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

Abstract

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 3 mm TEGDMA after a 6 h 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.

Introduction

Triethylene glycol dimethacrylate (TEGDMA) is a monomer of dental resin materials that causes specific stress responses in eukaryotic cells [1]. The substance induced cell death via apoptosis in various cell types including pulp and gingiva cells, and it appears as if cell death was mediated, at least in part, by the generation of reactive oxygen species (ROS), thus disturbing the redox balance [2], [3], [4], [5], [6], [7]. Furthermore, genotoxic and mutagenic effects caused by TEGDMA are probably to some extent a consequence of ROS-induced DNA damage. It has been shown that elevated numbers of micronuclei, which indicate chromosomal damage, decreased in the presence of the ROS scavenger N-acetylcysteine (NAC) [8], [9]. Upon DNA damage mammalian cells will activate functional cell cycle checkpoints through the coordinated activities of sensor, transducer, and effector proteins [10], [11]. The activated checkpoint will block or delay the cell cycle in order to initiate repair of DNA damage or to activate programmed cell death. The dental resin monomers TEGDMA and HEMA induced a cell cycle delay in various mammalian cells after a 24 and 48 h exposure period that was also inhibited in the presence of NAC [8], [9], [12].

Recently, it has been suggested that the adaptive cellular response to exogenous stimuli like dental monomers was the activation of pathways fundamental to the regulation of cell survival and apoptosis [1]. It was reported that TEGDMA and HEMA were able to activate the coordinated network of the mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI-3K) [7], [13]. Likewise, the activation of the nuclear factor-κB (NF-κB) might play a protective role against the cytotoxicity of the dental monomer HEMA mediated by an increased formation of ROS [5], [6]. These, as well as most likely other complex biological processes that have yet to be identified, are mechanisms for the immediate response primarily through the fast and reversible covalent posttranslational modification of constitutively expressed proteins. Nevertheless, the few activated proteins that have been detected so far are involved in signal transduction pathways related to the regulation of specific transcription factors [14]. Thus, we assumed that an upregulation and downregulation of gene expression would also contribute to the cellular protective response to TEGDMA exposure. Defining the inducible and supposedly protective genes may provide further insight into the fundamental multiple molecular mechanisms of biological responses to dental monomers. DNA microarrays are able to simultaneously monitor the expression of a large number of genes involved in a wide variety of biological regulatory pathways. In addition, knowledge databases have been developed to identify the gene functions and relationships [15]. Finally, microarray studies may lead to the production of a list of differentially expressed genes and the generation of regulatory networks formed by these genes. In the present study, we analysed and identified functions, pathways and networks of genes regulating the cell response to the dental resin monomer TEGDMA.