托福松生殖毒性之研究

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標題:	托福松生殖毒性之研究 The study for reproductive toxicity of Terbufos
作者:	陳佳筠
貢獻者:	嘉南藥理科技大學：生物科技系暨研究所王姿文
關鍵字:	不孕症基因毒性生殖毒性有機磷 Organophosphorus cytotoxicity genotoxicity infertility
日期:	2011
上傳時間:	2011-10-25 15:30:01 (UTC+8)
摘要:	近年來有機磷被大量使用，成為全球使用廣泛的化學農藥製劑，隨著有機磷抗藥性的產生，促使使用的濃度加強。然而有機磷的使用，可能會影響男性及女性生殖系統，導致生育能力降低、性功能障礙或不孕。農耕用藥種類繁多，對於生殖毒性及機能分歧，因此本研究以台灣常用的農藥托福松最為研究對象。針對托福松的農藥細胞毒性(cytotoxicity)及基因毒性(genotoxicity)進行分析。將睪丸GC-1細胞株處理於托福松不同劑量下處理，以探討托福松對於睪丸細胞影響與基因毒性。先以MTT assay判斷托福松對於睪丸細胞GC-1的存活率及影響濃度，後續利用流式細胞儀探討GC-1生殖細胞氧化壓力、粒線體膜電位的變化，進一步以TUNEL assay判斷托福松對於睪丸細胞GC-1 DNA damage程度，結果發現托福松確實會引起GC-1生殖細胞DNA damage，進而分析托福松對GC-1生殖細胞的細胞週期及細胞凋亡分析，最後則以western blot進行蛋白表現檢測。結果方面發現；MTT分析睪丸細胞GC-1存活率的結果經過不同時間處理過後，結果顯示有dose- and time- dependent趨勢，而睪丸細胞GC-1經過托福松的處理，生存率有顯著降低，相對表示托福松對於GC-1細胞的毒性較強。ROS檢測結果顯示，GC-1細胞在處理1-3小時後即有ROS大量產生，相較control組具有顯著性差異(p<0.001)，經過托福松3小時處理50、100、200μM分別增加1.9、2.1、1.5倍，經過10mM NAC前處理後ROS確實有抑制的趨勢。粒線體膜電位檢測結果方面，發現處理100μM托福松對於GC-1細胞MMP的產生粒線體膜電位升高比control高1.5倍。TUNEL assay分析結果，睪丸細胞GC-1處理托福松100μM 12小時與control相比TUNEL訊號增加2.8倍，有明顯DNA damage顯著性差異(p<0.01)。因此再以PI檢測GC-1細胞經過0、50、100、200μM 托福松處理24小時後，100μM、200μM 托福松Sub-G1分別明顯增加為4.76%(p<0.01)與22%(p<0.001)的凋亡細胞。而有G1期停滯的現象，再以100μM 托福松處理12、24、48、72小時後，發現GC-1細胞都停滯於G0/G1 phase，比例分別為39.71%、48.56%、54.31%、53.57%。同時使用Annexin V分析細胞凋亡，發現處理100μM托福松48、72小時，GC-1凋亡細胞分別顯著增加為24.89%與30.82%，隨著時間增加呈time-dependent現象。但經過前處理10mM NAC再加100μM托福松處理72小時後，凋亡細胞即顯著減少為22.49%。caspase-3檢測方面，GC-1細胞處理0、50、100、200μM托福松24小時後，caspase-3活性會隨著托福松劑量增加而活性上升，100、200μM分別顯著增加為123.71%(p<0.001)與187.06% ，western blot分析p53，p21等G0/G1停滯的上游相關基因表現，結果發現100μM托福松明顯造成p53和p21表現明顯增加，因而造成G0/G1停滯甚至造成細胞凋亡。因此本研究認為暴露在的托福松環境中，會影響精蟲發育及精蟲數目，很有可能會引起睪丸細胞發育不完全，甚至可能造成不孕。 Organophosphorus pesticides were widely use of agrochemicals in global, since resistant is increased. Organophosphorus pesticides is an endocrine disruptors of endocrine disrupting chemicals which effects on male endocrine function, reproductive system, induced sexual dysfunction, reproducibility decreased and infertility. In this study, we investigated the genotoxicity and cytotoxicity of organophosphorus pesticides of terbufos for testicular cell line. At the first, we determine the cell toxicity and cell viability for GC1 cells by MTT assay. Secondary, we determined the the cell viability effective, DNA damage degree, oxidative stress, mitochondrial membrane potential, cell cycle, apoptosis, caspase-3 activity and gene expression of terbufos for GC-1 cells. We found that exposure to various concentrations of terbufos for 6, 12, 24, 48 hours caused significant death in GC-1 cells in a dose- and time- dependent manner. The result of ROS relatived activity exposured to 50, 100, 200μM terbufos caused significant ROS level increased 1.9, 2.1, 1.5 fold(p<0.001) of control, respectivelyafter short-term exposure for 3 hours, in contras the ROS decreased after treated 10mM NAC as measured using flowcytometry. At the same time, we found that the GC-1 cell MMP which was increased 1.5 fold compared with control treated with 100μM terbufos for 3 hours. The 100μM Terbufos exposure for 12 hours caused significant DNA damage increased 2.8 fold(p<0.01) of TUNEL signal compared with control cell. The 100, 200μM terbufos were incubated with GC-1 24 hours induced significant Sub-G1 increased 4.76%(p<0.01) and 22% (p<0.001), respectively. Cell treated with 100μM terbufos caused GC-1 cell arrested in G1 phase after 12, 24, 48, 72 hours exposure. GC-1 cell expose 100μM terbufos induced significant apoptosis vai 48 and 72 hours in a time-depent manner which increased apoptotic cell 24.89%(p<0.001) and 30.82%(p<0.0001), respectively. But when GC-1 cell treatment with 10mM NAC are decreased apoptotic cell 22.49%(p<0.01) by Annexin V assay. The caspase-3 activity was significant increased after treatment with 50, 100, 200μM terbufos for 24 hours. The caspase-3 activity was in a dose-dependent manner which observed caspase-3 increased 123.71%(p<0.0001) and 187.06%(p<0.0001) and also observed the p53 and p21 genes protein expression increased after treatment with 50, 100, 200μM terbufos for 24 hours. In these results we realized the terbufos effect on mouse testicular cell GC-1. This study result help us to understand what the male reproductive effect of terbufos and also aid the implement of policy and regulatory measure of the organophosphorus pesticides of farmer’s safety knowledge.
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