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RESEARCH REPORTS |
B Protection against Apoptosis Induced by HEMA
1 Department of Oral and Maxillofacial Sciences,
2 Department of Cellular and Molecular Biology and Pathology,
3 Department of Neuroscience, Unit of Physiology, and
4 Department of Clinical and Experimental Medicine, University of Naples "Federico II", via S. Pansini 5, 80131-Naples, Italy; and
5 Department of Operative Dentistry and Periodontology, University of Regensburg, 93042-Regensburg, Germany;
* corresponding author, gspagnuo{at}unina.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B activation after HEMA exposure. Blocking of ROS production by anti-oxidants had no direct influence on apoptosis caused by HEMA, but inhibition of NF-B increased the fraction of apoptotic cells. Accordingly, mouse embryonic fibroblasts (MEF) from p65–/– mice were more susceptible to HEMA-induced apoptosis than were wild-type controls. Our results indicate that exposure to HEMA triggers apoptosis and that this mechanism is not directly dependent upon redox signaling. Nevertheless, ROS induction by HEMA activates NF-B, which exerts a protective role in counteracting apoptosis.
KEY WORDS: NF-B • HEMA • apoptosis • ROS • human fibroblasts
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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HEMA is frequently used in dental bonding resins as a wetting agent. It competes with water for penetration and infiltration into dentin, and it copolymerizes with other monomers of resin composites (Peutzfeldt, 1997). HEMA has been shown to diffuse rapidly across dentin toward the pulp, and this may cause pulp irritation (Bouillaguet et al., 1996).
Biological parameters such as cellular proliferation, vitality and cell death, mitochondrial activity, and protein or nucleic acid synthesis have been used as indicators of cellular responses to resin components (Hanks et al., 1991; Kostoryz et al., 2001; Schweikl et al., 2001; Noda et al., 2002). So far, few studies have assessed the type of cell death caused by dental monomers (Janke et al., 2003; Engelmann et al. 2004; Spagnuolo et al., 2004). Two modes of cell death have been described: apoptosis and necrosis. Apoptosis is an active and physiological mode of cell death, which permits the organism to eliminate unwanted or sub-lethally damaged cells without triggering inflammatory reactions. In contrast, necrosis generally sets off a tissue inflammation associated with clinical symptoms (Majno and Joris, 1995).
The execution of apoptosis is mediated by caspases, a family of cysteine proteases that plays a central role in the disassembly of the cell (Villa et al., 1997). Caspase-9 is the initiator caspase of the mitochondrial pathway of apoptosis, while caspase-8 is an initiator caspase of death-receptor-induced apoptosis (Thornberry and Lazebnik, 1998). Reactive oxygen species (ROS)—including hydrogen peroxide, superoxide anion, and the hydroxyl radical—are involved in apoptosis. ROS may induce apoptosis directly or act as intracellular messengers induced by various kinds of stimuli (Mates and Sanchez-Jimenez, 2000).
Numerous studies suggest that ROS serve as common intracellular mediators of IB degradation and subsequent NF-B activation (Baeuerle and Henkel, 1994). The NF-B family of proteins is an inducible transcription factor that regulates the expression of genes involved in disparate processes such as immunity and inflammation, growth, development, cell-death regulation, and protection from apoptosis (Karin and Ben-Neriah, 2000). NF-B is known to be one of the major pro-survival factors in many cells (Beg and Baltimore, 1996). In mammalian cells, there are 5 NF-B proteins: p50, p52, p65 (RelA), RelB, and c-Rel. NF-B is composed of homodimers and heterodimers of these proteins, typically p65:p50, which are held in the cytoplasm by the inhibitory IB proteins. Several IB proteins have been identified, including IB
, IBß, IB
, and IB
proteins, of which IB and IBß have been the best-studied (Karin and Ben-Neriah, 2000). Activation of NF-B is most often mediated by IB degradation, which permits NF-B to enter the nucleus (Karin, 1999).
Recent studies were focused on the type of cell death caused by dental materials and monomers (Cimpan et al., 2000; Janke et al., 2003; Spagnuolo et al., 2004) and on a possible link between an increase in ROS levels and the toxicity of monomers (Stanislawski et al., 2003). So far, there are no similar data available on HEMA toxicity. We hypothesized that HEMA causes an increase in the production of ROS, leading to apoptosis. We found that HEMA induced apoptosis and increased ROS levels in human primary skin fibroblasts. Unexpectedly, HEMA-induced apoptosis was not reduced but was enhanced by an anti-oxidant. Since several anti-oxidants are NF-B inhibitors (Brennan and O’Neill, 1995), and considering that NF-B is a pro-survival factor involved in protection from apoptosis, we further investigated the hypothesis that HEMA induces NF-B activation by increasing ROS, speculating that HEMA-induced apoptosis is counteracted by NF-B activation.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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For each experiment, HEMA (Sigma, St. Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO) (1 M stock solution) and then diluted in culture medium. Cells cultured in the presence of 0.3% DMSO alone served as the control in all experiments. A 10-µM quantity of pyrrolidine dithiocarbamate (PDTC; Sigma), a low-molecular-weight thiol compound, was used as an anti-oxidant (Schreck et al., 1992).
