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研究生:范秋婷
研究生(外文):Chiu-Ting Fan
論文名稱:亞砷酸鈉誘發產生之一氧化氮抑制人類纖維母細胞
論文名稱(外文):Inhibition of glutathione reductase by sodium arsenite-induced nitric oxide in human fibroblast
指導教授:黃海美
指導教授(外文):Haimei Huang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
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:52
中文關鍵詞:亞砷酸鈉麩胱甘還原酵素一氧化氮
外文關鍵詞:sodium arseniteglutathione reductasenitric oxide
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呈等比級數生長期的人類纖維母細胞(Human fibroblast, HF)培養於亞砷酸鈉(sodium arsenite, SA)的培養液中4小時,用SRB方式檢測細胞存活率,其50%的致死濃度介於30μM - 40μM,若濃度超過80μM 時,人類纖維母細胞存活率則低於 20%,表示亞砷酸鈉對人類纖維母細胞具有細胞毒性。
亞砷酸鈉處理會提高細胞培養液中亞硝酸的含量,間接代表一氧化氮(nitric oxide, NO)形成,亞砷酸鈉濃度愈高,產生愈高量一氧化氮,另外,同樣的處理,也會提高細胞內nitrotyrosine 含量代表peroxynitrite(NO與superoxide反應而成的產物)伴隨著一氧化氮生成而增加,此物質會硝化細胞內的蛋白質(造成對蛋白質的破壞)。NAME 3.2mM與亞砷酸鈉10μM 共同處理組,與單獨處理亞砷酸鈉10μM比較,細胞存活率高了15﹪,顯示一氧化氮生成是亞砷酸鈉引起的細胞死亡的原因之一。
以亞砷酸鈉處理人類纖維母細胞2小時或4小時後,麩胱甘還原酵素(glutathione reductase, GR)活性隨亞砷酸鈉的濃度以及處理時間增加而降低,4小時亞砷酸鈉40μM處理下,麩胱甘還原酵素活性只剩57%,亞砷酸鈉濃度大於80μM時,無論4小時或2小時處理,麩胱甘還原酵素活性皆低於50%。以4小時亞砷酸鈉處理後,繼續無藥培養12小時及24小時,觀察麩胱甘還原酵素活性回復程度,亞砷酸鈉40μM,12小時與24小時,麩胱甘還原酵素活性回升20 - 30%,亞砷酸鈉濃度大於120μM時,後培養24小時僅回復30%,結果並顯示,人類纖維母細胞在24小時內無法100﹪恢復麩胱甘還原酵素活性。分別用亞砷酸鈉40μM,亞砷酸鈉40μM與NAME1.6mM,亞砷酸鈉40μM與NAME 3.2mM處理人類纖維母細胞4小時後測量麩胱甘還原酵素活性,亞砷酸鈉與NAME 1.6mM共同處理,與單獨處理亞砷酸鈉比較,麩胱甘還原酵素活性回升 4% - 10%,亞砷酸鈉加NAME 3.2mM則回升 4% - 30%,表示麩胱甘還原酵素活性因亞砷酸鈉所引發的NO被抑制。
人類纖維母細胞經亞砷酸鈉4小時培養後,麩胱甘(glutathione, GSH)含量呈現出下降的趨勢。亞砷酸鈉 40μM處理組之GSH降低了16%,亞砷酸鈉160μM,其含量只剩44%。 與GSH對照的是GSSG含量,和控制組相比,亞砷酸鈉40μM處理組 已增至2.6倍,細胞內的GSH總量(GSH與GSSG含量相加),則沒有明顯的變化 --指出GSH的合成在4小時培養時間內,無顯著改變。麩胱甘還原酵素與亞砷酸鈉 10μM共同處理組,和單獨處理亞砷酸鈉10μM比較,細胞存活率高20﹪,顯示麩胱甘還原酵素活性的降低是亞砷酸鈉引起的細胞死亡的原因之一。

Human fibroblasts were exposed to sodium arsenite for 4h. The cytotoxicity of sodium arsenite treatment was determinted by SRB assay. The IC50 was 30μM- 40μM. Here, the cell survival was only 20﹪of control in the exposure of 80μM sodium arsnite. This study has shown that sodium arsnite influence the cytotoxicity of HF.
