Released dental composite components can damage human gingival fibroblasts (HGFs) and their DNA. The cytotoxicity, chromatin condensation and the induction of DNA double strand breaks (DSBs) by different compounds of dental composites was investigated using an improved γ-H2AX focus assay.
HGFs were incubated with the monomers: bisphenol-A-ethoxylate-dimethacrylate (Bis-DMA), bisphenol-A-glycerolate-dimethacrylate (BisGMA), ethyltriethylen glycol methacrylate (ETEGMA), glycidyl methacrylate (GMA), 1,6-hexandiol-dimethycrylate (HDDMA), trimethylolpropane ethoxylate triacrylate (TMPTA), and acrylamide (ACR). DSBs were determined by enumerating γ-H2AX and 53BP1 foci colocalized at DSBs.
A concentration-dependent induction of DSBs was found in the order: GMA > BisGMA > ACR > Bis-DMA > HDDMA > TMPTA > ETEGMA. HGFs exposure to GMA (0.3 mM) and to BisGMA (0.09 mM) induced the highest rate of DSB foci, i.e. 12-fold and 8-fold, respectively, relative to control (0.33 DSB foci/cell). At the highest concentrations (EC 50 ) prominent changes in the chromatin morphology of HGF cell nuclei, i.e. compaction of nuclear chromatin and reduction of the area covered by the ovoid fibroblast nuclei, were observed. Nuclear condensation was significantly induced by GMA (1.7-fold at 0.3 mM) and BisGMA (1.6-fold at 0.09 mM), which correlated with the highest numbers of induced DSB foci (GMA, BisGMA, 3.9 and 2.6 foci/cell, respectively).
The improved γ-H2AX/53BP1 focus assay revealed a concentration-dependent increase in DSBs for all tested substances. Furthermore, concentration-dependent changes in HGF cell nucleus morphology was noted, demonstrating genotoxic effects of the substances tested.
The use of tooth-colored composite materials in dentistry, as a replacement for amalgam, has significantly increased since 1990 albeit there are questions about their biocompatibility and toxicity. The increased use of polymer composite material has been linked to allergic reactions and adverse effects on gingiva and pulp homeostasis . Modern dental composite materials consist of monomers and co-monomers whose chemical curing by UV or halogen light leads to composite polymerization. However, polymerization is often incomplete because not all of the methacrylate groups of the (co)monomers do react . Consequently, residual monomers can be leached out of tooth fillings by saliva, food, or drinks and are consequently swallowed and may expose gingiva and the gastrointestinal tract . Besides intestinal absorption, dental monomer composites can diffuse into the pulp chamber or be absorbed through the lungs by inhalation and so reach the systemic circulation where they are further metabolized . It has been demonstrated that cellular or liver microsomal degradation of (co)monomers leads to formation of the epoxy compound 2,3-epoxymethacrylic acid . Epoxides are considered cytotoxic substances , since they are chemically very reactive compounds that can induce DNA single and DNA double-strand breaks (DSBs) . Genotoxic effects of dental compounds were observed in human lymphocytes for bisphenol-A-glycerolate-dimethacrylate (BisGMA), 2-hydroxyethyl-methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) . (Co)monomers have the potential to increase the amount of reactive oxygen species (ROS) in the cell . ROS cause, besides other effects, oxidative DNA damage that involves DNA double strand breaks (DSBs), which can lead to apoptosis in mammalian cells . ROS can be genotoxic, mutagenic, and have been implied in teratogenic effects and cancer formation .
Here, we examined the following dental composite components/monomers for their ability to induce DSBs: bisphenol-A-ethoxylate-dimethacrylate (Bis-DMA), BisGMA, ethyltriethylen glycol methacrylate (ETEGMA), glycidyl methacrylate (GMA), 1,6-hexandiol-dimethycrylate (HDDMA), trimethylolpropane ethoxylate triacrylate (TMPTA). Acrylamide (ACR) was also implemented in this study, since its metabolization can lead to the generation of an epoxide derivative, glycidamid, which is highly reactive and can mediate genotoxic and mutagenic effects . In our previous studies, we demonstrated the ability of some dental (co)monomers to induce DSBs in human gingiva fibroblasts (HGFs) as detected by the γ-H2AX focus assay . This assay visualizes DSBs by immunostaining the phosphorylated form of the histone H2AX (then called γ-H2AX) , which leads to microscopically visible “foci” that can be enumerated and correlated to the number of induced DSBs . In this study, an improved γ-H2AX focus assay was applied that uses γ-H2AX and 53BP1 colocalization to mark DSBs . The 53BP1 (tumor suppressor p53 binding protein 1) protein localizes to the chromatin surrounding a DSB and rapidly forms foci at DSBs that direct ATM DNA damage signaling and the DNA damage response .
The purpose of this study was to investigate the formation (co)monomer-induced DSBs characterized by colocalization of the two DSB indicator proteins using an improved γ-H2AX/53BP1 focus assay. Furthermore, this study explored whether the exposure to the tested substances additionally induced changes in the nuclear chromatin of HGF cell nuclei.
Materials and methods
The composite components BisGMA (CAS-No. 1565-94-2), ETEGMA (Cas-No. 39670-09-2), and HDDMA (Cas-No. 6600-59-3) were obtained from Evonik Röhm (Essen, Germany), Bis-DMA (Cas-No. 41637-38-1), GMA (Cas-No. 106-91-2), TMPTA (Cas-No. 28961-43-5), and the ACR (Cas-No. 79-06-1) were obtained from Sigma–Aldrich (Steinheim, Germany). The studied substances are listed in Tables 1 and 2 .
|Abbreviation||Molecular formula||Trivial name||Molecular weight (MW)|
|ACR||C 3 H 5 NO||Acrylamide||71.08|
|GMA (monomer)||C 7 H 10 O 3||Glycidyl methacrylate||142.15|
|TMPTA (monomer)||C 11 H 24 O 7||Trimethylolpropane ethoxylate triacrylate||692.00|
|ETEGMA (monomer)||C 12 H 22 O 5||Ethyltriethylen glycol methacrylate||246.30|
|HDDMA (monomer)||C 14 H 22 O 4||1,6-Hexandiol dimethacrylate||254.00|
|Bis-DMA (monomer)||C 29 H 36 O 7||Bisphenol-A-ethoxylate-dimethacrylate||660.00|
|BisGMA (monomer)||C 29 H 36 O 8||Bisphenol-A-glycerolate-dimethacrylate||512.60|
|Substance||EC 50 (mmol/L)||1 3 × EC 50 (mmol/L)||1 10 × EC 50 (mmol/L)||1 25 × EC 50 (mmol/L)|