氧化壓力的增加會誘發粒線體增生及功能損性傷。葉酸為一抗氧化營養分子，但粒線體葉酸擔任保護粒線體的角色尚未被了解。本研究選用中國大頰鼠卵巢細胞粒線體葉酸代謝正常野生株CHOK1，與粒線體serine hydroxylmethyltransferase酵素 (mSHMT) 缺陷突變株GLYA及粒線體葉酸轉運蛋白缺陷突變株GLYB為研究模式。探討粒線體葉酸代謝如何保護粒線體功能、DNA與調節粒線體生合成機制。投與外來氧化壓力t-butylhyhroperoxide (t-BH) 處理24、48 與72小時後，以細胞流式儀偵測胞內活性氧物種含量、粒線體質量與粒線體膜電位變化;即時定量PCR測定調節粒線體生合之轉錄因子表現、粒線體DNA (mtDNA) 拷貝數與4834bp大片斷損量 (ΔmtDNA4.8 kb)；並以HPLC測量DNA氧化傷害指標8-Hydroxydeoxyguanosine (8-OHdG) 含量；以微生物法分析葉酸含量。結果顯示，GLYB突變株細胞質與粒線體葉酸含量顯著低於CHOK1。胞內超氧陰離子含量顯著高於CHOK1與GLYA。t-BH處理後，顯著消耗細胞質與粒線體葉酸含量分別下降44%與28%，且胞內過氧化氫與超氧陰離子顯著增加4倍。t-BH處理24小時後，GLYB之粒線體生合成相關轉錄因子NRF-1、NRF-2、mtTFA與mtDNA複製因子pol γ皆向上調節，啟動粒線體生合成提早，並伴隨mtDNA拷貝數與粒線體質量增加。ROS大量產生，促使GLYB細胞內DNA氧化損傷8-OHdG增加3倍，mtDNA 4834 bp大片段斷損 (ΔmtDNA4.8 kb) 顯著累積，mtDNA轉譯之COX I、II及III基因發生點突變機率增加，導致粒線體膜電位去極化比例增加1.7倍。GLYA突變株，在t-BH處理後，顯著消耗細胞質與粒線體葉酸含量 (22%與48%)，且胞內ROS增加3倍，促使粒線體生合成相關轉錄因子NRF-1、mtTFA向上調節，並伴隨mtDNA拷貝數與粒線體質量增加，但其粒線體生合成比GLYB晚被啟動。ROS的產生，促使GLYA細胞內8-OHdG含量增加2倍，ΔmtDNA4.8 kb累積，導致粒線體膜電位去極化比例增加，但其氧化傷害介於CHOK1與GLYB之間。葉酸補充可顯著增加GLYA與GLYB細胞質與粒線體中葉酸濃度，並降低細胞因外因性氧化壓力誘發大量ROS產生，顯著降低胞內8-OHdG含量、ΔmtDNA4.8kb累積與點突變之發生，達到保護mtDNA之功能，恢復粒線體膜電位。並調節ROS誘發粒線體生合成相關轉錄因子表現，mtDNA拷貝數與粒線體質量隨之降低。縱合上述，粒線體葉酸具有保護粒線體DNA抗氧化傷害及維持粒線體膜電位正常功能。並平衡外來氧化壓力、調整細胞核啟動粒線體增生機制，以補償氧化壓力對粒線體傷害。補充葉酸可改善粒線體葉酸代謝酵素基因變異或生化缺陷，以補足粒線體葉酸不足，降低外因性氧化壓力誘發之氧化傷害。