Treatment of HF with SA40-160μM for 4h increase the nitrite content released into cell cultured medium,indicating the elevated level of nitric oxide (NO). Peroxynitrite is a powerful modifier of intracellular proteins. The nitration of tyrosine , nitrotyrosine , is a useful marker for detecting peroxynitrite in biological samples.HF cells exposure to SA40-160μM for 4h increase nitrotyrosine level by 10-20﹪,indicating the elevated level of peroxynitrite. Cotreament with 3.2mM Nω-nitro-L-arginine methyl ester ( NAME) and 10μM SA for 4h, the survival rose from 65﹪to 78﹪. These data suggest that NO was involed in SA-induced cell death.
HF cells incubated with 40-160μM SA showed glutathione reductase activity significantly decrease to less than 29-60﹪after 2h and less than 43-65﹪after 4h. After HF cells were exposed to 40μM SA for 4h and followed a postincubation in drug-free medium for 12 or 24h. Glutathione reductase(GR) activity rose from 20﹪to 30﹪after 12 and 24 h of recovery. However,GR activity did not recover to the control level wthin 24h. Cotreament of HF cells with SA and NAME(1.6 or 3.2mM) for 4h , partly restore the GR activity. At 1.6mM NAME,the percentage of the recovered GR activity was 4﹪-10﹪.At 3.2mM NAME,the percentage of the recovered GR activity was 4﹪-30﹪. The data showed that treatment with SA increased nitrite production in HF cells and that NAME could not significantly recover GR activity.
HF cells exposure to SA40-160μM for 4h decrease GSH level by 16-56nmol /mg protein. This decrease in GSH content was accompanied by a simultaneous increase in GSSG level, with a GSH disappearance and GSSG production.4h after an incubation with 40μM SA, GSSG increase about 2.6-fold.Intracellular total glutathione concentration were almost unchanged suggesting that GSH synthesis did not differ from control cells after 4h. Cotreament with GR and 10μM SA for 4h, the survival rose from 65﹪to 82﹪. These data suggest that decreased glutathione reductase induced by sodium arsenite, lead to cell loss of viability.

目錄
中文摘要 1
英文摘要 3
縮 寫 5
緒 論 6
實驗方法 14
結 果 21
討 論 26
參考文獻 33
圖 表 43

Anderson, B.B., Carandia, G., Lucci, M. (1987) Red cell GSH regeneration and glutathione reductase activity in G6PD variants in the Ferrana area. Br J Haematol, 67, 459-466.
ATSDR, (1992). Toxicological Profile for Arsenic, Atlanta: Agency for Toxic Substnces and Disease Registry.
Aposhian, H.V., Zakharyan, R.A., Wu, Y., Healy, S., Aposhian, M.M. (1997) Enzymatic methylation of arsenic compounds.II. An overview. In arsenic exposure and human health, vol 2, ed. C Abernethy, R Calderon, W Chappell. London:Chapman and Hall. In press
Barker, J.E., Heales, S.J.R., Cassidy, A., Bolanos, J.P., Land, J.M. and Clark, J.B. (1996) Depletion of brain glutathione results in a decrease of glutathione reductase activity; an enzyme susceptible to oxidative damage. Brain Res, 716, 118-122.
Bau, D.T., Gurr, J.R. and Jan, K.Y. (2001) Nitric oxide is involved in arsenite inhibition of pyrimidine dimer excision. Carcinogenesis, 22, 709-716.
Becker, K., Savvides, S.N., Keese, M., Schirmer, R.H.and Karplus, P.A. (1998) Enzyme inactivation through sulfhydryl oxidation by physiologic NO-carriers. Nat. Struct. Biol, 5, 267-271.
Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A. (1990) Apparent hydroxyl radical production by peroxynitrite:implications for endothelial injury from nitric oxide and superoxide. Proc. Natl.Acad. Sci. USA, 87, 1620-1624.
