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研究生:林裕萍
研究生(外文):Yu-Ping Lin
論文名稱:四氯對苯二酚誘發細胞凋亡的作用機轉及抗氧化劑在四氯對苯二酚誘發基因毒性及細胞傷害過程中扮演角色之探討
論文名稱(外文):Study on the apoptosis mechanisms in NIH/3T3 cells induced by tetrachlorohydroquinone(TCHQ) and elucidate the role of antioxidants during the process of TCHQ-induced genotoxicity and cytotoxicity
指導教授:王應然王應然引用關係
指導教授(外文):Ying-Jan Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境醫學研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:57
中文關鍵詞:抗氧化劑細胞凋亡四氯對苯二酚五氯酚
外文關鍵詞:Tetrachlorohydroquinonepentachlorophenolantioxidantsapoptosis
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Tetrachlorohydroquinone(TCHQ)四氯對苯二酚為Pentachlorophenol(PCP)五氯酚的主要毒性代謝產物之一;PCP被廣泛使用於木材防腐劑及生物殺菌劑上,為目前重要的環境污染物之一。PCP的主要代謝產物TCHQ已知會造成細胞之DNA單股斷裂(single-strand breaks, SSB),而具有基因毒性及細胞毒性,且TCHQ亦被證實其產生的氧化性壓力及毒性均較PCP來得高。因此推測PCP致癌的能力來自於TCHQ的毒性所致。
在前趨的動物二階段致癌實驗中證實,PCP誘發及促進腫瘤產生的能力,高於TCHQ,推測這可能是因為,TCHQ會導致細胞走上細胞凋亡(apoptosis)的能力高於PCP,因而降低了TCHQ致癌的能力。本研究會對TCHQ 是否可誘發細胞進行細胞凋亡及其作用機轉作進一步的研究。
研究中發現NIH/3T3纖維母細胞在TCHQ8小時50μM劑量的作用下,有明顯細胞凋亡的現象;而在一些控制細胞凋亡的重要基因中發現Bcl-2蛋白的表現量有上升的趨勢,並且會隨著時間的增加而降低;p53蛋白及cytochrome c蛋白的表現,隨著時間的增加有上昇的趨勢;此外亦發現隨著劑量及時間的增加,caspase 9與caspase 3的活性亦有增加的趨勢,另外在3T3 over-expression Bcl-2細胞株中發現其caspase 9與caspase 3並無上升的趨勢,證實了TCHQ造成3T3細胞凋亡是經由mitochondria-mediated的路徑。
在抗氧化劑方面篩檢出captopril、penicillamine、dihydrolipoic acid (DHLA)及2,3-dimercaptosuccinic acid (DMSA)會抑制TCHQ的細胞毒性及基因毒性,而這些化合物皆屬於thiol group;另一方面發現lipoic acid(LA)雖然無法抑制TCHQ的毒性但其還原態的化合物DHLA卻可以保護3T3細胞免於TCHQ的毒性,顯示這些抗氧化劑的保護作用可能來自於-SH group可以與ROS作用而降低其對DNA的傷害所致。
Tetrachlorohydroquinone (TCHQ) has been identified as a major toxic metabolite of the widely used wood preservative pentachlorophenol and also implicated in its genotoxicity.
In our preliminary two-stage carcinogenesis experiment, it was proved that the tumor promotion activity of PCP was higher than TCHQ but the toxicity of TCHQ was higher than PCP. We assumed that TCHQ, but not PCP, may cause apoptotic cell death and thus decrease the tumor promotion activity and tumorigenecity. This study was designed to test the hypothesis that TCHQ induced apoptotic cell death in 3T3 cells and to investigate the mechanisms of apoptosis induced by TCHQ. Moreover, we identified some antioxidants with the ability to reduce TCHQ-induced ROS, and defined whether these agents may inhibit cytotoxicity and genotoxicity induced by TCHQ.
