三種牙科樹脂單體之毒性效應及其機制探討

Abstract

中文摘要 樹脂包含材料在牙科醫療的應用極為廣泛。但此類材料所釋出的樹脂單體，卻可能導致各種不良的生物效應。雙甲基丙烯酸三縮乙二酯（TEGDMA），甲基丙烯酸2-羥基乙酯（HEMA）和雙甲基丙烯酸氨基甲酸乙酯（UDMA），是牙科樹脂材料中三項重要的成分，也是被釋放單體的主要來源。為增進此類材料在臨床上使用的安全性，必須對這些樹脂單體可能造成的毒性作用和機制有足夠的瞭解。因此，我們以細胞生長、細胞週期進行、活性氧(ROS)產生和和穀胱甘肽(GSH)的耗損為主要方向，對個別單體造成的毒性進行觀察。 首先,我們觀察TEGDMA對初代人類牙髓母細胞(HPF)及人類牙齦表皮細胞株Smulow-Glickman cells (S-G cells)的影響。TEGDMA會抑制HPF細胞的生長，效應的強度與TEGDMA濃度呈現正相關。此時HPF細胞內GSH耗盡程度和ROS產生也隨之升高，並且發生細胞週期干擾的現象。1和2.5 mM的TEGDMA會造成G2/M期休止，而5至10 mM則造成S期休止，在2.5，5和10 mM的濃度下有Sub-G0/G1峰顯現，可能表示細胞凋亡的發生。TEGDMA對於S-G cells造成的生長抑制模式與HPF類似，2.5-10 mM的TEGDMA 也會造成sub-G0/G1高峰發生，但沒有明顯的細胞週期休止現象。2.5-10 mM的TEGDMA也同時造成與濃度相關的GSH耗竭，但1 mM TEGDMA卻使S-G cells中GSH濃度明顯升高，可能是S-G cells在此濃度下產生的適應性反應。在ROS的產生方面，1mM TEGDMA即造成S-G cells內ROS濃度升高，2.5-5 mM之間漸次降低，但仍較控制組為高，而在10 mM 時達到最高峰。由此可見，TEGDMA 對S-G cells所造成的ROS的增加並非GSH耗竭所造成。

HEMA對HPF及S-G細胞造成之生長抑制模式與TEGDMA相似，但毒性程度明顯較低。對於HPF而言，HEMA造成的抑制生長現象與GSH消耗，ROS生產和細胞週期擾動明顯相關。5和10 mM HEMA會使HPF產生G2/M細胞週期休止，同時伴隨著細胞內GSH耗盡和ROS的堆積。HEMA對S-G cells的影響與HPF則不盡相同。2.5和5 mM HEMA 使S-G cells發生S期休止，10 mM HEMA則造成sub-G0/G1高峰發生，可能顯示細胞凋亡的進行。5和10 mM HEMA會造成S-G cells中GSH耗竭，但1mM HEMA 就使ROS過度產生，在1-5 mM 之間ROS濃度隨HEMA劑量增加而上升，然後在10 mM降低。如同TEGDMA，HEMA對S-G cells造成的GSH耗竭並非ROS升高的主要因素。 UDMA 對CHO-K1細胞的生長抑制情形與前述實驗相似，但產生毒性的濃度比TEGDMA與 HEMA低許多。0.1 mM的UDMA誘導 S期細胞週期阻滯，同時細胞內ROS明顯增加，並開始造成細胞凋亡。然而GSH 耗竭只發生在0.25 mM UDMA處理組，此時大量的細胞發生凋亡或壞死，顯示GSH 的耗盡可能為決定CHO-K1 cells存亡的關鍵因素。抗氧化劑NAC 和Catalase可降低UDMA的毒性效應。0.5-10 mM NAC和250-1000 U/ml的Catalase明顯減弱UDMA引起的生長抑制，並且減少ROS的產生和細胞週期的干擾，其效應與劑量相關。 本研究有助於瞭解三種樹脂單體毒性發生的機制。雖然本實驗所發現產生毒性的樹脂單體濃度，在正常的牙科治療程序下應不易產生。但是，在缺乏足夠厚度牙本質或材料未適當聚合的狀況下，被釋放的樹脂單體將可能導致本實驗所觀察到的毒性影響，臨床治療應審慎避免。 關鍵字： TEGDMA，HEMA，UDMA，細胞毒性，細胞週期，GSH，ROS

