Carola Bolay

Differential gene expression involved in oxidative stress response caused by triethylene glycol dimethacrylate.

Triethylene glycol dimethacrylate (TEGDMA) is a comonomer that is released from dental resin-based materials into hydrophilic solvents. The compound reduces cell vitality, and causes genotoxicity in mammalian cells in vitro. Here, we used gene expression profiling, combined with pathway analysis tools, to identify the molecular events associated with TEGDMA cytotoxicity in human fibroblasts using Affymetrix HG-U133A 2.0 GeneChip arrays. Increased ROS production and a cell cycle delay caused by 3mm TEGDMA after a 6h exposure were related to a cell response at the transcriptional level. The predominant biological processes associated with the genes that were differentially expressed in untreated and treated cell cultures included oxidative stress, cellular growth, proliferation and morphology, cell death, gene expression as well as DNA replication and repair. The most significantly upregulated genes were GEM (17-fold), KLHL24, DDIT4, TGIF, DUSP5 and ATF3, which are all related to the regulation of the cell structure, stress response, and cell proliferation. TXNIP was the most downregulated transcript (five-fold), whose gene product regulates the cellular redox balance. The downregulation of NRG1, ASPM, FBXO5, and PLK2 is linked to the regulation of cell proliferation and cell structure. The underlying mechanisms of the up- and downregulation of genes seem to be activated by the production of ROS, and the related regulation of the cellular redox balance disturbed in the presence of TEGDMA appears to be of the utmost importance. The coordinated induction of genes coding for oxidative stress response and antioxidant proteins is a critical mechanism of protection against TEGDMA-induced cell damage.

TEGDMA-induced oxidative DNA damage and activation of ATM and MAP kinases

The development of strategies for the protection of oral tissues against the adverse effects of resin monomers is primarily based on the elucidation of underlying molecular mechanisms. The generation of reactive oxygen species beyond the capacity of a balanced redox regulation in cells is probably a cause of cell damage. This study was designed to investigate oxidative DNA damage, the activation of ATM, a reporter of DNA damage, and redox-sensitive signal transduction through mitogen-activated protein kinases (MAPKs) by the monomer triethylene glycol dimethacrylate (TEGDMA). TEGDMA concentrations as high as 3-5 mM decreased THP-1 cell viability after a 24h and 48h exposure, and levels of 8-oxoguanine (8-oxoG) increased about 3- to 5-fold. The cells were partially protected from toxicity in the presence of N-acetylcysteine (NAC). TEGDMA also induced a delay in the cell cycle. The number of THP-1 cells increased about 2-fold in G1 phase and 5-fold in G2 phase in cultures treated with 3-5 mM TEGDMA. ATM was activated in THP-1 cells by TEGDMA. Likewise, the amounts of phospho-p38 were increased about 3-fold by 3 mM TEGDMA compared to untreated controls after a 24h and 48h exposure period, and phospho-ERK1/2 was induced in a very similar way. The activation of both MAPKs was inhibited by NAC. Our findings suggest that the activation of various signal transduction pathways is related to oxidative stress caused by a resin monomer. Signaling through ATM indicates oxidative DNA damage and the activation of MAPK pathways indicates oxidative stress-induced regulation of cell survival and apoptosis.

TEGDMA Reduces Mineralization in Dental Pulp Cells

Direct application of dentin bonding agents onto the exposed pulp has been advocated, but in vivo studies indicate a lack of reparative dentin formation. Our objective was to investigate the role of triethylene glycol dimethacrylate (TEGDMA), a commonly used compound in dentin bonding agents, as a potential inhibitor of mineralization. Human pulp cells were exposed to different concentrations of TEGDMA, and expression of the mineralization-related genes collagen I, alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, and dentin sialophosphoprotein was analyzed. Gene expression studies by real-time polymerase chain-reaction revealed a concentration- and time-dependent decrease of mineralization markers. A subtoxic TEGDMA concentration (0.3 mM) reduced expression levels by 5 to 20% after 4 hrs and by 50% after 12 hrs. Furthermore, alkaline phosphatase activity and calcium deposition were significantly lower in dental pulp cells treated with TEGDMA over 14 days. These findings indicate that even low TEGDMA concentrations might inhibit mineralization induced by dental pulp cells, thus impairing reparative dentin formation after pulp capping with dentin bonding agents.

Inhibition of cytokine and surface antigen expression in LPS-stimulated murine macrophages by triethylene glycol dimethacrylate.

Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) cause a shift in the cellular redox balance which influences redox-sensitive signaling pathways. The immediate response of the innate immune system to inflammatory challenges is controlled by related pathways. Therefore, the influence of TEGDMA on the expression of the pro- and anti-inflammatory cytokines TNF-alpha, IL-6, and IL-10 and surface antigens (CD14, CD40, CD80, CD86, CD54, MHC class I and II) was analyzed in RAW264.7 macrophages. No significant change in cytokine production or surface antigen expression was detected after the macrophages were treated with increasing TEGDMA concentrations for 6, 24, and 48h. However, co-stimulation with the bacterial endotoxin lipopolysaccharide (LPS) and TEGDMA resulted in a concentration-dependent inhibition of LPS-induced release of TNF-alpha, IL-6, and IL-10 by about 90% as detected by ELISA. Flow-cytometric analyses indicated an LPS-stimulated expression of all surface antigens. The LPS-induced expression of CD14 was inhibited by high TEGDMA concentrations. CD40 and CD80 expressions were down-regulated by TEGDMA in LPS-stimulated cells, and CD86 as well as MHC class I expression was inhibited to a lesser extent. The LPS-stimulated expression of CD54 (ICAM-1) was increased about twofold by increasing TEGDMA concentrations after a 24 and 48h exposure. Thus, the ability of macrophages to induce an appropriate immune response is inhibited by TEGDMA which reduces cytokine production and expression of surface antigens.

2-Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway.

Resin monomers of dental composites like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation of dental pulp-derived cells, or induce cell death via apoptosis. The induction of apoptosis is related to the availability of the antioxidant glutathione, although a detailed understanding of the signaling pathways is still unknown. The present study provides insight into the causal relationship between oxidative stress, oxidative DNA damage, and the specific signaling pathway leading to HEMA-induced apoptosis in RAW264.7 mouse macrophages. The differential expression of the antioxidative enzymes superoxide dismutase, glutathione peroxidase, and catalase in HEMA-exposed cells indicated oxidative stress, which was associated with the cleavage of pro-caspase 3 as a critical apoptosis executioner. A 2-fold increase in the amount of mitochondrial superoxide anions after a 24 h exposure to HEMA (6-8 mM) was paralleled by a considerable decrease in the mitochondrial membrane potential (MMP). Additionally, expression of proteins critical for the signaling of apoptosis through the intrinsic mitochondrial pathway was detected. Transcription-dependent and transcription-independent mechanisms of p53-regulated apoptosis were activated, and p53 was translocated from the cytosol to mitochondria. HEMA-induced transcriptional activity of p53 was indicated by increased levels of PUMA localized to mitochondria as a potent inducer of apoptosis. The expression of Bcl-xL and Bax suggested that cells responded to stress caused by HEMA via the activation of a complicated and antagonistic machinery of pro- and anti-apoptotic Bcl-2 family members. A HEMA-induced and oxidative stress-sensitive delay of the cell cycle, indicating a DNA damage response, occurred independent of the influence of KU55399, a potent inhibitor of ATM (ataxia-telangiectasia mutated) activity. However, ATM, a protein kinase which responds to DNA double-strand breaks, and the signaling pathway downstream were activated in HEMA-exposed cells. Likewise, expression and phosphorylation of the ATM targets H2AX and p53 was reduced in the presence of KU55399. Moreover, the percentage of cells undergoing apoptosis drastically decreased in HEMA-exposed cell cultures pre-treated with KU55933. These findings demonstrate that HEMA-induced apoptosis is mediated through the intrinsic mitochondrial pathway as a consequence of p53 activation via ATM signaling upon oxidative DNA damage.

Activation of stress-regulated transcription factors by triethylene glycol dimethacrylate monomer