In the present study, we found that apoptotic cell death can be induced in 3T3 cells treated with 50μM TCHQ for 8 hrs. The generation of ROS and collapse of mitochodrial transmembrane potrntial has been observed to occur in early apoptosis induced by TCHQ, and the process is related to bcl-2 protein expression. The activity of caspase9 and caspase3 subsequently increased with a time- and dose- dependent pattern. Bcl-2 over-expressed 3T3 cells provided marked protection against the cytotoxicity induced by TCHQ. This result suggested that Bcl-2 protein play an important role in the process of apoptosis in 3T3 cells, which also further indicated that apoptotic cell death in 3T3 cells occurs through the mitochondria-mediated pathway.
Captopril, penicillamine, dihydrolipoic acid (DHLA) and 2,3-dimercaptosuccinic acid (DMSA) are antioxidants with thiol groups. All of them were found to counteract the TCHQ-induced cytotoxicity. No protective ability was found in lipoic acid, oxidized form of DHLA, indicated that the protection comes from the –SH group which could bond with ROS and reduces the ROS-mediated DNA damage. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage induced by TCHQ. Using this method, we found that captopril provided marked protection against genotocixity induced by TCHQ.
第一章 序論
第一節 研究動機1

第二章 文獻探討
第一節 五氯酚2
第二節 四氯對苯二酚2
第三節 活性氧物種3
第四節 細胞凋亡4
4.1.1 p 53蛋白5
4.1.2 Bcl-2蛋白及Bax蛋白6
4.1.3 cytochrome c、caspase 9、caspase 36
第五節、抗氧化劑
5.1.1 Lipoic acid (LA),dihydrolipoic acid(DHLA)7
5.1.2 Captopril.8
5.1.3 penicillamine.8
5.1.4 meso-2,3-dimercaptosuccinic acid(DMSA)8

第三章 研究目的9

第四章 研究材料與方法
第一節 研究材料10
第二節 研究方法
細胞培養13
細胞氧化性壓力偵測分析13
DNA萃取及瓊膠電泳偵測細胞凋亡14
細胞存活率偵測分析(MTT assay)15
細胞基因毒性偵測分析(Comet assay)15
Mitochondrial transmembrane potential之偵測分析17
西方墨點法18
Caspase 活性分析.21

第五章 實驗架構.22

第六章 實驗結果
第一節、Tetrachlorohydroquinone(TCHQ)造成3T3細胞形態上的改變23
第二節、TCHQ引起3T3細胞凋亡23
第三節、 TCHQ造成3T3細胞內氧化性壓力增加24
第四節、抗氧化劑抵抗TCHQ對3T3細胞造成的細胞毒性25
第五節、抗氧化劑抵抗TCHQ對3T3細胞造成的基因毒性25
第六節、TCHQ造成3T3細胞mitochondria膜電位不穩定..27
第七節、Bcl-2蛋白在TCHQ造成細胞凋亡過程中的重要性27
第八節、3T3細胞內與細胞凋亡有關基因的表現28

第七章 討論
第一節、 TCHQ對NIH/3T3細胞之細胞凋亡機轉30
第二節、抗氧化劑抵禦TCHQ所造成細胞毒性及基因毒性31
1.Roberts, H. J. Pentachlorophenol-associated aplastic anemia, red cell aplasia, leukemia and other blood disorders, Journal of the Florida Medical Association. 77: 86-90, 1990.
2.McConnel, E. E. Toxicology and carcinogenesis studies of two pentachlorophenol technical-grade mixtures in B6C3F1 mice. NIH Publication 1989.
3.Juhl, U., Witte, I., and Butte, W. Metabolism of pentachlorophenol to tetrachlorohydroquinone by human liver homogenate, Bulletin of Environmental Contamination & Toxicology. 35: 596-601, 1985.
4.Zhu, B. Z., Kitrossky, N., and Chevion, M. Evidence for production of hydroxyl radicals by pentachlorophenol metabolites and hydrogen peroxide: A metal-independent organic Fenton reaction, Biochemical & Biophysical Research Communications. 270: 942-6, 2000.