Abstract Resin-containing products are extensively applied in dental practice. Monomers released from these materials may cause various adverse biological effects. Among which triethylene glycol dimethacrylate (TEGDMA), 2-hydroxyethyl methacrylate (HEMA) and urethane dimethacrylate (UDMA) are three principal resin monomers and also the major components released from various dental resin materials. The toxic effects and mechanisms of these resin monomers should be elucidated, thereby to improve the safety of utilization. Investigations of cell cycle progression, reactive oxygen species production and glutathione alteration are valuable directions to reveal the mechanism, thus were applied in the presented studies to get insights into the monomer-induced toxicity. Cytotoxic effect was first evaluated. TEGDMA induced growth inhibition in primary human pulp fibroblast (HPF) in a dose-dependent manner, which may be partially explained by induction of cell cycle perturbation. G2/M phase arrest was noted after exposure of HPF to 1 and 2.5 mM of TEGDMA, and S-phase arrest occurred at 5 and 10 mM. Glutathione depletion and ROS production were concomitantly observed. Sub-G0/G1 peaks were noted when HPF were treated with 2.5, 5 and 10 mM of TEGDMA, indicating the potential induction of apoptosis. TEGDMA also induced growth retardation in human Smulow-Glickman gingival cells (S-G cells) in a dose-related pattern, but no obvious phase arrest phenomenon was present. However, sub-G0/G1 peaks occurred in S-G cells when with 2.5, 5 and 10 mM of TEGDMA, similar to what we noticed in HPF. GSH depletion was marked in S-G cells at concentrations of 2.5, 5 and 10 mM, in a dose-relative manner, but at 1 mM TEGDMA exposure, excessive GSH production was noted, which seemed to be an adaptive reaction. ROS production in S-G cells got high at concentration of 1 mM TEGDMA exposure, then decrease with dose increase between 1-5 mM, then went highest at 10 mM. This suggested that the increase of ROS in S-G cells was not mainly caused by GSH depletion. HEMA also produced dose-dependent growth inhibition of HPF and S-G cells, but in a less toxic pattern compared to TEGDMA. In HPF, the cell growth suppression induced by HEMA may well be related to induction of GSH depletion, ROS production, and cell cycle perturbation. When treated with 5 and 10 mM HEMA, G2/M phase arrest was noted, which was concomitant with intracellular glutathione depletion and ROS production. While in S-G cells, the effects induced by HEMA were different. S-phase arrest occurred in S-G cells when treated with 2.5 and 5 mM, while at 10 mM a sub-G0/G1 peak was noted, which might indicate an apoptotic process. Glutathione depletion was marked in S-G cells only at concentrations of 5 and 10 mM, but ROS overgeneration was obvious since 1 mM and rose with dose increase between 1-5 mM, then lessened at 10mM. This suggested the increase of ROS in S-G cells was not mainly caused by depletion of GSH. UDMA elicited growth inhibition of CHO-K1 cells in a clearly dose-dependent manner, and in a much lower concentration compared to TEGDMA and HEMA. Cell cycle perturbation and ROS overproduction were also observed. 0.1 mM UDMA induced S-phase cell cycle arrest, simultaneously the ROS accumulated, and the apoptosis became significant. The effect of glutathione depletion only occurred at cells treated with 0.25 mM UDMA, a highly cytotoxic concentration at which point myriad cells were under apoptosis or necrosis. Thus glutathione depletion might be crucial for the death of CHO-K1 cells. 0.5-10 mM NAC and 250-1000 U/ml catalase obviously attenuated the UDMA-induced toxicity by reducing ROS generation and reverse cell cycle disturbance, and the effects were dose-related. The presented studies helped to elucidate the toxic mechanism of these resin monomers. Although the toxic concentration reported here might not be reached in prudent application, however, in a clinical situation lacking sufficient sound dentin and/or with poorly polymerized material, the unbound monomers may well lead to potential toxic effects as we addressed. Key words: TEGDMA, UDMA, HEMA, cytotoxicity, cell cycle, ROS, GSH