烏腳病 (Blackfoot disease)是一種閉塞性血栓及閉塞性動脈硬化的末梢血管疾病，流行於臺灣西南沿岸地區。井水高含量的腐植酸 (Humic acid，HA)被認為是烏腳病的致病因子之一。本研究以井水純化之腐植酸 (HA)及單一酚酸 (Protocatechuic acid)聚合而成之人工腐植酸 (Synthetic HA，SHA)，作用培養之人類臍帶靜脈血管內皮細胞後觀察內皮細胞產生一氧化氮 (Nitric oxide，NO)及內皮細胞死亡 (Cell death)的現象。結果顯示腐植酸會誘導生成一氧化氮；接著，會造成內皮細胞死亡。本實驗深入研究顯示，腐植酸所誘導內皮細胞死亡即為細胞凋亡 (Apoptosis)，包括細胞萎縮 (Cell shrinkage)，細胞膜泡狀突起 (Membrane blebbing）和DNA斷裂（DNA fragmentation）等特徵。加入NOS抑制劑 (L-NAME及L-NMA)、鈣離子抑制劑 (BAPTA)、抗氧化劑 (SOD、vitamin C、vitamin E)及蛋白激酵素抑制劑 (H7)等，可不同程度抑制腐植酸誘導內皮細胞產生一氧化氮及細胞凋亡的情形。利用西方點墨法 (Western blotting)發現腐植酸誘導eNOS蛋白的表現；此外，內皮細胞Bcl-2蛋白受到抑制而Bax、p53蛋白增加。由結果顯示，氧化性壓力和細胞內鈣離子增加為腐植酸誘導細胞凋亡的可能原因。
總結，內皮細胞死亡被認為是烏腳病誘發原因；因此腐植酸所誘導內皮細胞產生一氧化氮及細胞凋亡的結果，可能是烏腳病的致病原因。

Blackfoot disease (BFD) is a peripheral arterial occlusive disease found among the inhabitants of the southwest coast of Taiwan. Humic acid (HA) in well water used by the inhabitants for drinking is one possible etiological factors for BFD. In this study, the ability of natural HA, purified from the drinking well water of BFD endemic areas, and synthetic HA (SHA) to induce NO production and apoptosis, was studied in cultured human umbilical vein endothelial cells (HUVECs). Results show that HA could NO production and apoptosis in a dose-dependent manner. The characteristics of apoptosis in cultured endothelial cells include cell shrinkage, chromatin condensation, plasma membrane blebbing and DNA fragmentation. NO production and apoptosis of endothelial cells induced by HA were inhibited significantly by NOS inhibitor (L-NAME and L-NMA), free radical scavengers (SOD, vitamin C, and vitamin E), Ca2+ chelator (BATPA) and Protein kinase inhibitor (H7). These results suggest that Ca2+ and oxidative stress are mediators of apoptosis caused by HA.
Destruction of endothelial cells has been implicated in the process of the onset of BFD. The induction of excessive NO and apoptosis in endothelial cells may contribute the etiology of HA-induced vascular disorder of BFD in humans.

1. Tseng WP, Chen WY, Sung JL, Chen JS. A clinical study of blackfoot disease in Taiwan, an endemic peripheral vascular disease. Memorie College of Medicine National Taiwan University. 7:1-8, 1961.
2. Tseng WP. Effects and dose response relationship of skin cancer and blackfoot disease with arsenic. Environmental Heatth Perspectives. 19:109-119, 1977.
3. Lu FJ. Blackfoot disease: arsenic or humic acid. The Lancet. 336:115-116, 1990.
4. Lu FJ, Yang CK, Lin KH. Physicochemical characteristics of drinking water in blackfoot endemic areas in Chial and Taiwan Hsiens. Journal of the Formosan Medical Association. 74:596-605, 1975.