5.Lin, P. H., Nakamura, J., Yamaguchi, S., La, D. K., Upton, P. B., and Swenberg, J. A. Induction of direct adducts, apurinic/apyrimidinic sites and oxidized bases in nuclear DNA of human HeLa S3 tumor cells by tetrachlorohydroquinone, Carcinogenesis. 22: 635-9, 2001.
6.Seiler, J. P. Pentachlorophenol, Mutation Research. 257: 27-47, 1991.
7.Hattemer-Frey, H. A. and Travis, C. C. Pentachlorophenol: environmental partitioning and human exposure, Archives of Environmental Contamination & Toxicology. 18: 482-9, 1989.
8.Kutz, F. W., Cook, B. T., Carter-Pokras, O. D., Brody, D., and Murphy, R. S. Selected pesticide residues and metabolites in urine from a survey of the U.S. general population, Journal of Toxicology & Environmental Health. 37: 277-91, 1992.
9.Leet, T. L. and Collins, J. J. Chloracne and pentachlorophenol operations, American Journal of Industrial Medicine. 20: 815-8, 1991.
10.Jorens, P. G. and Schepens, P. J. Human pentachlorophenol poisoning, Human & Experimental Toxicology. 12: 479-95, 1993.
11.Anonymous Pentachlorophenol, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 53: 371-402, 1991.
12.Purschke, M., Jacobi, H., and Witte, I. Differences in genotoxicity of H(2)O(2) and tetrachlorohydroquinone in human fibroblasts. Mutation Research. 513: 159-67, 2002.
13.Wang, Y. J., Ho, Y. S., Jeng, J. H., Su, H. J., and Lee, C. C. Different cell death mechanisms and gene expression in human cells induced by pentachlorophenol and its major metabolite, tetrachlorohydroquinone, Chemico-Biological Interactions. 128: 173-88, 2000.
14.Ehrlich, W. The effect of pentachlorophenol and its metabolite tetrachlorohydroquinone on cell growth and the induction of DNA damage in Chinese hamster ovary cells, Mutation Research. 244: 299-302, 1990.
15.Carstens, C. P., Blum, J. K., and Witte, I. The role of hydroxyl radicals in tetrachlorohydroquinone induced DNA strand break formation in PM2 DNA and human fibroblasts, Chemico-Biological Interactions. 74: 305-14, 1990.
16.Witte, I., Zhu, B. Z., Lueken, A., Magnani, D., Stossberg, H., and Chevion, M. Protection by desferrioxamine and other hydroxamic acids against tetrachlorohydroquinone-induced cyto- and genotoxicity in human fibroblasts, Free Radical Biology & Medicine. 28: 693-700, 2000.
17.Jansson, K. and Jansson, V. Induction of micronuclei in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol, Mutation Research. 279: 205-8, 1992.
18.Dahlhaus, M., Almstadt, E., and Appel, K. E. The pentachlorophenol metabolite tetrachloro-p-hydroquinone induces the formation of 8-hydroxy-2-deoxyguanosine in liver DNA of male B6C3F1 mice, Toxicology Letters. 74: 265-74, 1994.
19.Fridovich, I. Fundamental aspects of reactive oxygen species, or what's the matter with oxygen?, Annals of the New York Academy of Sciences. 893: 13-8, 1999.
20.Marnett, L. J. Oxyradicals and DNA damage, Carcinogenesis. 21: 361-70, 2000.
21.Kamata, H. and Hirata, H. Redox regulation of cellular signalling, Cellular Signalling. 11: 1-14, 1999.
22.Kroemer, G., Dallaporta, B., and Resche-Rigon, M. The mitochondrial death/life regulator in apoptosis and necrosis, Annual Review of Physiology. 60: 619-42, 1998.
23.Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease, Science. 267: 1456-62, 1995.
24.Corcoran, G. B., Fix, L., Jones, D. P., Moslen, M. T., Nicotera, P., Oberhammer, F. A., and Buttyan, R. Apoptosis: molecular control point in toxicity, Toxicology & Applied Pharmacology. 128: 169-81, 1994.