Beckmann, J.S., Ye, Y.Z., Anderson, P.G., Chen, J.., Accavitti, M.A., Tarpey, M.M.and Roger White, C. (1994) Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol. Chem, Hoppe-Seyler, 375, 81-88.
Buchet JP, Lauwerys R, Roels H. (1981) Comparison of the urinary excretion of arsenic metabolites after a single oral dose of sodium arsenite, monomethylarsonate, or dimethylarsinate in man. Int Arch Occup Environ Health; 48,pp71-9
Chance, B., Sies, H., Boveries, A (1979) Hydroperoxide metabolism in mammalians organs. Physiol Rev, 59, 527-605.
Chen C.J., Chen, C.W., Wu, M.M. and Kuo, T.L. (1992) Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br J Cancer, 66, 888-892.
Chiou HY, Hsueh YM, Liaw KF, Horng SF, Chiang MH, Pu YS, Lin JS, Huang CH, Chen CJ. (1995) Incidence of internal cancers and ingested inorganic arsenic: a seven-year follow-up study in Taiwan. Cancer Res, 55, 1296-300
Cohn, V.H. and Lyle J. (1966) A fluorometric assay for glutathioe. Anal.Biochem, 14, 434 — 440.
Dai, J., Weinberg, R.S., Waxman, S.and Jing, Y. (1999) Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the gluthione redox system. Blood, 93, 268-277.
Dansette, P., Sassi, A., Deschamps, C., Mansuy, D (1990). Sulfur containing compounds as antioxidants. In Emerit I, Parker L, Auclair C(eds)Antioxidants in therapy and preventive medicine. Plenum Press,New York, NY, 209-215.
De Vera, M.E., Kim, Y.M., Wong, H.R., Wang, Q., Billiar, T.R. and Geller, D.A. (1996) Heat shock response inhibits cytokine-inducible nitric oxide synthase expression in rat hepatocytes. Hepatology, 24, 1238-1245.
Dong, H.Q., Wang, K.L., Ma, Y.J. (1993). A clinical analysis of 117 cases of acute arsenic poisoning. Chung Hua Nei Ko Tsa Chih 32, 813.
Flohe L (1982) .Glutathione peroxidase brought into focus. In:Mason RP(ed)Free radicals in biology,. 5 ,Academic Press, New York,NY, 223-254.
Francescutti, D., Baldwin, J., Lee, L and Mutus, B. (1996) Peroxynitrite modification of glutathione reductase:modeling studies and kinetic evidence suggest the modification of tyrosines at the glutathione disulfide site. Protein Engineering, .9, 189-194.
Frischer, H.and Ahmad, T. (1977) Severe generalized glutathione reductase deficiency after antitumor chemotherapy with BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea). J Lab Clin Med, 89, 1080-1091.
Garcia-Vargas GG, Garcia-Rangel A, Aguilar-Romo M, Garcia-Salcedo J, del Razo LM, Ostrosky-Wegman P, Cortinas de Nava C, Cebrian ME. (1991) A pilot study on the urinary excretion of porphyrins in human populations chronically exposed to arsenic in Mexico. Hum Exp Toxicol; 10, 189-193
Goebel, H.H., Schmidt, P.F., Bohl, J., Teltenborn, B., Kramer, G.,Gutmann, L., (1990). Polyneuropathy due to arsenic intoxication: biopsy studies. J. Neuropathol. Exp. Neurol, 49, 137
Gomez-Arroyo, S., Hernandez-Garcia, A. and Villalobos-Pietrini, R. (1988) Induction of sister-chromatid exchanges in Vicia faba by arsenic-contaminated drinking water. Mutat Res, 208, 219-214.
Gurr, J.R., Liu, F., Lynn S.and Jan, K.Y. (1998) Calcium-dependent nitric oxide production is involved in arsenite-induced micronuclei. Mutat Res, 416, 137-48.