Detection of Apoptosis
Cells were exposed to different concentrations of HEMA (0–10 mM) for 24 hrs at 37°C, harvested by centrifugation, and washed once with phosphate-buffered saline (PBS). Then, 1x 105–106 cells were suspended in 500 µL of binding buffer. Untreated and treated cells were stained with annexin V-FITC and propidium iodide (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). 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 (Vermes et al., 1995). Analysis of the data was performed by means of the WinMDI 2.8 program.
Measurement of Intracellular ROS
Generation of reactive oxygen species was measured with the use of an oxidation-sensitive fluorescent probe, 2'7'-dichlorofluorescin diacetate (DCFH-DA; Molecular Probe, Inc., Eugene, OR, USA), whose oxidized form (2'7'-dichlorofluorescein, DCF) is highly fluorescent (Myhre et al., 2003). Cells from routine cell cultures were harvested by centrifugation, washed twice with PBS, and suspended in 1 mL PBS. The cells were then loaded with 10 µM DCFH-DA and incubated at 37°C for 15 min. After the addition of HEMA (from 0 to 10 mM), cells were incubated at 37°C for various times (from 0 to 60 min). We used a FACScan flow cytometer to measure ROS generation by the fluorescence intensity (FL-1, 530 nm) of 10,000 cells. Mean fluorescence intensity was obtained by histogram statistics with use of the WinMDI 2.8 program.
SDS-PAGE and Western Blotting
Human skin fibroblasts were grown until 70–80% confluent and exposed to 0–10 mM HEMA for different time periods. Cytosolic extracts were then prepared with the use of a lysis buffer (20 mM HEPES, 1% Triton X-100, 150 mM NaCl, 10% glycerol), supplemented with protease inhibitors (Boeringher Mannheim, Mannheim, Germany) and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4). Equivalent amounts of proteins (40 µg) were resolved on a 10% SDS-PAGE. Proteins were transferred to an Immobilion-P membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 10% non-fat dry milk in TBS/Tween-20 and probed with rabbit polyclonal anti-caspases antibodies (–8, –9, and –3) and anti-IB (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The secondary antibody was a horseradish-peroxidase-linked anti-rabbit IgG (Amersham Biosciences, Little Chalfont, Buckinghamshire, England). Signals were detected by the ECL system (Amersham Biosciences, Little Chalfont, Buckinghamshire, England).
EMSA (Electromobility shift assay)
Cell were treated with 10 mM HEMA for various time periods, and total cell extracts were prepared by the use of a detergent lysis buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 0.5% Nonited P-40, 50 mM NaF, 1 mM Na3VO4 and complete protease inhibitor mixture [Roche]). Cells were harvested by centrifugation, washed once in cold PBS, and then re-suspended in lysis buffer (30 µL 5 x 106 cells). The cell lysate was incubated on ice for 30 min and then centrifuged for 10 min at 10,000 x g at 4°C. The protein content of the supernatant was determined, and equal amounts of proteins (5 µg) were added to a reaction mixture containing 20 µg BSA, 2 µg poly (dI-dC), 10 µL binding buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol), and 100,000 cpm of a 32P-labeled -B oligonucleotide in a final volume of 20 µL. For competition assay, a 100-ng quantity of non-radioactive -B oligonucleotide was added to the reaction mixture. Samples were incubated at room temperature for 30 min and then run on a 4% acrylamide gel.
Statistical Analysis
The statistical analyses of the data were performed by t tests or one-way ANOVA, followed by the Bonferroni procedure for multiple comparisons. The level of significance was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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) (n = 4).
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). Caspase-3 activation started 6 hrs after treatment with 10 mM HEMA cells (Fig. 1C). Moreover, the processing of procaspases-9 and -8 appeared to be complete, as shown by the disappearance of the precursor forms (Fig. 1D).
HEMA Induces ROS and NF-B Activation
HEMA increased ROS levels in a dose-dependent manner; treatment of cell culture with 10 mM HEMA produced a two-fold increase in fluorescence units compared with untreated controls (Fig. 2A) (n = 5). The time-course of ROS showed that ROS were produced immediately after HEMA treatment at all concentrations used. The increase of ROS in untreated cell cultures indicated high metabolic activity (Fig. 2B) (n = 5).