Increased oxidative stress results in mitochondrial (mt) DNA damage, mt dysfunction and enhanced mt biogenesis. The aims of the study is to investigable the mt folate metabolism to protect against oxidant stress-induced mtDNA injuries, enhanced mt biogenesis and mt dysfunction. Several Chinese hamster ovary (CHO) cells including wild type CHOK1 with normal mt folate metabolism, GLYA mutants with defective mt serine hydroxyl- methyltransferase defective, and GLYB mutant with defective mt folate transport protein defective were used as experimental models to explore molecular mechanisms by which mt folate may have impact on mt biogenesis and function. CHO cells were treated with t-butylhyhroperoxide (t-BH) for 24, 48, and 72 hr. Intracellular reactive oxygen species, mt mass and mt membrane potential were measured by flow cytometry. Gene expressions of transcription factors for mt biogenesis, mt DNA (mtDNA) copy numbers and 4834bp large deletion (ΔmtDNA4.8) of mtDNA were assayed by real-time polymerase chain reaction. Levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured by HPLC complete with ECD. Folate levels were measured by L. cassia method. Cytosolic and mt folate of GLYB was significantly lower than those of CHOK1. Intracellular superoxide of GLYB was significantiy higher than those of CHOK1 and GLYA. After t-BH treatment, the cytosolic and mt folate levels of GLYB decreased by 44 and 28%, respectively. ROS in GLYB increases by 4 fold of it’s untreated-values. This markedly elevated oxidative stress in GLYB appeared to induce expressions of transcription factors for mt biogenesis including NRF-1, NRF-2, mtTFA and Pol γ at 24 hr-treat of t-BH, which accompanied increased mtDNA copy numbers and mt mass. In parallel, t-BH treatment for 24hr induced higher levels of 8-OHdG and ΔmtDNA4.8 accumulated in GLYB compared to those in CHOK1. Frequencies of point mutations in mtDNA encoded for COX I, II and III subunits of cytochrome c oxidase were increased. Ratio of t-BH-treated GLYB with depolarized mt membrane potential was increased by 1.7 fold. T-BH-treated GLYA showed the intermediate responses of mtDNA injuries, mt biogenesis and mt dysfunction compared to those of t-BH treated GLYB. Pre-incubation of mutants cells with folate supplements significant increase both cytosolic and mt folate, and reduced the t-BH-induced ROS. Folate supplementation could reduce in t-BH treated GLYA and GLYB levels of 8-OHdG, ΔmtDNA4.8 accumulateion and point mutation frequencies. The expressions of transcription factors for mt biogenesis by ROS induced were partially modulated by folate supplement, which accompanied reduced mtDNA copy number and mt mass. In summary, the mt folate had antioxidant capability to protect against oxidative stress-induced ROS generation, mtDNA damage, and mt membrane potential depolaritation, and enhanced mt biogenesis. Folate supplementation could offset mt deficiency as the result of defective mt folate metabolism and protect against t-BH-induced mtDNA damage and dysfunction.

