UDMA-induced oxidative stress, cell cycle arrest and apoptosis in human dental pulp cells
Date Issued
2012
Date
2012
Author(s)
Wang, Hsin-Hui
Abstract
Aim: Urethane dimethacrylate (UDMA) is one of the monomers usually be included in dental resin materials. The purpose of our study is to investigate the effects of UDMA on cytotoxicity to human dental pulp cells, involving the relationship between ROS formation and UDMA toxicity. The influences UDMA on the expression of cell cycle- and apoptosis-related genes and proteins were also evaluated.
Materials and methods: Primary-cultured human dental pulp cells were obtained from human extracted premolars and third molars. Cells were treated with different concentrations of UDMA (from 0.025 to 0.35 mM), incubated 24 hours and then observed the changes of cell morphology in phase contrast microscope. Cell proliferation was evaluated by MTT assay. Cell cycle analysis was investigated by flow cytometry. Influences in mRNA expression were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and the changes in protein production were determined by Western blot. For the purpose of link the rule of ROS and antioxidant mechanism, pulp cells were pre-treated for 30 minutes with NAC (1, 3mM) and catalase (1000, 2000 U/ml), esterase (2, 4 U/ml) to check their ability of removing overproduced ROS. And Nrf2, HO-1, CES1A1, CES2 and CES3 mRNA expression can be the evidences of cells facing oxidative stress. By adding HO inhibitor Zn-P (1, 2.5 μM), CES inhibitor BNPP (0.5, 1 mM) and CES2 inhibitor Loperamide (10, 20 μM) 30 minutes before co-incubation with 0.1mM UDMA to evaluate the influences to cell viability by MTT assay. And the testing of expression of COX-2 gene and protein was to link UDMA and chronic inflammatory effect. One-way ANOVA and post hoc Tukey test were used to analyze differences between experimental and control groups.
Results: In human dental pulp cells, UDMA induced morphological changes and a significant decrease of cell viability at the concentration of 0.1mM UDMA to about 70%, and at the concentration of 0.25 and 0.35 mM UDMA to about 40%. At the concentrations of 0.35mM UDMA, it could lead to increase of G2/M cell percentage and decrease of G0/G1 cell percentage. The expression of production of cdc2, cyclinB1, cdc25C was inhibited, while that of p21 was slightly promoted in both PCR and Western blot examination. For apoptosis analysis, Bax, Bad were promoted, and Bcl2 was inhibited. Besides, the increase of HO-1, CES2 and COX-2 expression was noted after 24 hours. The reduction of cell viability caused by UDMA can be inhibited by NAC, catalase and esterase pre-treatment, and can be promoted by BNPP, loperamide and Zn-P pre-treatment.
Conclusion: UDMA at a concentration higher than 0.1 mM had a significant cytotoxicity. Under the concentration higher than 0.1 mM, UDMA can cause changes of cell morphology, reduction of cell viability, and 0.35mM UDMA can cause increase of G2/M phase cell percentage. These changes may be related to ROS overproduction, which can cause expressional variations of many genes, such as cdc2, cyclin B1, cdc25C, p21, Bax as well as Bcl2. As for HO-1 and CES2, the induction of its expression may play a role in cell protection. The finding of COX-2 gene and protein expression suggest UDMA can be a critical factor of inflammation related to resin-based dental biomaterials, and that COX-2 is involved in the inflammatory reaction of the resin monomers. We know the concentration of UDMA in dental resin product is not very high, but the concentration of UDMA elute can be high enough to damage pulp cells once the residual UDMA diffuses to pulp chamber with a relatively small volume. Therefore, we should use resin materials carefully in daily practice and should select proper case and the situation of enough residual dentin thickness to do the resin restoration procedure.
Subjects
Human dental pulp cell (HDP cell)
Urethane dimethacrylate(UDMA)
Reactive oxygen species(ROS)
Antioxidant
Apoptosis
Cell cycle
Cytotoxicity
Type
thesis
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