巴拉刈對正常大鼠肝臟細胞毒性機制探討

Study of Cell Toxicology by Paraquat in Rat Liver Epithelial Cell Line

許仁譯(Jen-Yi Hsu)

指導教授 ： 蔡錦蓮

高雄醫學大學/健康科學院/職業安全衛生研究所/碩士(2007年)

https://doi.org/10.6832/KMU.2007.00087

摘要

Paraquat(PQ)是極度廣泛使用的除草劑，也常被使用來當作自殺的藥劑，PQ的急性暴露會造成肝、腎衰竭；慢性暴露可能引起肺癌、腎臟疾病與帕金森氏病，多為流行病學或動物實驗，極少數的學者使用正常的肝臟細胞進行研究有關巴拉刈對肝臟所造成的傷害，因此我們藉由PQ作用在正常大鼠肝臟細胞(WB-F344 cell)，對細胞間隙交流(Gap junctional intercellular communication)、細胞凋亡機制(Apoptosis)與細胞週期(Cell cycle)產生的影響來探討其對肝臟細胞的毒性機制。
細胞毒性測試(MTT & Colony Forming Efficiency)實驗中，PQ對細胞的增殖造成了干擾，並對細胞的正常功能產生了毒殺效應，暴露24與48小時在40 μg/mL達到IC50，且有Dose & Time dependent。細胞間隙交流(GJIC)實驗，細胞間溝通作用因暴露PQ，於25 μg/mL開始對Connexon產生破壞而導致溝通作用喪失，此一溝通喪失根據先前的研究有致癌的可能。PQ進入細胞後的毒性機制，可由Immunoblot analysis & Western Blot幾個實驗中得知，影響Caspase 3 、Caspase 8、 Caspase 9、MDM2、Bax、Cyto-C、P53等蛋白造成Apoptosis。細胞週期則由Flow Cytometry觀察，影響P21、P27、P53造成細胞無法正常分裂。DNA fragmentation實驗，可觀察出細胞暴露PQ後實驗組較控制組有較多的DNA碎裂。
實驗結果證實，WB-F344細胞暴露PQ後，影響細胞膜cytosol 釋出FADD，激起caspase 8的增加，caspase 3因caspase 8的增強而活躍，活躍的caspase 3使DNA fragmentation產生，造成細胞凋亡。暴露PQ使P53不穩定，不穩定的P53蛋白易與MDM2結合，使P53喪失腫瘤抑制基因的功能；Bcl-2減弱，Bax從cytosol移動到粒線體，粒線體因局部與Bax結合改變通透性，同一時間Cytochrome-C從通透性改變的部位釋出，活化caspase 9，使caspase 3被激發，造成DNA fragmentation 而Apoptosis。P53的激發與增加使得P21與P27受到影響導致細胞週期的停滯。

關鍵字

巴拉刈 ； 細胞間隙交流 ； 細胞凋亡 ； WB-F344

並列摘要

Paraquat (PQ) not only is commonly used as a method of committing suicide, but also is excessively used as herbicides. Acute exposures of Paraquat will result in liver and kidney failure while chronic exposures may cause lung caner, nephritic disease and Parkinson disease. Epidemiology and animal experimentation is more widely examined than experimentation on normal liver cells for researching on how Paraquat causes harm to the liver. Therefore, we test Paraquat on normal WB-F344 cell, Gap junctional intercellular communication, Apoptosis and Cell cycle to analyze the effect of Paraquat on liver cells and the mechanism of the toxicity.
During the MTT & Colony Forming Efficiency experimentation, Paraquat interferes with cell breeding, and also kills the normal function of cells with toxic. The exposure of Paraquat between 24 to 48 hours at 40µg/mL reached to IC50 and also had Dose & Time dependent. During the GJIC experiment, because of Paraquat exposures at 25µg/mL, the cell’s Connexon communication therefore is destroyed. Thus, based on previous studies, lost of communication may be the cause of cancer. When Paraquat enters cells, the toxic mechanism can be examined by Immunoblot analysis & Western Blot experimentation. The influence of these proteins: Caspase 3, Caspase 8, Caspase 9, MDM2, Bax, Cyto-C and P53 will cause Apoptosis. Cell cycle will be analyzed by Flow Cytometry, and the influence of P21, P27 and P53 will cause the division of cell to not function properly.
Experimental results confirmed that, after the exposure of WB-F344 cells to Paraquat, affected plasma memberane cytosol releases FADD, which in turn stimulates the increase of caspase 8. Caspase 3 is activated by the elevated level of caspase 8, thereby cause DNA fragmentation and cell death. The exposure of Paraquat causes P53 to become unstable, the unstable P53 in turn binds to MDM2, and loses the ability to control cancerous genes. Accompanied by the weakening of Bcl-2, Bax moves from cytosol to mitochondrion; mitochondrion partially binds to BAX and become permeable. Simultaneously, cytochrome-C is released from the permeable spot and activates caspase 9 and stimulates caspase 3, thereby causes DNA fragmentation and apoptosis. The increasing, stimulated P53 will affect P21 and P27 and result in the Cell Cycle to stagnate.

並列關鍵字

Paraquat ； WB-F344 ； GJIC ； Apoptosis

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