| Summary: | 碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 103 === Aim: Camphorquinone (CQ) is one of the most commonly used photoinitiators in current light-curing resin-based materials. The purposes of this study focused on the various enzymes which take part in the metabolism of quinones, and their expression in human dental pulp cells. The hypothesis was that those enzymes may be induced in response to exposure CQ, and further modulate the toxicity of CQ. Besides, since the toxicity of CQ resulted in cell cycle arrest at G2/M phase, with a concomitant inhibition of CDK1 (cdc2), cyclin B1, and cdc25C, the role and activation of upstream checkpoints proteins was also evaluated.
Materials and methods: Primary-cultured human dental pulp cells were treated with different concentrations (0.1-3 mM) of CQ for 24 hours. The expression and distribution of upstream cell cycle checkpoint proteins, including ATM, Chk1, Chk2, p53, p21, GADD45α, were evaluated through immunofluorescence. The enzymes involved in quinone metabolism were screened by reverse transcription polymerase chain reaction (RT-PCR) for mRNA expression and by western blotting for protein alterations. Those enzymes can be classified into one-electron reduction enzymes of quinone (cytochrome P450 oxidoredutase, cytochrome b5 reduatases, thioredoxin reductase, etc.), two-electron reduction enzymes of quinone (NQOs), glutathione-S-transferases, and antioxidant enzymes (SODs, catalase, GPx). Besides, evaluation of AhR and Nrf2 expression by RT-PCR and western blot can further clarify if the induction of those enzymes is via AhR or Nrf2 pathways.
Results: Human dental pulp cells exposed to CQ for 24 hours up-regulated the p-ATM and p-Chk2 through 0.25-2 mM of CQ, while the subsequent p-p53 was mildly increased. The downstream p21 elevation from 0.5-2 mM of CQ and GADD45α boosted at 1-2 mM of CQ simultaneously contributed to the G2/M cell cycle arrest. As for quinone metabolizing enzymes, two-electron quinone reduction enzymes NQO1 and NQO2, were enhanced from low concentration until 2-3 mM of CQ, whereas GST-p1 remained unchanged. Speaking to antioxidant enzymes, the expression of CuZnSOD, MnSOD, catalase, and GPx1 all increased from 0.1-1 mM of CQ and started to decline at 2-3 mM of CQ. At last, those detoxifying enzymes expression can be modulated by AhR and Nrf2 pathways, which specifically enhanced at 1-2 mM of CQ exposure.
Conclusions: Human dental pulp cells exposed to increased concentrations of CQ will stimulate p-ATM, p-Chk2, p-p53, p21, and GADD45α expression, which consequently lead to G2/M cell cycle arrest. As for two-electron quinone reduction enzymes NQO1 and NQO2, the enhanced expression from low concentration of CQ in human dental pulp cells indicated that they may serve as a defense mechanism against the quinone structure of CQ. Speaking to CQ-generated ROS, the corresponding expression of CuZnSOD, MnSOD, catalase, and GPx1 may reduce the oxidative stress on human dental pulp cells. At last, those antioxidant and detoxifying enzymes expression can be modulated by AhR and Nrf2 pathways. In conclusion, exposure to CQ of human dental pulp cells induced multiple detoxifying enzymes expression. Difference in enzyme expression of dental pulp may potentially affect the treatment outcome of operative restoration in patients.
|
|---|