5. Lu FJ, Yamamura Y, Yamauchi H. Studies on fluorescent compounds in water of a well in blackfoot disease endemic areas in Taiwan: Humic substances. Journal of the Formosan Medical Association. 87:66-75, 1988.
6. Huang TS, Lu FJ, Tsai CW, Chopra IJ. Effect of humic acids on thyroidal function. Journal of Endocrinological Investigation. 17:787-791, 1994.
7. Lu FJ, Liu TM. Fluoresent compounds in drinking water of Blackfoot disease endemic areas. Animal experimental model. Journal of the Formosan Medical Association. 85:352-358, 1986.
8. Huang TS, Lu FJ, Tsai CW. Tissue distribution of absorbed humic acids. Environmental Geochemistry and Health. 17;1-4, 1995.
9. Jan IR, Chen JY, Lu FJ, Ling KH. Studies on fluorescent compounds in drinking water of blackfoot disease endemic areas. 4. The cytotoxic effects of fluorescent compounds on cell cultures. Journal of the Formosan Medical Association. 77:361-3, 1978.
10. Lu FJ, Tsai MH, Ling KH. [Studies on fluorescent compound in drinking water of blackfoot disease endemic areas. (1) Toxic effects of the fluorescent compound on chick embryos]. [Chinese]. Journal of the Formosan Medical Association. 76:58-63, 1977.
11. Hong CL, Huang PC. Analysis of body weight gain and estimation of additional energy costs during normal pregnancy in Taiwan and Japan. Journal of the Formosan Medical Association. 86:649-56, 1987.
12. Chen JY, Lu FJ, Lin KH. Studies on fluorescent compounds in drinking water of blackfoot disease endemic areas. 5. Effect of fluorescent compounds on protein and nucleic acid synthesis in growing HeLa cells. Chinese Journal of Microbiology. 10:115-7, 1977.
13. Lu FJ, Tsai SC, Chen JY. Studies on natural toxicants occuring in artesian well water of blackfoot disease endemic areas in Taiwan: Chromsome aberrations in cultured lyphocytes exposed to fluorescent compounds. 9:49-54, 1980.
14. Lu FJ, Lee YS. Humic acid: inhibitor of plasmin. Science of the Total Environment. 114:135-9, 1992.
15. 施信如. 營光物質對血漿中凝固因子及protein C之影響.台大醫學院 生化科碩士論文 (呂鋒洲教授指導) 1991.
16. Chiu HC, Shih SR, Lu FJ, Yang HL. Stimulation of endothelin production in cultured human endothelial cells by fluorescent compounds associated with blackfoot disease. Thrombosis Research. 69:139-51, 1993.
17. Chiu HC, Shih SH, Lu FJ. The Fluorescent compounds from drinking water and the vascular discorders of Blackfoot disease. FASEB. A523:884, 1991.
18. Yang HL, Lu FJ, Wung SL, Chiu HC. Humic acid induces expression of tissue factor by cultured endothelial cells: regulation by cytosolic calcium and protein kinase C. Thrombosis & Haemostasis. 71:325-30, 1994.
19. 呂鋒洲, 劉東明. 烏腳病患區飲水中螢光物質動物實驗模式. 台灣醫誌. 85:352-58, 1986.
20. 劉東明. 烏腳病患區井水中螢光物質之研究:螢光物質對小白鼠肝臟內脂質過氧化反應後醛類產物之影響. 台大醫學院. 生化科碩士論文 (呂鋒洲教授指導) 1986.
21. James AP, Linda KG, Garvey TE, Hamm J, James AS. Lack of hepatic promoting activity by the peroxisomal proliferating hepatocarcinogenesis di-(2-ethylhexyl) phthalate. Carcinogenesis. 6:141-4, 1985.
22. 黃蘭如. 烏腳病井水中抽物質誘導小白鼠肝臟中過氧化體增泩之研究. 台大醫學院. 生化科碩士論文 (呂鋒洲教授指導) 1991.
