI+/annexin V+ or PI+ alone) were detected and quantified as a percentage of the entire population [18]. The sum of apoptotic and necrotic cells were considered as cell death population [19]. Analysis of the data was performed by means of the WinMDI 2.8 program.

HEMA, 3-(4,5 dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (MTT) and NAC were purchased from Sigma Chemical Co, St. Louis, MO. 20 , 70 -dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes, Eugene, OR; Annexin V and Propidium Iodide (PI) Kit from MBL Medical & Biological Laboratories Co., Ltd., Nagoya, Japan. Medium and reagents were from Gibco, Life Technologies, NY, USA.

Values were expressed as the mean7SD and the data were analyzed by one-way analysis of variance followed by Bonferroni for multiple comparisons. The level of significance was set at po0.05.

2.2. Cell culture

3. Results

Human gingival fibroblasts were obtained from four healthy 20–23year-old patients, with informed consent. The protocol was reviewed and approved by the Institutional Review Board (University of Napoli ‘‘Federico II’’). Small samples of gingival tissues were dissected out and explanted into tissue culture dishes. Cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 250 mg/mL of fungizone. In all experiments, cells were pooled and used between passage 2 and passage 8.

3.1. Cytotoxicity of HEMA

2.3. Effect of HEMA on mitochondrial dehydrogenase activity Cytotoxic concentrations of HEMA were identified measuring mitochondrial dehydrogenase activity by MTT, as previously reported [16]. HGF were plated in a 96-well tissue culture dish at 10,000 cells/well. After 24 h of incubation, the medium was removed and cell monolayer was incubated in absence (control) or presence of various concentrations of HEMA (with or without NAC) for 6, 12 and 24 h. The medium was replaced by 100 mL/well of a solution of MTT (0.5 mg/mL) in phosphatebuffered saline (PBS), and the cells were incubated at 37 1C for 1 h in a 5% CO2 atmosphere. The MTT solution was removed and replaced with 100 mL/well of DMSO and gently swirled for 10 min. The optical density was immediately measured in a Bio-Rad ELISA reader at 540 nm. The results were expressed as the percentage of untreated cells. Each experiment was performed in triplicate.

2.6. Statistical analysis

After a 6 h incubation period to various concentrations of HEMA, exposed HGF did not show differences in mitochondrial dehydrogenase activity compared to control cell cultures, while an inhibition was found after 12 h with 12 mM HEMA (Fig. 1a). After a 24 h exposure period the mitochondrial activity was reduced in a dose-dependent manner, and significant differences were observed between cell cultures treated with 10 or 12 mM HEMA and control cells (Fig. 1a). HEMA caused a decrease in cell viability and an increase of the population of dead cells after a 24 h incubation period (Fig. 1b). A concentration of 10 mM HEMA showed a significant reduction in cell viability and an increase in the necrotic cell population compared with control cells (Fig. 1b). An even higher concentration (12 mM) reduced the HGF viability to less than 50% showing also a slight but significant increase of apoptotic cells associated with a large increase of necrotic cell population (Fig. 1b). Untreated HGF showed a typical spindle-shaped

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Fig. 2. Morphological alterations of HGF after 10 mM HEMA exposition in presence or absence of NAC for 24 h. (A) Untreated; (B) 10 mM HEMA; (C) 10 mM HEMA with 1 mM NAC; (D) 10 mM HEMA with 5 mM NAC (100
magnification).

observed after 2 h, but was less pronounced after 4 and 6 h. The significant reduction in intracellular ROS between 2 and 6 h after treatment may represent the recovery of reduced intracellular antioxidant molecules (e.g., glutathione, thioredoxin) and it may indicate that the cells are neutralizing the ROS increase. Mean DCF fluorescence reached a maximum after 2 h with 12 mM HEMA treatment showing a 14-fold increase compared to the control cells (Fig. 3a).

