Effects of antioxidants on DNA-double strand breaks in human gingival fibroblasts exposed to methacrylate based monomers

Abstract

Objective

(Co)monomers from dental resin composites have cytotoxic and genotoxic potential. In previous studies it has been demonstrated that antioxidants can decrease the cytotoxicity of various dental (co)monomers. In this study the effects of the antioxidants N -acetylcysteine (ACC) and ascorbic acid (Asc) on the number of DNA double-strand breaks (DSBs) in human gingiva fibroblasts (HGFs) were tested.

Methods

HGF was incubated with the (co)monomers bisphenol-A-glycidyl methacrylate (BisGMA), urethandimethacrylate (UDMA), ethylene glycol dimethacrylate (EGDMA) or 1,3-glyceroldimethacrylate (GDMA) with and without addition of antioxidants ACC and Asc. DNA-DSBs were determined using the γ-H2AX assay.

Results

Asc induced at 500 μM significant more DNA-DSBs in HGFs compared with controls (4.92 (1.28) vs . 1.62 (0.67); foci/cell mean (standard deviation), n = 3). Most DNA-DSBs were found after incubation of HGFs with 90 μM BisGMA (4.05 (0.56)) and 2720 μM EGDMA (5.36 (1.59)). The addition of 100 μM Asc or 500 μM ACC leaded to a statistical significant reduction of DNA-DSBs in HGFs for all tested (co)monomers. After incubation of HGFs with 2720 μM EGDMA and 500 μM ACC the foci/cell decrease from 5.36 (1.59) to 1.9 (1.17) (controls: 1.12 (0.24)). After incubation of HGFs with 90 μM BisGMA and 100 μM Asc the foci/cell decrease from 4.05 (0.56) to 1.96 (0.59) (controls: 1.12 (0.24)).

Significance

All tested (co)monomers can induce DNA-DSBs but addition of antioxidants (Asc or ACC) leads to reduction of DNA-DSBs.

Introduction

Methacrylate based dental (co)monomers are widely used in contemporary dental restorative materials. The conversion of (co)monomers can be induced by light and/or by autopolymerisation. However the polymerization is incomplete meaning that (co)monomers and additives can diffuse into the oral cavity or into the pulp. From here the released substances can enter the organism via the bloodstream . Moreover the methacrylates methyl methacrylate (MMA), ethylene glycol dimethacrylate (EGDMA), and triethylene glycol dimethacrylate (TEGDMA) were identified in the air of dental technicians’ workplaces .

(Co)monomers and additives released from resin-containing products can cause in persons, who are exposed to these substances, various adverse effects such as allergic contact dermatitis and bronchial asthma . In vitro studies have also shown that some dental methacrylates can cause cytotoxic, estrogenic and mutagenic reactions . Thereby reactive oxygen species (ROS) and epoxides play an important role in the metabolism of dental methacrylates . During the metabolism of these xenobiotics ( e.g. MMA, TEGDMA) the amount of ROS and epoxides increases while the amounts of (physiological) radical scavengers such as glutathione or vitamin C decrease.

Most epoxides as well as ROS are regarded as very toxic agents reacting with different cellular molecules and cellular structures such as desoxyribonucleic acid (DNA) .

In this context it is interesting to note that the number of different cancers of the oral mucosa is increasing in adults of 45 years and older with a simultaneous decrease in tobacco and alcohol consumption . Different factors such as human papillomaviruses (HPV), xenobiotics from different sources and their metabolic products are discussed .

DNA double-strand breaks (DNA-DSBs) caused by mutagenic agents like epoxides and ROS are considered as the most toxic type of DNA lesion . If they are left unrepaired they can cause cell death; if they are misrepaired they may lead to chromosomal translocations and genomic instability . Using the γ-H2AX-assay, a previous study has shown that methacrylate based dental monomers can induce DNA-DSB in HGF . H2AX, a protein from the H2A family and a component of the histone octamer in nucleosomes, can be phosphorylated by different kinases to γ-H2AX. This phosphorylation recruits and localizes DNA repair proteins at the foci . The foci represent DNA-DSBs and can be used as a biomarker for DNA damage. A labeled antibody against γ-H2AX can be used to label the foci which can then be visualized using an immunofluorescence microscope (for visualization of foci see ).

Many studies have dealt with the toxicology of (co)monomers and other substances from dental resins. Less is known about how they prevent cell damage. In some studies it could be demonstrated that the addition of antioxidant substances such as the vitamins C (ascorbic acid, Asc) and E or N -acetylcysteine (ACC) can reduce the cytotoxic effects of dental monomers such as TEGDMA, 2-hydroxyethyl methacrylate (HEMA) or poly-methyl methacrylate (PMMA) . It is not known whether antioxidants lead to a reduction of DNA-DSB in human oral cells. Human oral cells ( e.g. , gingival and/or pulp fibroblasts) in this physiological situation are among the first to come into contact with eluted substances.

The aim of this study was therefore to test the hypothesis that the antioxidants Asc or ACC can reduce the number of DNA-DSBs caused by the dental methacrylates BisGMA, UDMA, GDMA and EGDMA in HGF. The number of foci of DNA-DSBs were investigated using γ-H2AX focus assay, which is a direct marker for DNA-DSBs.

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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Effects of antioxidants on DNA-double strand breaks in human gingival fibroblasts exposed to methacrylate based monomers
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