25.Gerschenson, L. E. and Rotello, R. J. Apoptosis: a different type of cell death, FASEB Journal. 6: 2450-5, 1992.
26.Kerr, J. F., Wyllie, A. H., and Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics, British Journal of Cancer. 26: 239-57, 1972.
27.Thompson, E. B. Apoptosis and steroid hormones, Molecular Endocrinology. 8: 665-73, 1994.
28.Buja, L. M., Eigenbrodt, M. L., and Eigenbrodt, E. H. Apoptosis and necrosis. Basic types and mechanisms of cell death, Archives of Pathology & Laboratory Medicine. 117: 1208-14, 1993.
29.Schwartz, L. M., Smith, S. W., Jones, M. E., and Osborne, B. A. Do all programmed cell deaths occur via apoptosis, Proceedings of the National Academy of Sciences of the United States of America. 90: 980-4, 1993.
30.Wyllie, A. H., Morris, R. G., Smith, A. L., and Dunlop, D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis, Journal of Pathology. 142: 67-77, 1984.
31.Wyllie, A. H. What is apoptosis?, Histopathology. 10: 995-8, 1986.
32.Cotter, T. G., Lennon, S. V., Glynn, J. G., and Martin, S. J. Cell death via apoptosis and its relationship to growth, development and differentiation of both tumour and normal cells, Anticancer Research. 10: 1153-9, 1990.
33.Kastan, M. B., Radin, A. I., Kuerbitz, S. J., Onyekwere, O., Wolkow, C. A., Civin, C. I., Stone, K. D., Woo, T., Ravindranath, Y., and Craig, R. W. Levels of p53 protein increase with maturation in human hematopoietic cells, Cancer Research. 51: 4279-86, 1991.
34.Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L., and Wyllie, A. H. Thymocyte apoptosis induced by p53-dependent and independent pathways, Nature. 362: 849-52, 1993.
35.Shaw, P., Bovey, R., Tardy, S., Sahli, R., Sordat, B., and Costa, J. Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line, Proceedings of the National Academy of Sciences of the United States of America. 89: 4495-9, 1992.
36.Vander Heiden, M. G. and Thompson, C. B. Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis?, Nature Cell Biology. 1: E209-16, 1999.
37.Shimizu, S., Eguchi, Y., Kamiike, W., Funahashi, Y., Mignon, A., Lacronique, V., Matsuda, H., and Tsujimoto, Y. Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux, Proceedings of the National Academy of Sciences of the United States of America. 95: 1455-9, 1998.
38.Jurgensmeier, J. M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., and Reed, J. C. Bax directly induces release of cytochrome c from isolated mitochondria, Proceedings of the National Academy of Sciences of the United States of America. 95: 4997-5002, 1998.
39.Budihardjo, I., Oliver, H., Lutter, M., Luo, X., and Wang, X. Biochemical pathways of caspase activation during apoptosis, Annual Review of Cell & Developmental Biology. 15: 269-90, 1999.
40.Stadtman, E. R. and Levine, R. L. Protein oxidation, Annals of the New York Academy of Sciences. 899: 191-208, 2000.
41.Furchgott, R. F. A research trail over half a century, Annual Review of Pharmacology & Toxicology. 35: 1-27, 1995.
42.Bustamante, J., Lodge, J. K., Marcocci, L., Tritschler, H. J., Packer, L., and Rihn, B. H. Alpha-lipoic acid in liver metabolism and disease, Free Radical Biology & Medicine. 24: 1023-39, 1998.
43.Packer, L., Witt, E. H., and Tritschler, H. J. alpha-Lipoic acid as a biological antioxidant, Free Radical Biology & Medicine. 19: 227-50, 1995.
44.Teichert, J. and Preiss, R. HPLC-methods for determination of lipoic acid and its reduced form in human plasma, International Journal of Clinical Pharmacology, Therapy, & Toxicology. 30: 511-2, 1992.
