Resin-containing products are widely applied in modern dentistry. Chemical substances released from the materials may cause cytotoxicity and adverse biological effects. N,N-Dimethyl-p-toluidine (DMPT), 2-Dimethylaminoethyl methacrylate (DMAEMA) and 4-Dimethylaminobenzoic acid ethyl ester (DMABEE) are three chemicals released from composite resins. Because of moderate toxicity, they were less investigated in previous studies. However, these three chemicals do not bond to composite resin after curing and are released to oral cavities for a long time. Thus, it is necessary to examine their cytoxicity and toxic mechanism. Investigations of cell growth, cell cycle progression, reactive oxygen species (ROS) production and DNA damage are valuable direction to realize the mechanism, thus were applied in the present studies to get insight into the chemicals-induced toxicity. First of all, we evaluated the effect of DMPT on CHO-K1 cells. DMPT induced both short-term and long-term growth inhibition of CHO-K1 cells. In the mean time, the ratio of micronuclei (MNi) increased in a dose-dependent manner. However, ROS production was not elevated and N-acetyl-cysteine (NAC) could not rehabilitate the growth potential. On cell cycle analysis, after treating with DMPT, there were no obvious arrest at different phases of cell cycle compared to control group. Moreover, the mode of cell death most accumulated in necrosis. According to the results from above, it suggested that ROS production was not the main cause of CHO-K1 cell death and we needed to examine other pathways to explain of DNA damage and cell death. DMAEMA also produced growth inhibition of CHO-K1 cells in the same pattern compared to DMPT. But, the effect of DNA damage of DMAEMA was weaker than DMPT. Only under 3.5 mM DMAEMA, the amount of MNi rised slightly. Similarly, ROS production obviously increased when CHO-K1 cells exposed to the highest concentration DMAEMA. However, we found that the cell cycle arrested at S and Sub-G0/G1 phase started from 1.5 mM DMAEMA. This result implied that the cells might get into apoptosis. Thus, first, we could state that DMAEMA could induce CHO-K1 cells death by ROS production, but the ability of self-repairing might alleviate the toxicity of DMAEMA. Secondary, due to different results of DNA damage between DMPT and DMAEMA, we hypothesize that they induced cell death by different mechanism. Among three drugs, DMABEE was the most toxic to CHO-K1 cells. It elicited growth inhibition of CHO-K1 cells in a dose-dependent manner, and at a much lower concentration compared to DMPT and DMAEMA. In short-term inhibition, 0.75 mM DMABEE induced about 50% down-regulation of growth capacity; in long-term inhibition, there were obvious decrease of growth potential from 0.25 mM DMABEE. Further, in morphology, CHO-K1 cells changed from cuboid- or round-shape to fibroblastic variation under high concentrations of DMABEE. However, the morphologic change could be recovered by pre-treating with carboxylesterases (CES). Similarly, after co-incubating with CES, the growth inhibition of CHO-K1 cells by DMABEE could be prevented. In CBMN assays, the percentage of MNi elevated started from 0.25 mM DMABEE. The cells were severely broken and could not be calculated by treating with 1.0 mM DMABEE. ROS production of CHO-K1 cells also increased in a dose-related manner of DMABEE. But, there was no prevention of growth inhibition after pre-incubating with NAC. We believed that NAC played a role of pro-oxidant instead of anti-oxidant. Thus, the growth inhibition of CHO-K1 cells dramatically increased. The cell cycle got arrested on G0/G1 phase started from treating with 0.25 mM DMABEE and the cell residing in the quadrant of necrosis and late apoptosis would increase. Theses results indicated that CHO-K1 cells would death through DNA damage by ROS attack and cause cell necrosis or late apoptosis. The present studies helped us to elucidate the cytotoxic mechanism of these three chemicals leached from composite resins. Although the cytotoxic concentration reported by us might not reached in prudent application. while lacking sufficient thickness of dentin or poor polymerization, the unbound chemical substances could lead potential toxic effect to the pulp tissue.
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