樟腦醌的毒性對於人類牙髓細胞不同代謝酵素表現的影響

Abstract

目的：樟腦醌為目前最常使用於牙科樹脂材料的光啟始劑。本研究目的著重在各種參與醌類代謝的酵素，是否會表現在人類牙髓細胞；若有，這些酵素是否會被樟腦醌誘發，並且影響其對於牙髓細胞的毒性。此外，已知樟腦醌的毒性會造成人類牙髓細胞的細胞週期停留在G2/M階段，因此我們也探討上游調控的蛋白質之表現是否會改變。 實驗方法：將人類牙髓細胞取出培養，並暴露於不同濃度(0.1-3 mM)的樟腦醌下24小時，以免疫螢光染色來觀察調控細胞週期的上游蛋白質的分布與表現。此外，以反轉錄聚合酶連鎖反應(RT-PCR)及西方點墨法(Western blot)來評估醌類代謝酵素於牙髓細胞的表現及隨著樟腦醌的濃度上升是否會有對應的變化，包含了醌類還原酵素(quinone reduction enzymes)、穀胱甘肽轉換酵素(glutathione-S-transferase)、及抗氧化酵素。最後探討這些解毒酵素的產生是否會經由AhR及Nrf2這兩個轉錄因子的途徑來調節。 實驗結果：人類牙髓細胞在暴露24小時的樟腦醌之後，會表現調控細胞週期的上游蛋白質，包含p-ATM、p-Chk2、p-p53，及較下游的p21、GADD45α，並且隨著樟腦醌的濃度增加而有上升的情形。而在醌類還原酵素的方面，NQO1、NQO2皆有隨著樟腦醌濃度上升而表現，而在2-3 mM時些微下降；而GST-p1則是沒有什麼改變。至於抗氧化酵素，CuZnSOD、MnSOD、catalase、GPx1也會隨著樟腦醌濃度上升而增加，並且也在2-3 mM時些許減少。最後，AhR及Nrf2這兩個轉錄因子在1-2 mM樟腦醌都有顯著的增加。 結論：樟腦醌會引發p-ATM、p-Chk2、p-p53、p21、GADD45α等調控細胞週期的上游調控蛋白質表現，導致人類牙髓細胞之細胞週期停頓在G2/M階段。而NQO1、NQO2醌類還原酵素隨著樟腦醌的濃度而表現上升，顯示它們可能是被樟腦醌的醌類結構所誘發，進而去降低樟腦醌的毒性。而抗氧化酵素CuZnSOD、MnSOD、catalase、GPx的表現增加可歸因於樟腦醌所產生的活性氧，並具將其代謝分解的功能。最後，這些解毒酵素的表現，因AhR及Nrf2這兩個轉錄因子的表現上升，顯示可能是由這兩條路徑來調控。本研究發現樟腦醌會誘發牙髓細胞表現多種解毒酵素，而這些酵素的表現可能會影響到患者以含樟腦醌的樹酯材料填補後的預後。

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.