Fig. 1. Effect of HEMA on human primary gingival fibroblasts. (A) Cells were exposed to HEMA for 6, 12 and 24 h and mitochondrial dehydrogenase activity (MTT) was measured as described in Section 2. (B) After 24 h HEMA exposition viable, necrotic and apoptotic HGF were detected and quantified by flow cytometry as described in Section 2. Results represent the means7SD of at least four independent experiments (n ¼ 426). * indicates significant differences between treated and untreated cell cultures (one-way ANOVA followed by Bonferroni post hoc test, po0.05).

morphology with extended cellular processes like filopodia and lamellopodia (Fig. 2a). However, the treatment of the cells with 10 mM HEMA alone or with 1 mM NAC for 24 h induced morphological alterations. The cells then appeared smaller, retracted and rounded (Fig. 2b,c), whereas HGF exposed for 24 h to 10 mM HEMA in the presence of 5 mM NAC were similar in appearance to untreated cells (Fig. 2d). 3.2. The effect of HEMA on ROS levels in HGF Treatment of HGF with HEMA (0–12 mM) induced a time- and concentration-dependent increase of ROS indicated by DCF fluorescence (Fig. 3a). A significant production of ROS for all HEMA concentrations was

3.3. The effect of NAC on ROS production and cytotoxicity caused by HEMA In order to investigate whether NAC had the ability of reducing HEMA cytotoxicity by decreasing the inducedROS levels, we treated HGF with HEMA in the presence and absence of NAC. HGF treated with NAC alone (1, 5, 10 mM) did not show any effects in terms of ROS production and cytotoxicity (Figs. 3b and 4). However, NAC was active at concentrations of 5 and 10 mM in the presence of HEMA (Fig. 4). It induced a full protection restoring the mitochondrial activity in cell cultures treated with HEMA concentrations lower than 12 mM (Fig. 4a). In addition, 5 mM NAC reduced HEMA-induced cell death except for 12 mM HEMA, whereas a complete protection with 10 mM NAC was observed with all HEMA concentrations (Fig. 4b, c). In contrast, 1 mM NAC enhanced HEMA-induced cytotoxicity reducing cell viability and amplifying cell death and mitochondrial activity inhibition (Fig. 4). In particular, after a 24 h exposure to 4–12 mM HEMA and 1 mM NAC, the mitochondrial activity of HGF was inhibited by 15.0–98.5% (Fig. 4a). Moreover, 1 mM NAC co-treatments significantly decreased the cell viability and increased cell death caused by 8–12 mM HEMA (Figs. 4b,c).

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ingly, after 2 h 1 mM NAC decreased ROS production induced by HEMA, whereas after 6 h a markedly increase of ROS levels was observed (Fig. 5). 4. Discussion

Fig. 3. Induction of ROS levels in HGF exposed to HEMA and NAC. ROS levels were determined by the DCFH-DA as described in Section 2. (A) HGF were incubated with HEMA, and ROS were calculated as -fold increase in fluorescence compared with that of untreated cells. ROS levels in the control group were arbitrarily assigned a fluorescence value of 1. (B) Cell cultures were treated with various NAC concentrations, and ROS production was measured as mean fluorescence intensity derived by WinMDI 2.8 program analysis. Results are expressed as means7SD (n ¼ 5); * indicates significant differences between treated and untreated cell cultures (one-way ANOVA followed by Bonferroni post hoc test, po0.05).

To determine whether HEMA-induced cytotoxicity was related to the generation of ROS, the effect of NAC on ROS levels was investigated. Treatment of HGF with 10 mM HEMA induced a marked increase in ROS production after a 2, 4 and 6 h exposure period compared with untreated cells (Fig. 3a). However, the ROS production induced by 10 mM HEMA was significantly inhibited in the presence of 1–10 mM NAC after a 2 h exposure period, while only 10 mM NAC induced a significant inhibition after a 4 h exposure (Fig. 5). Moreover, none of the NAC concentrations used here decreased HEMAinduced ROS levels after a 6 h exposure (Fig. 5). Interest-

