環境中特定有機與無機物質對細胞毒性交互作用之探討

中山醫學大學機構典藏 CSMUIR > 醫學院 > 醫學分子毒理學研究所 > 博碩士論文 > Item 310902500/951

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/951

Title:	環境中特定有機與無機物質對細胞毒性交互作用之探討 The Effects of Organic and Inorganic Environmental Chemicals on Cell Toxcity
Authors:	游維哲 Yu, Wei-Che
Contributors:	中山醫學院：毒理學研究所;陳文貴
Keywords:	有機物質;細胞毒性
Date:	1997
Issue Date:	2010-03-25T03:46:25Z (UTC)
Abstract:	現有對環境毒物、毒性之評估與規範多只針對單一化學物質，鮮少考量在數種環境特定化學物質共同曝露時對人體之危害。而事實上在現實環境中，人體極可能同時暴露與吸收數種環境毒物，而其間對人體之危害亦可能互相拮抗或加強之效應，有必要結合現在對環境毒物了解進一步探討在數種化學物質共同暴露下對生物體所造成之加成毒性機轉。本研究特別選取無機與有機之特定化學物質，一為氯化鎘：環境與勞工作業時常易接觸之有害污染物，另一為乙醛：環境與工業上常見之有機污染物，亦為酒精在生物體中之初步代謝物，以V79細胞株探討此二種化學物質，在共同暴露時對細胞與基因毒性之複合效應。本實驗先以Trypan blue exclusion assay 觀察到單一暴露在氯化鎘10~50μM或乙醛1~50mM下，V79細胞存活率均在80%以上，但在共同暴露下其存活率則降低為70%，再以MTT方法觀察對細胞活性之抑制作用亦有類似的結果。接著利用colony efficency assay觀察長時間下之毒性效應，發現乙醛和氯化鎘共同暴露下有明顯的細胞複合毒性，其致死率為預期致死率的2倍，而在HPRT assay（基因毒性檢驗）下亦表現明顯的複合效應。進一步觀察在單一與共同暴露乙醛和氯化鎘四小時後，對細胞生長的影響，發現細胞生長所受到之抑制作用，單一種暴露比共同暴露有較快之生長回復的現象，再利用flow cytometry觀察細胞週期變化，發現與控制組相比，共同暴露乙醛和氯化鎘會嚴重導致細胞週期的改變。又由重金屬含量測定得之，細胞內鎘之含量不會因共同暴露乙醛而有所增加。　　以DNA單股斷裂測定毒物對細胞DNA之傷害程度，顯示乙醛和氯化鎘能造成DNA單股斷裂的增加，但在共同處理下，並無明顯加強DNA傷害之程度。在脂質過氧化MDA實驗觀察到單一或共同暴露乙醛和氯化鎘會使細胞脂質過氧化程度增加，在共同暴露下乙醛會明顯加強鎘所造成的脂質過氧化程度，並有明顯的複合效應，如以天然物中之free radical scavenger quercetin 預處理則可抑制MDA之產生，而以甘草之主成份glycyrrhizin則無；由rhodamine 123螢光量在cytoplasma之變化則顯示在共同暴露下，對mitochondria plasma membrane 有明顯的損傷作用，但在預處理quercetin 和glycyrrhizin 則會抑制其損傷程度，由以上實驗結果顯示共同暴露乙醛和氯化鎘在許多層面上會對V79細胞產生明顯的複合毒性，而天然物Quercetin和Glycyrrhizin則對乙醛和氯化鎘所造成的細胞傷害，能透過不同的機制達到保護的效果。　　As one of the most potent hazard inorganic substances in our environment, cadmium has been found to have severe toxic and genotoxic effect in animal both in vivo and in vitro system, and even in human a well. On the other hand, acetaldehyde as the cigarette smoke contained substance and the primary metabolite of ethanol is also an extensive organic chemical widely contaminating the environmental hazardous substances, the synergistic/antagonistic effect of the mixture of environmental pollutants have to be investigated. The goals of this work focus on the coeffect of both cadmium and acetaldehyde (represented the inorganic and organic active environmental pollutants), respectively to V79 cell line in its toxicity and mutagenesis. In addition, because the oxidative stress is one of the main causes of genotoxicity, the in vitro assay of lipid peroxidation by TBA reaction has also been studied. The evaluation of shot-term performance of cell viability was studied using trypan blue exclusion and MTT assay. 　　In the single exposure to cadmium (10-50μM) or acetaldehyde (1-5mM) alone, both retain their viability above 80% comared to the control cell. There is no significant enhancement of cytotoxicity in dual exposure. However, the cloning efficiency assay for long-term observation for cell performance has showed a significant synergistic effect on cytotoxicity. Moreover, by counting the cell number growth within 0-72 hours after dual exposure cells from combined exposure have less recovery ability to normal cell growth after dual exposure. And the cell cycle study by flow cytometry assay has showed that the coexposure of cadmium and acetaldehyde can retain most cells in the G0/G1 states. 　　The mechanism of the synergistic effect is thought due to the enhancement of acetaldehyde to the cadmium entering the cells. We use the atomic absorption spectrometry is used to measure the cadmium in the cells. Results showed that the addition of acetaldehyde yield no enhancement of the entrance of cadmium to the cell. 　　Comparing with the singe exposure, the HPRT gene expression assay showed that the dual exp'osure enhance their mutagenesis and also exhibit a synergistic effect in certain dose range (cadmium 5-10μM, acetaldehyde 1-5mM). But in the assay for DNA single strand breakage had no significant synergistic response. In lipid peroxidation assay, the addition of acetaldehyde significantly increases the TBA formation of cadmium exposed cells. Finally, when introduce the natural antioxidant quercetin and traditional medicine Glycyrrhizin to the exposed cells, the quercetin showed a more potent inhibition to their cytotoxity and lipid peroxidation formation, while Glycyrrhizin was not.
URI:	http://140.128.138.153:8080/handle/310902500/951
Appears in Collections:	[醫學分子毒理學研究所] 博碩士論文

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