目錄

中文摘要............................................................I
英文摘要..........................................................III
縮寫表..............................................................V
圖目錄............................................................XIV
表目錄............................................................XIX
第一章 前言..........................................................1
第二章 文獻回顧.......................................................2
一、氧化壓力對粒線體之影響...........................................2
(一) 粒線體為內生性氧化壓力來源.....................................2
(二) 氧化壓力對粒線體之傷害........................................3
1. 氧化壓力與呼吸酵素複體IV ...................................4
2. 氧化壓力與粒線體DNA突變.....................................7
(1) 大片段斷損的產生..........................................7
(2) 8-hydroxy 2’-deoxyguanosine的產生........................8
(3) 點突變的產生..............................................10
3. 氧化壓力與粒線體膜電位......................................10
(三) 氧化壓力啟動粒線體之生合成機制...............................11
二、葉酸.........................................................15
(一)、葉酸之生化代謝............................................15
(二)、葉酸主導細胞質與粒線體之單碳代謝............................16
(三)、葉酸缺乏誘發氧化壓力產生....................................17
(四)、葉酸缺乏對粒線體之影響.....................................19
(五)、粒線體葉酸代謝異常對細胞變異之影響...........................20
1. GLYA......................................................21
2. GLYB...............................................................22
三、研究目的.................................................................24
第三章 實驗材料與方法................................................25
一、實驗材料......................................................25
(一)、細胞株.................................................25
(二)、培養基與儀器設備.........................................25
二、實驗方法......................................................26
(一)、細胞陪養.................................................26
(二)、氧化劑配製...............................................26
(三)、細胞內ROS含量之測定......................................26
(四)、細胞粒線體膜電位之測定....................................28
(五)、細胞粒線體膜質量測定......................................28
(六)、DNA氧化傷害測定..........................................29
(七)、粒線體DNA之分析..........................................31
(八)、Reverse transcription-polymerase chain reaction (RT-PCR).....36
(九) 即時定量聚合酶連鎖反應 (real-time PCR)....................39
(十) 葉酸含量分析............................................44
(十一) 統計分析..............................................46
第四章 結果 I .....................................................47
氧化壓力敏感之粒線體葉酸代謝異常細胞株誘發粒線體葉酸缺乏、粒線體DNA損傷、粒線體生合成與功能性損傷
ㄧ、t-BH處理對CHOK1，GLYA與GLYB之細胞內葉酸濃度之影響....................................................................................................47
二、不同時間點t-BH誘發CHOK1、GLYA與GLYB細胞內過氧化氫與超氧陰離子含量之影響........................................................47
三、不同時間點t-BH處理對CHOK1、GLYA及GLYB之粒線體生合成轉錄因子之調節....................................................................49
四、不同時間點t-BH處理對CHOK1、GLYA及GLYB之粒線體增生之影響........................................................................................51
五、不同時間點t-BH處理對CHOK1、GLYA及GLYB之mtDNA大片段斷損之影響........................................................................53
六、t-BH處理對粒線體葉酸缺乏之GLYB細胞粒線體COX I部分基因突變性之影響........................................................................54
七、t-BH處理對粒線體葉酸缺乏之GLYB細胞粒線體COX II部分基因突變性之影響........................................................................55
八、t-BH處理對粒線體葉酸缺乏之GLYB細胞粒線體COX III部分基因突變性之影響....................................................................56
九、不同時間點t-BH處理對CHOK1、GLYA及GLYB之細胞核DNA轉錄之粒線體呼吸鏈酵素基因mRNA表現量之影響....56
十、不同時間點t-BH處理對CHOK1、GLYA及GLYB之粒線體COX次單元及部份粒線體基因轉錄mRNA表現量之影響....57
第五章 結果 II.......................................................................................59
補充葉酸對外因性氧化壓力誘發粒線體葉酸代謝異常細胞株粒線體氧化傷害之調節
ㄧ、葉酸補充與t-BH處理對CHOK1、GLYA及GLYB之細胞質與粒線體葉酸濃度之調節.............................................................59
二、葉酸補充與t-BH處理誘發CHOK1、GLYA及GLYB細胞內過氧化氫含量之影響.....................................................................60
三、葉酸補充與t-BH處理對CHOK1、GLYA及GLYB細胞內超氧陰離子含量之影響.....................................................................60
四、葉酸補充與t-BH處理對CHOK1、GLYA及GLYB之DNA氧化傷害指標8-OHdG含量之影響..............................................61
五、葉酸補充與t-BH處理對CHOK1、GLYA及GLYB之mtDNA大片段斷損之影響.....................................................................62
六、葉酸補充與t-BH處理對粒線體葉酸缺乏之GLYB細胞粒線體COX I部分基因突變性之影響..................................................63
七、葉酸補充與t-BH處理對粒線體葉酸缺乏之GLYB細胞粒線體COX II部分基因突變性之影響.................................................63
八、葉酸補充與t-BH處理對粒線體葉酸缺乏之GLYB細胞粒線體COX III部分基因突變性之影響...............................................64
九、葉酸補充與t-BH處理對CHOK1、GLYA及GLYB之mtDNA及細胞核DNA轉譯之次單元mRNA表現量之影響...............65
十、葉酸補充與t-BH處理對CHOK1、GLYA及GLYB之粒線體膜電位之影響.................................................................................68
十ㄧ、葉酸補充與其他抗氧化劑對t-BH處理之CHOK1、GLYA及GLYB之DNA氧化傷害指標8-OHdG含量之影響.............69
十二、葉酸補充與其他抗氧化劑對t-BH處理之CHOK1、GLYA及GLYB之粒線體功能性之影響..................................................70
第六章 結果 III......................................................................................72
葉酸之補充對外因性氧化壓力誘發粒線體生合成之影響
ㄧ、葉酸補充與t-BH處理對CHOK1、GLYA及GLYB細胞內粒線體生合成轉錄因子之調節............................................................72
二、葉酸補充與t-BH處理對CHOK1、GLYA及GLYB細胞粒線體增生之影響....................................................................................76
第七章 討論............................................................................................79
第八章 結論..........................................................95
第九章 總結..........................................................97
第十章 參考文獻.....................................................147
附錄...............................................................160

