Lee Dong Hee, Lim Bum-Soon, Lee Yong-Keun, Ahn Sug-Joon, Yang Hyeong-Cheol
Department of Dental Biomaterials Science and Dental Research Institute, College of Dentistry, Seoul National University, 28 Yeonkun-dong, Chongro-ku, Seoul 110-749, South Korea.
Dent Mater. 2006 Dec;22(12):1086-92. doi: 10.1016/j.dental.2005.09.002. Epub 2006 Jan 10.
This investigation studied the possibility that apoptosis as well as mutagenicity induced by resin monomers are mediated by oxidative stress.
A range of dilutions of three resin monomers (GMA, TEGDMA, and HEMA) was added to culture medium (DMEM/10% FBS), of V79-4 fibroblasts and RPC-C2A pulp cells for 24 h. Their cytotoxic effects were measured by a colorimetric functional assay (MTT). Chromosomal aberration induced by the resin monomers was investigated by counting micronuclei in V79-4 cells. The effects of the resin monomers on DNA fragmentation were viewed by agarose gel electrophoresis of DNA, isolated from RPC-C2A pulp cells that were treated by resin compounds. Resin monomer-induced apoptosis was further confirmed by flow cytometry (staining with both annexin V-FITC and PI).
All monomers exhibited a dose-dependent cytotoxic effect, and the ranking of the cytotoxicity based on TC50 was GMA > TEGDMA > HEMA. The resin monomer-induced cytotoxicity was significantly decreased by co-treatment with N-acetylcystein (NAC), an antioxidant. The authors also confirmed a dose-dependent genotoxicity of the resin monomers that had induced micronucleated cells in V79-4 fibroblasts. Similar to the effects on cytotoxicity, NAC reduced the numbers of micronuclei in comparison with those generated by the resin monomers. The preventive effects of NAC were also observed in monomer-induced apoptosis in RPC-C2A cells. A DNA ladder pattern, characteristic of apoptosis, was shown at cytotoxic concentrations, but NAC blocked the resin monomer-mediated DNA fragmentation. The preventive effects of NAC on apoptosis were confirmed by Annexin V staining. Cells exposed to 300 microM GMA, 7 mM TEGDMA, or 14 mM HEMA for 24 h showed a significant increase in apoptotic cells, while NAC co-treatment caused a reduction in apoptotic cells compared to controls.
These findings suggest that glutathione depletion and oxidative stress are responsible for GMA, TEGDMA, and HEMA-induced mutagenicity and apoptosis.
本研究探讨树脂单体诱导的细胞凋亡及致突变性是否由氧化应激介导。
将三种树脂单体（甲基丙烯酸缩水甘油酯、双甲基丙烯酸三乙二醇酯和甲基丙烯酸羟乙酯）的一系列稀释液添加到含有V79-4成纤维细胞和RPC-C2A牙髓细胞的培养基（DMEM/10%胎牛血清）中，作用24小时。通过比色功能测定法（MTT）测量其细胞毒性作用。通过计数V79-4细胞中的微核来研究树脂单体诱导的染色体畸变。通过对经树脂化合物处理的RPC-C2A牙髓细胞分离的DNA进行琼脂糖凝胶电泳，观察树脂单体对DNA片段化的影响。通过流式细胞术（用膜联蛋白V-FITC和碘化丙啶染色）进一步证实树脂单体诱导的细胞凋亡。
所有单体均表现出剂量依赖性细胞毒性作用，基于半数毒性浓度（TC50）的细胞毒性排序为：甲基丙烯酸缩水甘油酯＞双甲基丙烯酸三乙二醇酯＞甲基丙烯酸羟乙酯。与抗氧化剂N-乙酰半胱氨酸（NAC）共同处理可显著降低树脂单体诱导的细胞毒性。作者还证实了树脂单体在V79-4成纤维细胞中诱导微核细胞的剂量依赖性遗传毒性。与对细胞毒性的影响类似，与树脂单体产生的微核数量相比，NAC减少了微核数量。在RPC-C2A细胞中，NAC对单体诱导的细胞凋亡也有预防作用。在细胞毒性浓度下出现了凋亡特有的DNA梯状条带，但NAC可阻断树脂单体介导的DNA片段化。通过膜联蛋白V染色证实了NAC对细胞凋亡的预防作用。暴露于300微摩尔/升甲基丙烯酸缩水甘油酯、7毫摩尔/升双甲基丙烯酸三乙二醇酯或14毫摩尔/升甲基丙烯酸羟乙酯24小时的细胞，凋亡细胞显著增加，而与NAC共同处理导致凋亡细胞比对照组减少。
这些发现表明，谷胱甘肽耗竭和氧化应激是甲基丙烯酸缩水甘油酯、双甲基丙烯酸三乙二醇酯和甲基丙烯酸羟乙酯诱导的致突变性和细胞凋亡的原因。