Previous in vitro investigations on HEMA showed that the concentration which caused a toxic effect in vitro depended on the type of assay and cell line used [5,8,20]. The concentration range of HEMA in our study was in line with previous in vitro investigations on dental resin monomers [6,10,21–24], although the effective HEMA concentrations which may reach in oral and pulp tissues are unknown until now. In some clinical trials, the toxic concentrations will depend on the procedures used (e.g. direct pulp capping) or on the remaining dentin thickness [25]. Here, as expected, the reduction of viability and the increase of cell death caused by HEMA were time- and dose-dependent. Ten and 12 mM HEMA caused a significant decrease of mitochondrial activity and viability with an increase of cell death compared to the untreated cells after 24 h (Fig. 1). In particular, HEMA cell death showed an increase of the necrotic cell population rather than the apoptotic population (Fig. 1b). Cell damage by 10 mM HEMA was also indicated by clear morphological alterations in HGF (Fig. 2). Cell death and life are controlled by a number of factors within the cell, including a balance between ROS production and antioxidants system [26]. Recently, it has been demonstrated that HEMA exposition caused glutathione (GSH) depletion and ROS production [6,10,27]. ROS are involved in regulating cell growth, cell differentiation and cell death. ROS may directly modulate signaling cascades by activating or inhibiting various transcription factors (i.e. NF-kB, AP-1 and Nf-r2), or more indirectly affecting such signaling by changing the cellular redox status (i.e. glutathione and thioredoxin) [28,29]. High ROS levels, if not counteracted by cellular antioxidants, cause acute injury and damage of important biomolecules including cellular proteins, lipids and DNA leading to cell death [30,31]. We have previously demonstrated that an increase in ROS levels caused by HEMA may activate NF-kB pathway, which in turn protected the cells from induced cell death [6]. In this study, we showed that HEMA cytotoxicity was associated with an increase of ROS levels in HGF and this result was in line with previous studies on human primary skin or pulp fibroblasts [6,10]. Chang et al. [10] reported that ROS production induced by HEMA is probably not caused by GSH depletion in human gingival epithelial cells, because GSH depletion was marked only at high concentrations, while an excessive ROS production was noted also at lower concentration. Likewise, a significant change of the GSH-GSSG ratio was not assessed in THP-1 human monocytic cells after treatment with HEMA sub-lethal concentrations [27]. Furthermore, one should consider the possibility that HEMA could induce ROS overproduction during the experimental

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Fig. 4. Effect of NAC on HEMA toxicity. HGF were treated with 0–12 mM HEMA in the presence or absence of various NAC concentrations for 24 h. (A) Mitochondrial dehydrogenase activity, (B) viability, and (C) cell death were quantified as described in Section 2. Results represent the means7SD of at least four independent experiments (n ¼ 426). * indicates significant differences between treated and untreated cell cultures (one-way ANOVA followed by Bonferroni post hoc test, po0.05).

Fig. 5. Effect of NAC on HEMA-induced ROS production. Cells were treated with 10 mM HEMA in the absence (control) or presence of NAC at indicated concentrations. Values represented are means7SD (n ¼ 5); asterisks indicate statistically significant differences between cell cultures treated with 10 mM HEMA and untreated controls (one-way ANOVA followed by Bonferroni post hoc test, po0.05).

preparation via redox cycling, especially in the presence of transition metals. However, more future studies are needed to clarify the source of ROS and the relationship between GSH depletion, monomers and ROS increase. It has been reported that antioxidants, such as NAC, ascorbate, and Trolox, might prevent cell toxicity induced