Haddad, I.Y., Pataki, G., Hu, P., Beckman, J.S. and Matalon, S. (1994) Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J. Clin. Invest, 94, 2407-2413.
Halliwill, B.and Gutteridge, J.M.C. (1990) Role of free radicals and iron in relation to biology and catalytic metals ions in human disease:an overview. Method Enzymol, 186B, 1-85.
Hirata, M., Tanaka, A., Hisanaga, A. and Ishinishi, N. (1990). Effects of glutathione depletion on the acute nephrotoxic potential of arsenite and on arsenic metabolism in hamsters. Toxicol Appl Pharmacol, 106, 469-481.
Huang, H., Huang, C.F., Wu, D.R., Jinn, C.M. and Jan KY (1993). Glutathione as a cellular defense against arsenic toxicity in cultured Chinese hamaster ovary cells. Toxicology , 79, 195-204.
Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J., Smith, C.D. and Beckman, J.S. (1992) Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biochem. Biophys, 298, 431-437.
Ischiropoulos H, al-Mehdi AB. (1995) Peroxynitrite-mediated oxidative protein modifications. FEBS Lett, 364, 279-82.
Kala, S.V., Neely, M.W., Kala,G., Pratert, C.I., Atwood, D.W., Rice, J.S.and Lieberman, M.W. (2000). The MRP2/cMOAT transporter and arsnic- glutathione complex formation are required for biliary excretion of arsenic. The journal of biological chemistry, 275, 33404-33408.
Kaur, K., and Halliwell, B. (1994) Evidence for nitric oxide-mediated oxidative damage in chronic inflammation;Nitrotyrosine in serm and synovial fluid from rheumatoid patients. FEBS Letters, 350, 9-12.
Kehrer, J.P. (1993) Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol, 23, 21-48.
Kooy, N.W., Royall, J.A., Ye, Y.Z., Kelly, D.R. and Beckman, J.S. (1995) Evidence for in vivo peroxynitrite production in human acute lung injury. Am. J. Resp. Crit. Care Med, 151, 1250-1254
.Lee, T.C., Wei, M.L., Chang, W.J., Ho, .IC., Lo, J.F., Jan, K.Y. and Huang, H (1989) Elevation of glutathione and gluthione s-transferse activity in arsenic resistant Chinese hamster ovary cells. In Vitro Cell Dev Biol, 25, 442-448.
Lee, T.C. and Ho, I.C. (1995) Modulation of cellular antioxidant defense activities by sodium arsenite in human fibroblasts. Arch Toxicol, 69, 498-504.
Liu, F. and Jan, K.Y (2000) DNA damage in arsenite- and cadmium-treated bovine aortic endothelial cells. Free Radic Biol Med, 28, 55-63.
Lu, S.C., (1999). Regulation of hepatic glutathione synthesis:current concepts and controversies. FASES J , 13, 1169-1183
Lynn, S., Shiung, J.N., Gurr, J.R. and Jan, K.Y. (1998) Arsenite stimulates poly(ADP-ribosylation)by generation of nitric oxide. Free Radic Biol Med, 24, 442-9.
Machlin, L.J.and Bendich, A. (1987) Free radical tissue damage:protective role of antioxidant nutrients. Faseb J, 1, 441-445.
Misko, T.P., Schilling, R.J., Salvemini, D., Moore, W.M., Currie, M.G. (1993) A fluorometric assay for the measurement of nitrite biological samles. Anal. Biochem, 214, 11-16.
Meister, A.and Anderson, M.E. (1983) Gluthione. Annu Rev Biochem, 52, 711-760.
Neiger, R.D. and Osweiler, G.K. (1989) Effect of subacute low level dietary sodium arsenite on dogs. Fundam. Appl. Toxicol, 13, 349.
Nordenson, I. And Beckman, L. (1991) Is the genotoxic effect of arsenic mediated by oxygen free radicals? Hum Hered, 41, 41-73.
Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions Biochemistry , 14, 2644-2651
Ochi, T. (1997) Arsenic compound-induced increases in glutathione levels in cultured Chinese hamster V79 cells and mechanisms associated with changes in gamma -glutamylcysteine synthetase activity, cystine uptake and utilization of cysteine. Arch Toxicol, 71, 730-740.
