Objective. Oxidative stress induced by compounds of dental composites like 2-hydroxyethyl methacrylate (HEMA) due to excess formation of reactive oxygen species (ROS) disturbs vital cell functions leading to apoptosis. The sources of ROS in cells exposed to resin monomers are unknown. The present study investigates functions of flavin-containing ROS and RNS (reactive nitrogen species) producing enzymes in cells exposed to HEMA.Methods. The formation of oxidative stress in RAW264.7 mouse macrophages exposed to HEMA (0-6-8 mM) was determined by flow cytometry (FRCS) after staining of cells with 2'7'-dichlorodihydrofluorescin diacetate (H2DCF-DA), dihydroethidium (DHE) or dihydrorhodamine 123 (DHR123). Cells in apoptosis or necrosis were identified by annexin-V-FITC/propidium iodide labeling followed by FACS analysis. Expression of ROS/RNS producing enzymes was analyzed by Western blotting.Results. DCF fluorescence increased in cells exposed to HEMA for 1 h suggesting the production of hydroxyl radicals, H2O2, or nitric oxide and superoxide anions which form peroxynitrite (ONOO-). Increased DHR123 fluorescence after 24 h indicated the formation of mostly H2O2. The induction of apoptosis in the presence of HEMA was decreased by low concentrations of diphenylene iodonium (DPI), an inhibitor of flavin-containing enzymes. Expression of p47(Phox), a regulatory subunit of the superoxide producing Nox2, was downregulated, and the expression of NOS which produces nitric oxide (NO) was possibly inhibited by feedback loop mechanisms in HEMA-exposed cultures. Inhibition of HEMA-induced apoptosis by VAS2870 or apocynin further suggested a crucial function of Nox2.Significance. The present findings show the physiological relevance of flavin-containing enzymes in monomer-induced oxidative stress and apoptosis. (C) 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Objective. Resin monomers such as 2-hydroxyethyl methacrylate (HEMA) induce apoptosis because of the excess formation of reactive oxygen species (ROS). The portion of ROS including superoxide anions, hydrogen peroxide or hydroxyl radicals in monomer-induced apoptosis is unknown. Here, the effectiveness of superoxide anions or hydroxyl radicals was analyzed using tempol or sodium formate as radical scavengers.Methods. RAW264.7 mouse macrophages were exposed to HEMA (0-6-8 mM) in the presence of tempol (0-0.05-0.5-5.0 mM) or sodium formate (0 1 5 10 mM). The formation of ROS using DCFH2-DA or dihydrorhodamine 123 (DHR123) as fluorescent dyes and the induction of apoptosis was determined by flow cytometry after 1 h or 24 h exposure periods. Expression of enzymes related to ROS metabolism was detected by Western blotting.Results. DCF fluorescence significantly increased after short exposure (1 h) while DHR123 fluorescence was enhanced after a long exposure period (24h) in cells treated with HEMA. Although no influence was detected on the formation of ROS, tempol or sodium formate protected cells from HEMA-induced apoptosis. The number of cells in late apoptosis or necrosis induced with 6 or 8 mM HEMA was reduced in the presence of tempol or low concentrations of sodium formate. HEMA-induced expression of catalase, indicating oxidative stress, decreased in the presence of tempol.Significance. Superoxide anions and hydroxyl radicals contribute to HEMA-induced apoptosis. The current findings support the development of strategies based on the pharmacological inhibition of enzymes producing superoxide anions finally converted to hydroxyl radicals to compensate for potential adverse tissue reactions associated with dental composites. (C) 2016 Published by Elsevier Ltd on behalf of The Academy of Dental Materials.

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.