馬逸興 (1996) 具有A8344G突變粒線體DNA的cybrid在氧化性壓力下的反應之研究。國立陽明大學生物化學系碩士論文。

劉秀娟 (2002) 粒線體葉酸代謝異常對氧化劑誘發細胞程式凋亡的影響。輔仁大學食品營養學系碩士論文。

吳明芳 (2003) 受到過氧化氫對具有粒線體 DNA A8344G 突變的細胞質融合細胞中抗氧化酵素表現的影響。國立陽明大學生物化學系碩士論文。

詹雅淳 (2004) 粒線體葉酸代謝異常對細胞抗氧化酵素系統與程式凋亡粒線體訊息傳遞機制之影響。輔仁大學食品營養學系碩士論文。

張純敏 (2004) 葉酸營養狀態對大白鼠肝臟粒線體基因斷損，功能性及氧化傷害的影響。輔仁大學食品營養學系碩士論文。

陳淑貞 (2005) 粒線體葉酸代謝異常對氧化壓力與粒線體基因突變性及功能性之影響。輔仁大學食品營養學系碩士論文。

余朱青 (2006) 葉酸調節肝臟粒線體呼吸酵素複體IV功能性及其機制探討。輔仁大學食品營養學系碩士論文。

周宜芳 (2006)葉酸缺乏促進年輕大鼠組織老化指標粒線體DNA大片段斷損累積及其相關機制。輔仁大學食品營養學系碩士論文。

Anders D, Sanz N, Zaragoza A, Alvarez AM, Cascales M. Changes in antioxidant defence systems induced by cyclosporine A in cultures of hepatocytes from 2- and 12-month-old rats. Biochem Pharm. 2000;59:1091-1100.

Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJ, Staden R, Young IG. Sequence and organization of the human mitochondrial genome. Nature. 1981;290: 457-465.

Assanelli D, Bonanome A, Pezzini A, Albertini F, Maccalli P, Grassi M, Archetti S, Negrini R, Visioli F. Folic acid and vitamin E supplementation effects on homocysteinemia, endothelial function and plasma antioxidant capacity in young myocardial-infarction patients. Pharmacol Res. 2004;49:79-84.

Attardi G, Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988;4:289-333.

Baik S, Youn HS, Chung ME, Lee WK, Cho MJ, Ko GH, Park CK, Kasai H, Rhee KH. Increased oxidative DNA damage in Helicobacter pylori-infected human gastric mucosa. Cancer Research. 1996;56:1279-1282.

Barrientos A, Casademont J, Cardellach F, Estivill X, Urbano-Marquez A, Nunes V. Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging. Brain Res Mol Brain Res. 1997a;52:284-289.

Barrientos A, Casademont J, Cardellach F, Ardite E, Estivill X, Urbano-Marquez A, Fernandez-Checa JC, Nunes V. Qualitative and quantitative changes in skeletal muscle mtDNA and expression of mitochondrial-encoded genes in the human aging process. Biochem Mol Med. 1997b;62:165-171.

Beckman KB, Ames BN. Oxidative decay of DNA. J Biol Chem. 1997;272:19633-19636.

Bergeron R, Ren JM, Cadman KS, Moore IK, Perret P, Pypaert M, Young LH, Semenkovich CF, Shulman GI. Chronic activation of AMP kinase results in NRF-1 activation and mitochondrial biogenesis. Am J Physiol Endocrinol Metab. 2001;281:E1340-1346.

Branda RF, Brooks EM, Chen Z, Naud SJ, Nicklas JA. Dietary modulation of mitochondrial DNA deletions and copy number after chemotherapy in rats. Mutat Res. 2002;501:29-36.

Brookes PS, Baggott JE. Oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate by complex IV of rat mitochondria. Biochemistry. 2002;41:5633-5636.