23. Lu FJ, Lee TC, Pang VF, Huang TS. Humic acid-induced testicular morphological changes in rats. Bulletin of Environmental Contamination & Toxicology. 58:619-27, 1997.
24. Lee Y, Huang TS, Yang ML, Huang LR, Chen CH, Lu FJ. Peroxisome proliferation, adipocyte determination and differentiation of C3H10T1/2 fibroblast cells induced by humic acid: induction of PPAR in diverse cells. Journal of Cellular Physiology. 179:218-25, 1999.
25. Lu FJ, Yamamura Y, Yamauchi H. Studies on fluorescent compounds in water of a well in Blackfoot disease endemic areas in Taiwan: Humic substances. Journal of the Formosan Medical Association. 87:66-75, 1988.
26. Jezierski A, Czechowski F, Jerzykiewicz M, Chen Y, Drozd J. Electron paramagnetic resonance (EPR) studies on stable and transient radicals in humic acids from compost, soil, peat and brown coal. Spectrochimica Acta. 56:379-385, 2000.
27. Yang HL, Hesu YC, Lu FJ. Humic acid induces the generation of nitric oxide of human umbilical vein endothelial cells. Thrombosis and Haemostasis. 1998.
28. Hseu YC, Lu FJ, Engelking LR, Chen CL, Chen YH, Yang HL. Humic acid nduced echinocyte transformation in human erythrocytes: characterization of morphological changes and determination of the mechanism underlying damage. Journal of Toxicology & Environmental Health. 60:215-30, 2000.
29. Cheng ML, Ho HY, Chiu DTY, Lu FJ. Humic acid-mediated oxidative damages to human erythrocytes: a possible mechanism leading to anemia in blackfoot disease. Free Radical Biology and Medicine. 27:470-477, 1999.
30. Liang HJ, Tsai CL, Lu FJ. Oxidative stress induced by humic acid solvent extraction fraction in cultured rabbit articular chondrocytes. Journal of Toxicology & Environmental Health. 54:477-89, 1998.
31. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. Journal of Clinical Investigation. 52:2745-56, 1973.
32. Toss AS. Localization of fibrinolytic activity in tissue. British Medical Bulletin. 20:210, 1964.
33. Holemans R, Mann LS. Cope C. Fibrinolytic activity of renal venous and arterial blood. American Journal of the Medical Sciences. 254:330-3, 1967.
34. Yanagisawa M, Kurihara H, Kimura S, Goto K, Masaki T. A novel peptide vasoconstrictor, endothelin, is produced by vascular endothelium and modulates smooth muscle Ca2+ channels. Journal of Hypertension. 6:S188-91, 1988.
35. Miller WL, Redfield MM, Burnett JC Jr. Integrated cardiac, renal, and endocrine actions of endothelin. Journal of Clinical Investigation. 83:317-20, 1989.
36. Tseng WP. Blackfoot disease in Taiwan: a 30-year follow-up study. Angiology. 40:547-558, 1989.
37. Yu HS. Blackfoot disease and chronic arsenism in southern Taiwan. International Journal of Dermatology. 23:258-260, 1984.
38. Yu HS, Sheu HM, Ko SS, Chiang LC, Chien CH, Lin SM, Tserng BR, Chen CS. Studies on blackfoot disease and chronic arsenism in southern Taiwan: with special reference to skin lesions and fluorescent substances. Journal of Dermatology. 11:361-370, 1984.
39. Yu HS, Chang CH, Chen GS, Yang SA, Yu CL. Study of dramic microcirculatory problems in blackfoot diseases emphasizing its differences from arterioslerosis. Journal of Biomedical Science. 2:183-188, 1995.
40. Tseng CH, Chong CK, Chen CJ, Tai TY. Dose-response relationship between peripheral vascular disease and ingested inorganic arsenic among residents in blackfoot disease endemic villages in Taiwan. Atherosclerosis. 120: 125-33, 1996.