Triethylene glycol dimethacrylate (TEGDMA) is a resin monomer available for short exposure scenarios of oral tissues due to incomplete polymerization processes of dental composite materials. The generation of reactive oxygen species (ROS) in the presence of resin monomers is discussed as a common mechanism underlying cellular reactions as diverse as disturbed responses of the innate immune system, inhibition of dentin mineralization processes, genotoxicity and a delayed cell cycle. Yet, the signaling pathway through a network of proteins that finally initiates the execution of monomer-induced specific cell responses is unknown so far. The aim of the present study was to extend the knowledge of molecular mechanisms of monomer-induced cell death as a basis for reasonable therapy strategies. Thus, the monomer-induced expression and phosphorylation of stress-related transcription factors was analyzed in various cell lines. The time-related induction of apoptosis was investigated as well. The expression of p53 increased in HeLa cell cultures treated with camptothecin (positive control) for 24h, and the formation of p53Ser15 and p53Ser46 was detected in cell nuclei by Western blotting. TEGDMA (3 mm) appeared to stimulate p53 expression only slightly, but increased p21 expression was found in cell nuclei and cytoplasm. Both camptothecin and TEGDMA increased p53 expression to some extent in the nuclear fraction in human transformed pulp-derived cells (tHPC), and similar effects were detected in RAW264.7 macrophages. No clear induction of c-Jun and phospho-c-Jun by TEGDMA was detected in HeLa cell nuclei, and the expression of ATF-2 and phospho-ATF-2 was inhibited in the presence of the monomer. ATF-3 expression was found only in the nuclear fraction of camptothecin-treated HeLa cultures. TEGDMA seemed to inhibit the formation of phospho-c-Jun and phospho-ATF-2 in tHPC, and the monomer acted negatively on the expression of c-Jun, ATF-2 and ATF-3 in RAW264.7 macrophages. These changes in the expression and activation of stress-related transcription factors were time-related to the induction of apoptosis by TEGDMA in all cell lines. The present study provides experimental evidence that TEGDMA interferes with the regulation of cellular pathways through transcription factors activated as a consequence of DNA damage like p53 or initiated downstream of MAPK (mitogen-activated protein kinases) like c-Jun, ATF-2 and ATF-3. The direct causal correlation between DNA damage, activation or inhibition of MAPKs and transcription factors, and apoptosis is under current investigation. However, the induction of apoptosis in different cell lines in the presence of monomers like TEGDMA may be subject to a higher level of complexity than currently suggested by simple linear models.

Influence of TEGDMA on the mammalian cell cycle in comparison with chemotherapeutic agents

OBJECTIVES The dental resin monomer triethylene glycol dimethacrylate (TEGDMA) caused a cell cycle arrest in response to DNA damage. However, the underlying mechanisms are unclear. Therefore, the influence of TEGDMA on the cell cycle was analyzed in comparison with the chemotherapeutic agents adriamycin and mitomycin C (MMC), which arrest the cell cycle through different mechanisms. METHODS RAW264.7 mouse macrophages were exposed to TEGDMA, adriamycin, or MMC, and flow cytometry (FACS) was used for cell cycle analyses. In addition, the number of surviving cells was determined by a crystal violet assay, and viability in treated cultures was determined by FACS after staining of cells with trypan blue. Morphological changes in cells were interpreted using forward and side scatter (FSC/SSC) cell physical criteria. RESULTS The exposure of cells to 1mM TEGDMA resulted in a delay of the cell cycle in G1 phase since 85.3% of the cells were found in G1 compared with 47.4% in untreated controls. Adriamycin also increased the number of cells (72.1%) in G1 compared to controls. Caffeine, an inhibitor of the checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), had no effect on the TEGDMA and adriamycin-induced cell cycle arrest. In contrast, MMC delayed the cell cycle in G2 since cell numbers increased to 22.1% compared to 10.7% in controls. The effect of MMC on G2 was even increased by low caffeine concentrations (100-400muM), but 1000muM caffeine inhibited MMC activity. SIGNIFICANCE Our results suggest that the mechanism of a TEGDMA-induced arrest of the cell cycle is different from the effect of the direct-acting interstrand crosslinking agent MMC. Since TEGDMA produced oxidative stress, it probably acts indirectly on the cell cycle through reactive oxygen species, unless TEGDMA-DNA adducts are shown experimentally.

Function of MAPK and downstream transcription factors in monomer-induced apoptosis

The resin monomer triethylene glycol dimethacrylate (TEGDMA) disrupts vital cell functions, and the production of oxidative stress is considered a common underlying mechanism. The precise signaling pathways, however, that initiate monomer-induced effects, which disturb responses of the innate immune system, inhibit dentin mineralization processes, or induce apoptosis in target cells in vitro are still unknown. The present study provides insight into the causal relationship between TEGDMA-induced apoptosis and the activation of MAPK and transcription factors downstream using pharmacological inhibitors of the ERK1/2, p38 and JNK pathways. The endotoxin lipopolysaccharide (LPS; 0.1 μg/ml) was included as an inducer of MAPK activity in RAW264.7 mouse macrophages. Cell viability was decreased from 95% in untreated cultures to about 43% after a 24 h exposure to 3 mM TEGDMA. Inhibition of the ERK1/2 pathway by the MEK1/2 inhibitor PD98059 reduced cell viability to 84%. While apoptosis induced by TEGDMA remained unchanged, Western blot analyses revealed that the activation of ERK1/2 in the presence of TEGDMA was inhibited by PD98059. LPS-induced expression of activated transcription factors c-Jun, ATF-2, ATF-3 and phospho-Elk1 was decreased in cells co-treated with TEGDMA. This inhibition was more intense in the presence of PD98059, indicating that the MEK/ERK pathway is involved in the inhibition of the LPS-induced activation of transcription factors by TEGDMA. No clear effects of the p38 inhibitor SB203580 and the JNK inhibitor SP600125 on TEGDMA-induced apoptosis were detected. The antioxidant N-acetylcysteine (NAC) protected cells from TEGDMA-induced cell death, and inhibited the activation of ERK1/2, p38 and JNK by TEGDMA. Moreover, the TEGDMA-induced downregulation of the expression of the transcription factors c-Jun and ATF-2 was prevented as well. In conclusion, physiologically relevant concentrations of inhibitors differentially modified the expression of MAPK and transcription factors in cell cultures exposed to LPS and the monomer TEGDMA. The absence of a drastic effect of the MAPK pathway inhibitors on TEGDMA-induced apoptosis on the one hand, and the protective effect of NAC and PD98059 in particular on TEGDMA-induced MAPK activation and apoptosis on the other hand, leads to a new model for the role of MAPK in the regulation of cell homeostasis in monomer-exposed cells and tissues.