Objectives. Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) are able to cause an imbalance of the redox state in mammalian cells. The resulting oxidative stress originating from reactive oxygen species (ROS) has been associated with cytotoxicity. We hypothesized that ROS might contribute to the generation of genotoxicity by TEGDMA and HEMA as well. Therefore, we examined the formation of micronuclei in V79 cells by both resin monomers in the presence of the antioxidant N-acetylcysteine (NAC), which scavenges ROS. In addition, we analyzed the effects of TEGDMA and HEMA on the normal cell cycle in the presence of NAC.Methods. V79 fibroblasts were exposed to increasing concentrations of TEGDMA and HEMA in the presence and absence of NAC for 24 h. Genotoxicity was indicated by the formation of micronuclei. The modification of the normal cell cycle was analyzed by flow cytometry (FACS).Results. A dose-related increase in the number of micronuclei in V79 cells-induced by TEGDMA and HEMA indicated genotoxicity of both chemicals. However, the formation of micronuclei was reduced in the presence of 10 mmol/L NAC, indicating its protective role. A cell cycle delay in G2 phase caused by TEGDMA was absent when cells were co-treated with NAC. Similarly, the presence of NAC led to a reversion of the cell cycle delay in HEMA-treated cell cultures.Significance. Our results suggest that genotoxic effects and the modification of the cell cycle caused by TEGDMA and HEMA are mediated, at least in part, by oxidative stress. (C) 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Mutagenicity of single compounds of dental resinous materials has been investigated on many occasions before, but the induction of mutagenic effects by extracts of clinically used composites is still unknown. Here, cytotoxic effects and the formation of micronuclei were determined in V79 fibroblasts after exposure to extracts of modern composite filling materials (Solitaire, Solitaire 2, Tetric Ceram, Dyract AP, Definite). For cytotoxicity testing, test specimens were aged for various time periods (0, 24, and 168 h), and V79 cells were then exposed to dilutions of the original extracts for 24, 48, and 72 h. The ranking of the cytotoxic effects of the composites according to EC50 values after a 24-h exposure period was as follows: Solitaire (most toxic)=Solitaire 2<Tetric Ceram<Dyract AP<Definite (least toxic). Cytotoxicity was independent of the period of aging for each composite, but varied with exposure periods. The cytotoxic effect of Solitaire increased about two-fold between exposure periods of 24, 48, and 72 h, no changes were observed with Solitaire 2, and cytotoxicity of Tetric Ceram, Dyract AP, and Definite was reduced. Even eight-fold diluted original extracts of freshly mixed Solitaire test specimens increased the numbers of micronuclei about 10-fold, and Solitaire 2 was slightly less effective. The mutagenic effects of these materials were reduced in the presence of a rat liver homogenate (S9). Weak increases of the numbers of micronuclei were detected only with undiluted extracts of Tetric Ceram and Dyract AP, but Definite was not effective. Our findings suggest that mutagenic components of biologically active composite resins should be replaced by more biocompatible substances to avoid risk factors for the health of patients and dental personnel.

The induction of DNA damage by a genotoxic agent is a signal leading to cell cycle delay, and thereby enables and induces DNA repair prior to cell cycle progression. Triethylene glycol dimethacrylate (TEGDMA), a monomer of dental resinous materials, caused mutagenic effects in mammalian cells probably as a consequence of DNA damage. Therefore, we hypothesized that TEGDMA will induce a cell cycle delay in mammalian cells. Here, cell lines deficient and proficient of a functional p53 tumor suppressor protein were used to study the effects of TEGDMA on the various phases of the cell cycle. V79 Chinese hamster lung fibroblasts (p53 deficient), N1 human skin fibroblasts (p53 proficient), and primary human pulp fibroblasts (p53 proficient) were exposed to increasing TEGDMA concentrations (0–3mmol/l). Cell survival and vitality were determined after a 24-h exposure period and a 24-h recovery period, and the distribution of cells between the phases of the cell cycle in untreated and TEGDMA-treated cultures was analyzed by flow cytometry. The majority of the TEGDMA-treated V79 cells accumulated in G2 phase. In contrast, about 30% of human N1 fibroblasts were reversibly blocked in G1 phase by 0.5–3.0mmol/l TEGDMA. The fraction of G2-phase cells was increased only by high TEGDMA concentrations. The percentage of human pulp cells in G1 phase increased very slightly with 1mmol/l TEGDMA, but cell numbers in G1 phase were reduced by 10–20% by 1.5–3mmol/l TEGDMA. The percentage of pulp cells in G2 phase increased about 2-fold without any obvious effect of a 24-h recovery period. Therefore, TEGDMA caused cell cycle delays through p53-dependent and independent pathways in the various cell lines. From these results, we conclude that TEGDMA may influence physiological processes like cell growth and differentiation of human pulp cells in vivo.