41. Yang HL, Lu FJ, Wang SL, Chiu HC. Humic acid induces expression of tissue factor by cultured endothelial cells: regulation by cytosolic calcium and protein kinase C. Thrombosis and Haemostasis. 71:325-330, 1994.
42. Chiu HC, Shih SR, Lu FJ, Yang HL. Stimulation of endothelin production in cultured human endothelial cells by fluorescent compounds associated with blackfoot disease. Thrombosis Research. 69:139-151, 1993.
43. Yang HL, Chiu HC, Lu FJ. Effects of humic acid on the viability and coagulant properties of human umbilical vein endothelial cells. American Journal of Hematology. 51:200-206,1996.
44. Yang HL, Hseu YC, Lu FJ, Tsai HD. Humic acid reduces protein C-activating cofactor activity of thrombomomodulin of human umbilical vein endothelial cells. British Journal of Haematology. 101:16-23, 1998.
45. Sherr CJ. Cancer cell cycles. Science. 274:1672-1677, 1996.
46. Stephen IR. Fibronectin supports bronchial epithelial cell adhesion and survival in the absence of growth factor. The American Physiological society. L684-L693, 1997.
47. Guido M, Isabelle J. Apoptosis , oncosis and necrosis. American Society for Investigative Pathology. 146:3-15, 1995.
48. Hermann S. Mechanism and Genes of cellular suicide. Science. 267:1445-1448, 1995.
49. Dive C, Hickman JA. Drug-target interactions only the first step in the commitment to a programmed cell death? British Journal of Cancer. 64:192-6, 1991.
50. Kerr JFR, wyllie AH, Currie AR. Apoptosis : a basic biological phenomenon with wild ranging implications in tissue kinetics. British Journal of Cancer. 26 :1790-1794, 1972.
51. Zhang YH, Takahashi K, Jiang GZ, Kawai M, Fukada M, Yokochi T. In vivo induction of apoptosis (programmed cell death) in mouse thymus by adminstration of lipopolysaccaride. Infection & Immunity. 61:5044-5048, 1993.
52. Martin SJ, Green DR. Apoptosis and cancer: the failure of controls on cell death and cell survival. Critical Reviews in Oncology/Hematology. 16:137-153, 1995.
53. Li N, Karin M. Ionizing radiation and short wavelength UV activate NF-kappaB through two distinct mechanisms. Proceedings of the National Academy of Sciences of the United States of America. 95:13012-7, 1998.
54. Mastrangelo AJ, Betenbaugh MJ. Overcoming apoptosis: new methods for improving protein-expression systems. Trends in Biotechnology. 16:88-95, 1998.
55. Brown DG, Klrinr L, Tenniswood M. The biochemistry of cell death by apoptosis. Biochem, Cell Biology. 88:1071-1074, 1990.
56. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. International Review of Cytology. 68:251-306, 1980.
57. McConkey DJ. Biochemical determinants of apoptosis and necrosis. Toxicology Letters. 99:157-68, 1998.
58. Wyllie Andrew H. Apoptosis snd the regulation of cell numbers in normal and neoplastic tissues:an overview. Cancer and Metastasis Reviews. 11:95-103, 1992.
59. Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends in Biochemical Sciences. 22:299-306, 1997.
60. Widmann C, Johnson NL, Gardner AM, Smith RJ, Johnson GL. Potentiation of apoptosis by low dose stress stimuli in cells expressing activated MEK kinase 1. Oncogene. 15:2439-47, 1997.
61. Cardone MH, Salvesen GS, Widmann C, Johnson G, Frisch SM. The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell. 90:315-23, 1997.
62. Green DR, Reed JC. Mitochondria and apoptosis. Science. 281:1309-12, 1998.
63. Nagata S, Golstein P. The Fas death factor. Science. 267:1449-56, 1995.
64. Wyllie A. Apoptosis. Clues in the p53 murder mystery. Nature. 389:237-8, 1997.
65. Bates S, Vousden KH. p53 in signaling checkpoint arrest or apoptosis. Current Opinion in Genetics & Development. 6:12-8, 1996.
