RESUMO
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 (0-6-8mM) in the presence of tempol (0-0.05-0.5-5.0mM) or sodium formate (0-1-5-10mM). 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 1h or 24h exposure periods. Expression of enzymes related to ROS metabolism was detected by Western blotting. RESULTS: DCF fluorescence significantly increased after short exposure (1h) 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 8mM 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.
Assuntos
Apoptose , Radical Hidroxila , Metacrilatos , Superóxidos , Animais , Macrófagos , Camundongos , Espécies Reativas de OxigênioRESUMO
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.
Assuntos
Polpa Dentária/efeitos dos fármacos , Dentina Secundária/efeitos dos fármacos , Adesivos Dentinários/toxicidade , Polietilenoglicóis/toxicidade , Ácidos Polimetacrílicos/toxicidade , Agentes de Capeamento da Polpa Dentária e Pulpectomia/toxicidade , Calcificação de Dente/efeitos dos fármacos , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Dentina Secundária/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Expressão Gênica/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Sialoproteína de Ligação à Integrina/biossíntese , Sialoproteína de Ligação à Integrina/genética , Osteocalcina/biossíntese , Osteocalcina/genética , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Sialoglicoproteínas/biossíntese , Sialoglicoproteínas/genética , Estatísticas não ParamétricasRESUMO
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 24h. 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.