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B inhibitory subunit IB. The levels of IB in cell lysates were decreased 180 min after exposure to 10 mM HEMA (Fig. 3A). In addition, IB degradation was dose-dependent (Fig. 3B). To assess the DNA binding of NF-B, we performed an EMSA on total cell extracts. A 10-mM quantity of HEMA caused DNA binding after 180 min (Fig. 3C). The specificity of the protein-DNA complex was confirmed by a competition assay performed by a non-radioactive -B oligonucleotide (Fig. 3C).
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B Counteracts HEMA-induced Apoptosis). Paradoxically, PDTC alone did not affect basal apoptosis but significantly increased HEMA-induced apoptosis, notwithstanding the reduction of ROS (Fig. 4B). To identify the signals influenced by HEMA and PDTC, we treated cells with HEMA in the absence and in the presence of PDTC for 180 min and then determined IB degradation and DNA binding of NF-B. PDTC completely blocked the binding and IB degradation induced by 10 mM HEMA (Figs. 3C, 4C). Reduction of active NF-B resulted in an amplification of apoptosis (Fig. 4B) (n = 3). To investigate this hypothesis, we used MEF from p65–/– mice. MEF wild-type and p65–/– cells were treated with a sub-lethal concentration of 8 mM HEMA. Interestingly, p65–/– fibroblasts showed a significant increase (n = 3) of apoptosis (three-fold) and necrosis (two-fold) compared with that in wild-type cells (Fig. 4D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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ROS are involved in apoptosis as well as in cell survival (Mates and Sanchez-Jimenez, 2000). Here, elevated levels of ROS were observed immediately after HEMA treatment. The amounts of ROS due to treatment with HEMA may be underestimated related to in vivo situations, since the solvent DMSO has previously been reported to have ROS scavenger activity (Zegura et al., 2004). ROS generation was completely reduced by a free radical scavenger such as PDTC. However, reduction of ROS generation by PDTC did not prevent HEMA-induced apoptosis. This strongly suggested that ROS do not directly mediate HEMA-induced toxicity.
Many studies have suggested that ROS serve as intracellular mediators of IB degradation and subsequent NF-B activation (Baeuerle and Henkel, 1994). NF-B has been implicated as a key regulatory molecule that may function to mediate a cellular survival response (Beg and Baltimore, 1996). In our experimental system, IB was degraded after 3 hrs of HEMA treatment. This delayed timing of IB degradation and DNA binding of NF-B suggests that HEMA did not directly induce NF-B. It is likely that HEMA caused cell damage which, in turn, could be responsible for NF-B activation. The fact that PDTC can independently inhibit NF-B activation has been used in support of a general model in which NF-B activation is governed by oxidative stress (Schreck et al., 1991, 1992; Moellering et al., 1999). Evidence is emerging, however, that the major mode of the inhibitory action of PDTC may not be restricted to its anti-oxidant activity (Brennan and O’Neill, 1995). We have shown that inhibition of IB degradation and DNA binding of NF-B by PDTC significantly increased HEMA-induced apoptosis in human fibroblasts. This suggests that NF-B may be important for protection from HEMA-induced apoptosis. The role played by NF-B was further confirmed by the evidence that MEF from p65–/– mice are more susceptible to HEMA-induced apoptosis.
It has been shown that HEMA diffuses through dentin, and the risk of cytotoxic effects in pulp cells might be increased (Bouillaguet et al., 1996). Noda et al.(2002) estimated that HEMA leaching from dentin adhesives might reach concentrations up to 1.5–8 mM in the pulp. This concentration is within the range of our study. If used in a direct pulp-capping procedure, then this concentration is clearly higher than those used here. The balance of the apoptotic-necrosis response induced by HEMA may have time-concentration dependence. Therefore, if the HEMA concentration is high, then apoptosis would never occur. If low, then apoptosis could be more prominent. This might be a critical feature in pulp-capping (direct or indirect), where high-acute or low-chronic amounts of HEMA are exposed to pulpal cells.
In summary, we demonstrated that HEMA induces cell death through an apoptotic pathway in primary human fibroblasts, involving activation of caspase-8, -9, and -3. The HEMA-induced apoptosis is not directly dependent on the generation of ROS, because reduction of ROS by anti-oxidants had no effect on apoptosis. Moreover, we provided experimental evidence that NF-B plays a major role in protecting cells from apoptosis induced by HEMA.
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ACKNOWLEDGMENTS |
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Received October 27, 2003; Last revision August 6, 2004; Accepted August 24, 2004
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B in the immune system. Annu Rev Immunol 12:141–179.[ISI][Medline]
Beg AA, Baltimore D (1996). An essential role for NF-B in preventing TNF-alpha-induced cell death. Science 274:782–784.