45.Handelman, G. J., Han, D., Tritschler, H., and Packer, L. Alpha-lipoic acid reduction by mammalian cells to the dithiol form, and release into the culture medium, Biochemical Pharmacology. 47: 1725-30, 1994.
46.Suh, J. H., Shigeno, E. T., Morrow, J. D., Cox, B., Rocha, A. E., Frei, B., and Hagen, T. M. Oxidative stress in the aging rat heart is reversed by dietary supplementation with (R)-(alpha)-lipoic acid, FASEB Journal. 15: 700-6, 2001.
47.Anderson, B., Khaper, N., Dhalla, A. K., and Singal, P. K. Anti-free radical mechanisms in captopril protection against reperfusion injury in isolated rat hearts, Canadian Journal of Cardiology. 12: 1099-104, 1996.
48.Lapenna, D., De Gioia, S., Mezzetti, A., Ciofani, G., Di Ilio, C., and Cuccurullo, F. The prooxidant properties of captopril, Biochemical Pharmacology. 50: 27-32, 1995.
49.Srivastava, P. J., Chandra, S., Arif, A. J., Singh, C., and Panday, V. Metal chelators/antioxidants: approaches to protect erythrocytic oxidative stress injury during Plasmodium berghei infection in Mastomys coucha, Pharmacological Research. 40: 239-41, 1999.
50.Iciek, M., Polak, M., and Wlodek, L. Effect of thiol drugs on the oxidative hemolysis in human erythrocytes, Acta Poloniae Pharmaceutica. 57: 449-54, 2000.
51.Ercal, N., Treeratphan, P., Hammond, T. C., Matthews, R. H., Grannemann, N. H., and Spitz, D. R. In vivo indices of oxidative stress in lead-exposed C57BL/6 mice are reduced by treatment with meso-2,3-dimercaptosuccinic acid or N-acetylcysteine, Free Radical Biology & Medicine. 21: 157-61, 1996.
52.Sha, S. H. and Schacht, J. Antioxidants attenuate gentamicin-induced free radical formation in vitro and ototoxicity in vivo: D-methionine is a potential protectant, Hearing Research. 142: 34-40, 2000.
53.Longo, V. D., Ellerby, L. M., Bredesen, D. E., Valentine, J. S., and Gralla, E. B. Human Bcl-2 reverses survival defects in yeast lacking superoxide dismutase and delays death of wild-type yeast, Journal of Cell Biology. 137: 1581-8, 1997.
54.Wiseman, H., Kaur, H., and Halliwell, B. DNA damage and cancer: measurement and mechanism, Cancer Letters. 93: 113-20, 1995.
55.Wang, Y. J., Lee, C. C., Chang, W. C., Liou, H. B., and Ho, Y. S. Oxidative stress and liver toxicity in rats and human hepatoma cell line induced by pentachlorophenol and its major metabolite tetrachlorohydroquinone, Toxicology Letters. 122: 157-69, 2001.
56.Wispriyono, B., Matsuoka, M., and Igisu, H. Effects of pentachlorophenol and tetrachlorohydroquinone on mitogen-activated protein kinase pathways in Jurkat T cells, Environmental Health Perspectives. 110: 139-43, 2002.
57.Zhu, B. Z., Har-El, R., Kitrossky, N., and Chevion, M. New modes of action of desferrioxamine: scavenging of semiquinone radical and stimulation of hydrolysis of tetrachlorohydroquinone, Free Radical Biology & Medicine. 24: 360-9, 1998.
58.Suzuki, Y. J., Tsuchiya, M., and Packer, L. Thioctic acid and dihydrolipoic acid are novel antioxidants which interact with reactive oxygen species, Free Radical Research Communications. 15: 255-63, 1991.
59.Kawabata, T., Tritschler, H. J., and Packer, L. Reaction of (R,S)-dihydrolipoic acid and homologs with iron, Methods in Enzymology. 251: 325-32, 1995.
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