66. Cox LS, Lane DP. Tumour suppressors, kinases and clamps: how p53 regulates the cell cycle in response to DNA damage. Bioessays. 17:501-8, 1995.
67. Yin C, Knudson CM, Korsmeyer SJ, Van Dyke T. Bax suppresses tumorigenesis and stimulates apoptosis in vivo. Nature. 385:637-40, 1997.
68. Zhan Q, Fan S, Bae I, Guillouf C. Liebermann DA. O'Connor PM. Fornace AJ Jr. Induction of bax by genotoxic stress in human cells correlates with normal p53 status and apoptosis. Oncogene. 9:3743-51, 1994.
69. Araki S, Shimada Y, Kaji K, Hayashi H. Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation. Biochemical and Biophysical Research Communications. 69:1248, 1990.
70. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annual Review of Cell Biology. 7:663-98, 1991.
71. Lin JK, Chou CK. In vitro apoptosis in the human hepatoma cell line induced by transforming growth factor beta 1. Cancer Research. 52:385-8, 1992.
72. Lowe SW, Schmitt EM, Smith SW. Osborne BA. Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature. 362:847-9, 1993.
73. Walton MI, Whysong D, O'Connor PM, Hockenbery D, Korsmeyer SJ, Kohn KW. Constitutive expression of human Bcl-2 modulates nitrogen mustard and camptothecin induced apoptosis. Cancer Research. 53:1853-61, 1993.
74. Kyprianou N, English HF, Davidson NE, Isaacs JT. Programmed cell death during regression of the MCF-7 human breast cancer following estrogen ablation. Cancer Research. 51:162-6, 1991.
75. Bertrand R, Solary E, Jenkins J, Pommier Y. Apoptosis and its modulation in human promyelocytic HL-60 cells treated with DNA topoisomerase I and II inhibitors. Experimental Cell Research. 207:388-97, 1993
76. Yoshioka A, Tanaka S, Hiraoka O, Koyama Y, Hirota Y, Wataya Y. Deoxyribonucleoside-triphosphate imbalance death: deoxyadenosine induced dNTP imbalance and DNA double strand breaks in mouse FM3A cells and the mechanism of cell death. Biochemical & Biophysical Research Communications. 146:258-64, 1987.
77. Wagner BA, Buettner GR, Oberley LW, Darby CJ, Burns CP. Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells. Journal of Biological Chemistry. 275:22461-9, 2000.
78. Flynn TP, Allen DW, Johnson GJ, White JG. Oxidant damage of the lipids and proteins of the erythrocyte membranes in unstable hameoglobin disease. The Journal of Clinical Investigation. 71:1215-1223, 1983.
79. Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR, Harris CC. Hepatitis B virus protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proceedings of the National Academy of Sciences of the United States of America. 91:2230-2234, 1994.
80. Hollstein M, Sidransky D, Vogelstein B, Harris CC. P53 mutation in human cancers. Science. 253:49-53, 1991.
81. Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature. 362: 847-849, 1993.
82. Loignon M, Fetni R, Gordon AJ, Drobetsky EA. A p53-independent pathway for induction of p21waf1cip1 and concomitant G1 arrest in UV-irradiated human skin fibroblasts. Cancer Research. 57:3390-4, 1997.
83. Riva C, Chauvin C, Pison C, Leverve X. Cellular physiology and molecular events in hypoxia-induced apoptosis. Anticancer Research. 18:4729-36, 1998.
84. Miyamoto Y, Hosotani R, Wada M, Lee JU, Koshiba T, Fujimoto K, Tsuji S, Nakajima S, Doi R, Kato M, Shimada Y, Imamura M. Immunohistochemical analysis of Bcl-2, Bax, Bcl-X, and Mcl-1 expression in pancreatic cancers. Oncology. 56:73-82, 1999.