by TEGDMA or dental materials [11,12,32]. In addition, it has been provided evidence that antioxidant vitamins (A, C, E) may decrease cytotoxicity of HEMA and TEGDMA in immortalized cultured cell lines [22]. In a recent study [14], carried out on supercoiled double stranded plasmid DNA model, it has been shown that glutathione and NAC reduced oxidative DNA damage caused by resin-based dental materials initiators, whereas low concentrations enhanced the extent of DNA damage. Also, vitamin E and vitamin C treatments increased oxidative DNA damage in the presence of such initiators [14]. However, Pagoria et al. [15] confirmed the protective effect of high concentrations of NAC and ascorbic acid on 3T3 and OCCM.30 immortalized cell lines against damage induced by VLirradiated CQ/DMT. In the present study, we investigated the effectiveness of the ROS scavenger NAC to reduce HEMA-induced cytotoxicity and cell death. NAC is a precursor of glutathione and it may decrease cell oxidative stress directly, as a source of sulfhydryl groups which neutralize ROS, or indirectly, restoring the glutathione content [33]. Therefore, in our study we co-treated HGF with different concentrations of NAC and HEMA. Similar to the above studies [14,15], our results showed that high NAC concentrations (5 and 10 mM) reduced ROS production and toxicity induced by HEMA showing a complete and significant reversion of HEMA effect (Fig. 4). In contrast to high NAC concentration, 1 mM was able to reduce ROS production only after 2 h, while at 6 h the same concentration showed instead a pro-oxidant effect enhancing the production of ROS and cell damage induced by

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HEMA (Figs. 4 and 5). Thus, here high NAC concentrations acted as antioxidant reducing the overproduction of intracellular ROS thereby preventing cytotoxicity induced by HEMA. Such protective effect of NAC could be also related to a possible restoring of the reduced glutathione content [33] or to a modulation of redox-sensitive cellular signal transduction components including Ras, Raf-1, MEK, ERK and transcription factors such as AP-1, NFkB and Nr-f2 [29,34]. Obviously, further studies are needed to test this hypothesis. However, our result showed also a pro-oxidant effect of 1 mM NAC on HGF co-treated with HEMA. There is evidence that antioxidants might act as pro-oxidants [35,36]. NAC is an extracellular and intracellular antioxidant [37]. In antioxidant reactions, thiols undergo one-electron oxidation with the formation of thiyl radicals [38]. Thiyl radicals are produced in all reactions of thiols with other free radicals and with H2O2, and they can also be generated in the reactions of thiols with transition metal ions. Reduced ions become very often promoters of free radical reactions, such as in biological Fenton reaction [37,38]. Antioxidant action of thiol compounds is determined by both, efficient detoxification of free radicals and inactivation of the concurrently created thiyl radical. For this reason, thiol compounds, similarly as any compounds able to play a role of antioxidants, should immediately undergo regenerative reduction [38,39]. NAC could exert a pro-oxidative action because it might react with ROS generated by HEMA, leading both to the formation of thiyl radicals and to excessive generation of hydroxyl radical ions, but additional studies are needed to clarify this point. This indicates that thiol compounds, as NAC, in the presence of trace amounts of transition metal ions and oxygen might be oxidized to form thiyl radicals and superoxide radical anion, and, in consequence, also other ROS [40–43]. Therefore, in our experimental model, low NAC concentrations are not sufficient to counteract both the ROS derived from autooxidation of NAC and the ROS production induced by HEMA, resulting in a markedly increase of ROS and cell damage such as we found. 5. Conclusion In this study we show, on HGF, that HEMA induces ROS production which could be responsible, at least in part, for the cytotoxicity of this monomer. In fact, our results showed that high NAC concentrations had the ability, by decreasing ROS levels, to reduce cell damage and cytotoxicity caused by HEMA, although a low NAC concentration enhanced HEMA toxicity. For this reason we are in agreement with the previous studies in which was highlighted the importance to investigate, in future, the in vivo effective amount of NAC (or other antioxidants) able to prevent cell damage and cytotoxicity induced by dental monomers and initiators [14,15]. In addition, further in vitro investigations on pulpal fibroblasts or odontoblasts (odontoblast cell lines) should be focused on the relationship between monomers, antioxidants and intracellular

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