Origin of oxidative stress in cells exposed to dental resin monomers

Abstract

Event Abstract Back to Event Origin of oxidative stress in cells exposed to dental resin monomers Helmut Schweikl1, Carola Bolay1, Christine Petzel1*, Markus Forstner1*, Karl-Anton Hiller1*, Stephanie Krifka1* and Wolfgang Buchalla1* 1 University Hospital Regensburg, Department of Conservative Dentistry and Periodontology, Germany Residual monomers released from dental composite materials interact with tissues of the oral cavity [1]. Monomers like 2-hydroxyethyl methacrylate (HEMA) disrupt vital cell functions e.g. responses of the innate immune system, mineralization and cell differentiation, or induce cell death via apoptosis [2]. These effects are associated with the induction of oxidative stress due to the enhanced formation of reactive oxygen species (ROS) [3]. Finding the currently unknown origins of ROS levels, which increase in the presence of resin monomers such as HEMA, would lead to the development of new therapeutic strategies for protecting oral tissues including dental pulp from adverse reactions causally related to a monomer-disturbed redox homeostasis. RAW264.7 mouse macrophages as a model of the cellular innate immune system were exposed to HEMA (0-8 mM) for 1h or 24h. Phorbol myristate acetate (PMA, 0.0-1.0µM) was used as positive control. The activities of flavin-containing enzymes such as Nox (NADPH oxidase) isoforms, xanthine oxidase, or nitric oxide synthase (NOS) were inhibited by diphenyleneiodonium (DPI), and apocynin was included as a specific inhibitor of Nox2. The formation of ROS and the number of cells in the various stages of cell death (apoptosis and necrosis) were analysed by flow cytometry (FACS) using standard procedures. General oxidative stress was determined after staining of cells with 2′7′-dichlorodihydrofluorescin diacetate (H2DCF-DA) as a fluorescent probe, while dihydroethidium (DHE) or dihydrorhodamin123 (DHR123) was used to detect superoxide anions or hydrogen peroxide (H2O2), respectively. The expression of p47phox, a regulatory subunit of the superoxide producing Nox2 enzyme and NOS which forms nitric oxide (NO) was analyzed by routine Western blotting. Expression of Nrf2, a transcription factor that regulates cell responses to oxidative stress, and the stress-inducible HO-1 (heme oxidase-1) was investigated as well. General oxidative stress was significantly increased 1.5-fold in cells exposed to HEMA for 1h, and DHR123 fluorescence (formation of H2O2) was enhanced 2-fold after 24h exposure. DPI concentrations (0-0.1 µM) used here were physiologically relevant as demonstrated by the inhibition of a 2-fold increase in the formation of superoxide anions in PMA-stimulated cells. However, HEMA-induced DHR123 fluorescence (H2O2) was not inhibited by low DPI concentrations (0-0.1 µM) in contrast to DPI effects on apoptosis in HEMA-exposed cell cultures. The number of vital cells was increased by DPI from 70% found in cultures treated with 8mM HEMA to 84% in cultures exposed to 8mM HEMA plus 0.01 µM DPI while the percentage of cells in apoptosis decreased in parallel. Moreover, the specific Nox2 inhibitor apocynin reduced the number of cells in apoptosis about twofold. In addition, the expression of Nox2 (p47phox) was downregulated in HEMA-treated cell cultures, and NOS expression was completely inhibited. In contrast, expression of Nrf2 and HO-1 drastically increased in cells after exposure to HEMA. HEMA induced oxidative stress as indicated by ROS formation and upregulation of Nrf2 and HO-1. HEMA-induced cell death was inhibited by DPI but ROS formation was not affected. These seemingly contradictory observations with DPI suggest that controlling cellular redox imbalance is a complex process. In this control system, Nox2 (p47phox) and NOS are most likely sources of HEMA-induced oxidative stress because their expression is downregulated seemingly through a feed back-mechanismus caused by high levels of ROS. Thus, both ROS producing enzymes could be major players in HEMA-induced redox imbalance at least in part caused by the formation of peroxynitrite (ONOO-) from superoxide anions and nitric oxide. Supported by the Deutsche Forschungsgemeinschaft DFG (Schw 431/13-2)