85. Miyashita T, Reed JC Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 80:293-9, 1995.
86. Buckbinder L, Talbott R, Velasco-Miguel S, Takenaka I, Faha B, Seizinger BR, Kley N. Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature. 377:646-9, 1995.
87. Prokocimer M, Rotter V. Structure and function of p53 in normal cells and their aberrations in cancer cells: projection on the hematologic cell lineages. Blood. 84:2391-411, 1994.
88. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science. 226:1097-9, 1984.
89. Steinman HM. The Bcl-2 oncoprotein functions as a pro-oxidant. Journal of Biological Chemistry. 270:3487-90, 1995.
90. Hunter JJ, Parslow TG. A peptide sequence from Bax that converts Bcl-2 into an activator of apoptosis. Journal of Biological Chemistry. 271:8521-4, 1996.
91. Moley KH, Chi MM, Knudson CM, Korsmeyer SJ, Mueckler MM, Hyperglycemia induces apoptosis in pre-implantation embryos through cell death effector pathways. Nature Medicine. 4:1421-4, 1998.
92. Williams GT, Smith CA. Molecular regulation of apoptosis: genetic controls on cell death. Cell. 74:777-9, 1993.
93. Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell. 80:285-91, 1995.
94. Decaudin D, Geley S, Hirsch T, Castedo M, Marchetti P, Macho A, Kofler R, Kroemer G. Bcl-2 and. Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Research. 57:62-7, 1997.
95. Reed JC. Bcl-2 and the regulation of programmed cell death. The Journal of Cell Biology. 124:1-6, 1994.
96. Dypbukt JM, Ankarcrona M., Burkitt M, Sjoholm A, Strom K, Orrenius S, Nicotera P. Different prooxidant levels stimulate growth, trigger apoptosis, or produce necrosis of insulin-secreting RINm5F cells. The role of intracellular polyamines. Journal of Biological Chemistry. 269:30553-60, 1994
97. Saikumar P, Dong Z, Mikhailov V, Denton M, Weinberg JM, Venkatachalam MA. Apoptosis: definition, mechanisms, and relevance to disease. American Journal of Medicine. 107:489-506, 1999.
98. Bjorkerud S, Bjorkerud B. Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. American Journal of Pathology. 149:367-80, 1996.
99. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 288:373-6, 1980.
100. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 327:524-6, 1987.
101. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proceedings of the National Academy of Sciences of the United States of America. 84:9265-9, 1987
102. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews. 43:109-142, 1991.
103. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB Journal. 6:3051-3064, 1992.
104. Shen YH, Wang XL, Wilcken DE Nitric oxide induces and inhibits apoptosis through different pathways. FEBS Letters. 433:125-131, 1998.
105. Marletta. MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry. 27:8706-8711, 1988.
106. Simmons ML, Murphy S. Induction of nitric oxide synthase in glial cells. Journal of Neurochemistry. 59:897-905, 1992.
107. Ree DD, Palmer RM, Moncada S. Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proceedings of the National Academy of Sciences of the United States of America. 86:3375-3378, 1989.
108. Nava E, Luscher TF. Endothelium-derived vasoactive factors in hypertension: Nitric oxide and endothelin. Journal of Hypertension. Official Journal of the International Society of Hypertension. 13;839-848, 1995.
109. Huie RE, Padmaja S. The reaction of no with superoxide. Free Radical Research Communications. 18:195-199,1993.
110. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA, Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proceedings of the National Academy of Sciences of the United States of America. 87:1620-1624, 1990.
111. Noronha-Dutra AA, Epperlein MM, Woolf N. Reaction of nitric oxide with hydrogen peroxide to produce potentially cytotoxic singlet oxygen as a model for nitric oxide-mediated killing. FEBS Letters. 321:59-62, 1993.
112. Matheis G, Sherman MP, Buckberg GD, Haybron DM, Young HH, Ignarro LJ. Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. American Journal of Physiology. 1992;262:H616-620.