Ozolins, T.R.S.and Hales, B.F. (1999) Tissue-specific regulation of glutathione Homeostasis and the activator protein-1(AP-1)response in the rat conceptus. Biochemical Pharmacology, 57, 1165-1175.
Radi,R., Beckman, J.S., Bush, K.M. and Freeman, B.A. (1991) Peroxynitrite -induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys, 288, 481-487.
Savvides, S.N., Scheiwein, M., Bohme, C.C., Arteel, G.E., Karplus, P.A., Becker, K. and Schirmer, R.H. (2002) Crystal structure of the antioxidant enzyme glutathione reductase inactivated by peroxynitrite. J.B.C, 277, 2279-2784.
Skehan, Q.., Storeng, R Scudiero, D., Monks, A., McManhon, J., Vistica, D., Warren, J. T., Bodesch, Kenney, S. and Boyd, M.R. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J.Nat. Cancer. Inst, 82, 1107-1112
Styblo, M.and Thomas. (1995) In vitro inhibition of glutathione reductase by arsenotriglutathione. Biochemical Pharmacology, 49, 971-977.
Styblo, M., Serves, S.V., Cullen, W.R., Thomas, D.J. (1997) Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols. Chem Res Toxicol, 10, 27-33.
Van der Vliet, A., O’Neill, C.A., Halliwell, B., Cross, C.E. and Kaur, H. (1994) Aromatic hydroxylation and nitration of phenylalanine and tyrosine by peroxynitrite. Evidence for hydroxyl radical production from peroxynitrite.
FEBS Lett, 339, 89-92.
Vahter, M and Marafante, E. (1988) In vivio methylation and detoxication of arsenic.In the biological alkylation of heavy elements, special publication, ed PJ Craig, F Glocking , 66:105-19. London: R. Soc.Chem.
Vahter, M., Couch, R., Nermell, B., Nilsson, R. (1995) .Lack of methlyation of inorganic arsenic in the chimpanzee. Toxicol. Appl. Pharmacol, 133, 262-268
Wang, H.F. and Lee, T.C. (1993) .Glutathione S-transferase π facilitates the excretion of arsen from arsenic-resistant Chinese hamster ovary cells. Biochem Biophys Res Commun, 192, 1093-1099.
Wang, T.S.and Huang, H. (1994) Active oxygen species are involved in the induction of micronuclei by arsenite in XRS-5 cells. Mutagenesis, 9, 253-257.
Wang, T.S., Kuo, C.F., Jan, K.Y. and Huang, H. (1996) Arsenite induces apoptosis in Cinese hamster ovary cells by generation of reactive oxygen species. J Cell Physiol, 169, 256-268.
Wang, T.S., Shu, Y.F., Liu, Y.C Jan, K.Y. and Huang, H. (1997) Glutathione peroxidase and catalase modulate the genotoxicity of arsenite. Toxicology, 121, 229-237.
Wardman P (1988) Conjugation and oxidation of glutathione via thiyl free radicals. In:Sies H, Ketterer B(ends)Glutathione conjugathion, mechanisms and biological signficance. Academic Press Limited, New York, NY, pp43-72.
Wong, H.R., Finder, J.D., Wasserloos, K and Pitt, B.R. (1995) Expression of iNOS in cultured rat pulmonary artery smooth muscle cells is inhibited by the heat shock response. Am J Physiol, 269, L843-8
Yager, J.W. and Wiencke, J. K. (1993) Enhancement of chromosomal damage by arsenic: implications for mechanism. Environ. Health Perspect,101, 79-82.
Yamanaka, K., Hoshino, M., Okamoto, M., Sawamura, R., Hasegawa, A. and Okada, S. (1990) Induction of DNA damage by dimethylarsine, a metablite of inorganic arsenics, is for the major part likely due to its peroxyl radical. Biochem Biophys Res Commun, 168, 58-64.

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