Bouillaguet S, Wataha JC, Hanks CT, Ciucchi B, Holz J (1996). In vitro cytotoxicity and dentin permeability of HEMA. J Endod 22:244–248.[ISI][Medline]
Brennan P, O’Neill LA (1995). Effects of oxidants and antioxidants on nuclear factor kappa B activation in three different cell lines: evidence against a universal hypothesis involving oxygen radicals. Biochim Biophys Acta 1260:167–175.[Medline]
Cimpan MR, Cressey LL, Skaug N, Halstensen A, Lie SA, Gjertsen BT, et al. (2000). Patterns of cell death induced by eluates from denture base acrylic resins in U-937 human monoblastoid cells. Eur J Oral Sci 108:59–69.[ISI][Medline]
De Smaele E, Zazzeroni F, Papa S, Nguyen DU, Jin R, Jones J, et al. (2001). Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature 414:308–313.[Medline]
Engelmann J, Janke V, Volk J, Leyhausen G, von Neuhoff N, Schlegelberger B, et al. (2004). Effects of BisGMA on glutathione metabolism and apoptosis in human gingival fibroblasts in vitro. Biomaterials 25:4573–4580.[ISI][Medline]
Geurtsen W (2000). Biocompatibility of resin-modified filling materials. Crit Rev Oral Biol Med 11:333–355.[Abstract]
Hanks CT, Strawn SE, Wataha JC, Craig RG (1991). Cytotoxic effects of resin components on cultured mammalian fibroblasts. J Dent Res 70:1450–1455.
Janke V, von Neuhoff N, Schlegelberger B, Leyhausen G, Geurtsen W (2003). TEGDMA causes apoptosis in primary human gingival fibroblasts. J Dent Res 82:814–818.
Karin M (1999). How NF-B is activated: the role of the IB kinase (IKK) complex. Oncogene 18:6867–6874.[ISI][Medline]
Karin M, Ben-Neriah Y (2000). Phosphorylation meets ubiquitination: the control of NF-kappaB activity. Annu Rev Immunol 18:621–663.[ISI][Medline]
Kostoryz EL, Tong PY, Strautman AF, Glaros AG, Eick JD, Yourtee DM (2001). Effects of dental resin on TNF--induced ICAM-1 expression in endothelial cells. J Dent Res 80:1789–1792.
Majno G, Joris I (1995). Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 146:3–15.[Abstract]
Mates JM, Sanchez-Jimenez FM (2000). Role of reactive oxygen species in apoptosis: implications for cancer therapy. Int J Biochem Cell Biol 32:157–170.[ISI][Medline]
Moellering D, McAndrew J, Jo H, Darley-Usmar VM (1999). Effects of pyrrolidine dithiocarbamate on endothelial cells: protection against oxidative stress. Free Radic Biol Med 26:1138–1145.[ISI][Medline]
Myhre O, Andersen JM, Aarnes H, Fonnum F (2003). Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol 65:1575–1582.[ISI][Medline]
Noda M, Wataha JC, Kaga M, Lockwood PE, Volkmann KR, Sano H (2002). Components of dentinal adhesives modulate heat shock protein 72 expression in heat-stressed THP-1 human monocytes at sublethal concentrations. J Dent Res 81:265–269.
Peutzfeldt A (1997). Resin composites in dentistry: the monomer systems. Eur J Oral Sci 105:97–116.[ISI][Medline]
Schreck R, Rieber P, Baeuerle PA (1991). Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-B transcription factor and HIV-1. EMBO J 10:2247–2258.[ISI][Medline]
Schreck R, Meier B, Männel DN, Dröge W, Baeuerle PA (1992). Dithiocarbamates as potent inhibitors of nuclear factor-B activation in intact cells. J Exp Med 175:1181–1194.
Schweikl H, Schmalz G, Spruss T (2001). The induction of micronuclei in vitro by unpolymerized resin monomers. J Dent Res 80:1615–1620.
Spagnuolo G, Galler K, Schmalz G, Cosentino C, Rengo S, Schweikl H (2004). Inhibition of phosphatidylinositol 3-kinase amplifies TEGDMA-induced apoptosis in primary human pulp cells. J Dent Res 83:703–707.
Stanislawski L, Lefeuvre M, Bourd K, Soheili-Majd E, Goldberg M, Perianin A (2003). TEGDMA-induced toxicity in human fibroblasts is associated with early and drastic glutathione depletion with subsequent production of oxygen reactive species. J Biomed Mater Res 66(A):476–482.
Thornberry NA, Lazebnik Y (1998). Caspases: enemies within. Science 281:1312–1316.
Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods 184:39–51.[ISI][Medline]
Villa P, Kaufmann SH, Earnshaw WC (1997). Caspases and caspase inhibitors. Trends Biochem Sci 22:388–392.[ISI][Medline]
Zegura B, Lah TT, Filipic M (2004). The role of reactive oxygen species in microcystin-LR-induced DNA damage. Toxicology 200(1):59–68.[ISI][Medline]
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