113. Moncada, S. Nitric oxide gas: Mediator, modulator, and pathophysiologic entity. The Journal of Laboratory and Clinical Medicine. 1992;120:187-191.
114. Aiken NR, Thurman EM, Maleolm RL. Comparisom of XAD macroporous resins for the concentration of flulvic acid from aqueous solution. Analytical Chemistry. 51:1799, 1979.
115. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothlial cells derived from umbilical veins. The Journal of Clinical Investigation. 52: 2745-56, 1973.
116. Gree LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, and Tamnnenbaum SR. Analysis of nitrate, nitrite and [15N]nitrate in biological fliuds. Analytical Biochemistry. 126:131-138,1982.
117. Moldeus P, Hogberg J, Orrenius S. Isolation and use of liver cells. Methods in Enzymology. 52:60-71, 1978.
118. Sambrook J, Fritttsch EF, Maniatis T. Molecular Cloning: A Laboratory Manuuual, 2nd ed. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989.
119. Shimizu S, Nomoto M, Yamamoto T, Momose K. Reduction by NG-nitro-L-arginine of H2O2-induced endothelial cell injury. British Journal of Pharmacology. 113:564-8, 1994
120. Okayama N, Coe L, Itoh M, Alexander JS. Intracellular mechanisms of nitric oxide plus hydrogen peroxide-mediated neutrophil adherence to cultured human endothelial cells. Inflammation Research. 47:428-433, 1998.
121. Shen YH, Wang XL, Wilcken DE. Nitric oxide induces and inhibits apoptosis through different pathways. FEBS Letters. 433(1-2):125-31, 1998.
122. Daemen NARC, van’t Veer C, Denecker G, Heemskerk VH, Wolfs TGAM, Causs M, Vandenabeels P and Buurman WA. Inhibition of apoptosis induced by ischmia-reperfusion prevents inflammation. The Journal of Clinical Investigation. 104: 541-549, 1999.
123. Yang BC, Chang HM, Wang YS, Chen RF, Lin SJ. Transient induction of apoptosis in serum-starved glioma cells by insulin and IGF-1. Biochimica et Biophysica Acta. 1314: 83-92, 1996.
124. Wyllie AH. Kerr JF. Currie AR. Cell death: the significance of apoptosis. International Review of Cytology. 68: 251-306, 1980.
125. Wyllie AH, Morris RG, Smith AL, Dunlop D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. Journal of Pathology. 142: 67-77, 1984.
126. Richter C. Pro-oxidants and mitochondrial Ca2+: their relationship to apoptosis and oncogenesis. FEBS Letters. 325:104-7, 1993.
127. Brawn MK, Chiou WJ, Leach KL. Oxidant-induced activation of protein kinase C in UC11MG cells. Free Radical Research. 22:23-37, 1995.
128. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 75: 241-51, 1993.
129. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB. Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 74: 597-608, 1993.
130. Lu FJ, Huang TS, Lin YS, et al. Peripheral vasculopathy in rats induced by humic acids. Appl Organomet Chem 8:223-228, 1994.
131. Lohmann RD, Beyersmann D. Cadmium and zinc mediated changes of the Ca2+-dependent endonuclease in apoptosis. Biochemical & Biophysical Research Communications. 190:1097-103, 1993.
132. Nicotera P. Bonfoco E. Brune B. Mechanisms for nitric oxide-induced cell death: involvement of apoptosis. Advances in Neuroimmunology. 5:411-20, 1995.
133. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 75:241-51, 1993.
134. Oppenheim RW. Related mechanisms of action of growth factors and antioxidants in apoptosis: an overview. Advances in Neurology. 72:69-78, 1997.
135. Lotem J, Peled-Kamar M, Groner Y, Sachs L. Cellular oxidative stress and the control of apoptosis by wild-type p53, cytotoxic compounds, and cytokines. Proceedings of the National Academy of Sciences of the United States of America. 93: 9166-71, 1996.