Evaluation of cell responses toward adhesives with different photoinitiating systems

OBJECTIVES The photoinitiator diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) is more reactive than a camphorquinone/amine (CQ) system, and TPO-based adhesives obtained a higher degree of conversion (DC) with fewer leached monomers. The hypothesis tested here is that a TPO-based adhesive is less toxic than a CQ-based adhesive. METHODS A CQ-based adhesive (SBU-CQ) (Scotchbond Universal, 3M ESPE) and its experimental counterpart with TPO (SBU-TPO) were tested for cytotoxicity in human pulp-derived cells (tHPC). Oxidative stress was analyzed by the generation of reactive oxygen species (ROS) and by the expression of antioxidant enzymes. A dentin barrier test (DBT) was used to evaluate cell viability in simulated clinical circumstances. RESULTS Unpolymerized SBU-TPO was significantly more toxic than SBU-CQ after a 24h exposure, and TPO alone (EC50=0.06mM) was more cytotoxic than CQ (EC50=0.88mM), EDMAB (EC50=0.68mM) or CQ/EDMAB (EC50=0.50mM). Cultures preincubated with BSO (l-buthionine sulfoximine), an inhibitor of glutathione synthesis, indicated a minor role of glutathione in cytotoxic responses toward the adhesives. Although the generation of ROS was not detected, a differential expression of enzymatic antioxidants revealed that cells exposed to unpolymerized SBU-TPO or SBU-CQ are subject to oxidative stress. Polymerized SBU-TPO was more cytotoxic than SBU-CQ under specific experimental conditions only, but no cytotoxicity was detected in a DBT with a 200μm dentin barrier. SIGNIFICANCE Not only DC and monomer-release determine the biocompatibility of adhesives, but also the cytotoxicity of the (photo-)initiator should be taken into account. Addition of TPO rendered a universal adhesive more toxic compared to CQ; however, this effect could be annulled by a thin dentin barrier.

Interaction between LPS and a dental resin monomer on cell viability in mouse macrophages

OBJECTIVE Lipopolysaccharide (LPS) from cariogenic microorganisms and resin monomers like HEMA (2-hydroxyethyl methacrylate) included in dentin adhesive are present in a clinical situation in deep dentinal cavity preparations. Here, cell survival, expression of proteins related to redox homeostasis, and viability of mouse macrophages exposed to LPS and HEMA were analyzed with respect to the influence of oxidative stress. METHODS Cell survival of RAW264.7 mouse macrophages was determined using a crystal violet assay, protein expression was detected by Western blotting, and HEMA- or LPS-induced apoptosis (cell viability) was analyzed by flow cytometry. Cells were exposed to HEMA (0-8mM), LPS (0.1μg/ml) or combinations of both substances for 24h. The influence of mitogen-activated protein kinases (MAPK) was analyzed using the specific inhibitors PD98059 (ERK1/2), SB203580 (p38) or SP600125 (JNK), and oxidative stress was identified by the antioxidant N-acetylcysteine (NAC). RESULTS Cell survival was reduced by HEMA. LPS, however, increased cell survival from 29% in cultures exposed to 8mM HEMA, to 46% in cultures co-exposed to 8mM HEMA/LPS. Notably, LPS-induced apoptosis was neutralized by 4-6mM HEMA but apoptosis caused by 8mM HEMA was counteracted by LPS. Expression of NOS (nitric oxide synthase), p47phox and p67phox subunits of NADPH oxidase, catalase or heme oxygenase (HO-1) was associated with HEMA- or LPS-induced apoptosis. While no influence of MAPK was detected, NAC inhibited cytotoxic effects of HEMA. SIGNIFICANCE HEMA- and LPS-triggered pathways may induce apoptosis and interfere with physiological tissue responses as a result of the differential formation of oxidative stress.