Bohr VA, Dianov GL. Oxidative DNA damage processing in nuclear and mitochondrial DNA. Biochimie. 1999;81:155-160.

Chabi B, Adhihetty PJ, Ljubicic V, Hood DA. How is mitochondrial biogenesis affected in mitochondrial disease? Med Sci Sports Exerc. 2005;37:2102-2110.

Chasin LA, Feldman A, Konstam M, Urlaub G. Reversion of a Chinese hamster cell auxotrophic mutant. Proc Natl Acad Sci U S A. 1974;71:718-722.

Chern CL, Huang RF, Chen YH, Cheng JT, Liu TZ. Folate deficiency-induced oxidative stress and apoptosis are mediated via homocysteine-dependent overproduction of hydrogen peroxide and enhanced activation of NF-kappaB in human Hep G2 cells. Biomed Pharmacother. 2001;55:434-442.

Clayton DA. Replication and transcription of vertebrate mitochondrial DNA. Annu Rev Cell Biol. 1991;7:453-478.

Cook RJ, Blair JA. The distribution and chemical nature of radioactive folates in rat liver cells and rat liver mitochondria. Biochem J. 1979;178:651-659.

Cooper BA. Folate. Its metabolism and utilization. Clin Biochem. 1984;17:95-98.

Cooper JM, Mann VM, Schapira AH. Analyses of mitochondrial respiratory chain function and mitochondrial DNA deletion in human skeletal muscle: effect of ageing. J Neurol Sci. 1992;113:91-98.

Coskun PE, Beal MF, Wallace DC. Alzheimer's brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication. Proc Natl Acad Sci. 2004;101:10726-10731.

Cottrell DA, Blakely EL, Johnson MA, Ince PG, Turnbull DM. Mitochondrial enzyme-deficient hippocampal neurons and choroidal cells in AD. Neurology. 2001;57:260-264.

Cottrell DA, Blakely EL, Johnson MA, Ince PG, Borthwick GM, Turnbull DM. Cytochrome c oxidase deficient cells accumulate in the hippocampus and choroid plexus with age. Neurobiol Aging. 2001;22:265-272.

Corral-Debrinski M, Horton T, Lott MT, Shoffner JM, Beal MF, Wallace DC. Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age. Nat Genet. 1992;2:324-329.

Crott JW, Choi SW, Branda RF, Mason JB. Accumulation of mitochondrial DNA deletions is age, tissue and folate-dependent in rats. Mutat Res. 2005;570:63-70.

Devasagayam TPA, Steenken S, Obendrof MSW, Schulz WA, Sies H. Formation of 8-hydroxy (deoxy) guanine and generation of strsnd breaks at guranine residues in DNA by singlet oxygen. Biochemistry. 1991;30:6283-6289.

Dandona P, Thusu K, Cook S, Snyder B, Makowshi J, Armstrong D, Nicotera T. Oxidative damage to DNA in diabetes mellitus. The Lancet. 1996;347:444-445.

Di Simone N, Riccardi P, Maggiano N, Piacentani A, D'Asta M, Capelli A, Caruso A. Effect of folic acid on homocysteine-induced trophoblast apoptosis. Mol Hum Reprod. 2004;10:665-669.

Diaz F, Bayona-Bafaluy MP, Rana M, Mora M, Hao H and Moraes CT. Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control. Nucleic Acids Res. 2002;30:4626-4633.

Duan W, Ladenheim B, Cutler RG, Kruman II, Cadet JL, Mattson MP. Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease. J Neurochem. 2002;80:101-110.

Duthie SJ, Hawdon A. DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro. FASEB J. 1998;12:1491-1497.

Ekstrand MI, Falkenberg M, Rantanen A, Park CB, Gaspari M, Hultenby K, Rustin P, Gustafsson CM, Larsson NG. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum Mol Genet. 2004;13:935-944.

Endres M, Ahmadi M, Kruman I, Biniszkiewicz D, Meisel A, Gertz K. Folate deficiency increases postischemic brain injury. Stroke. 2005;36:321-325.

Farinati F, Cardin R, Degan P, Rugge M, Di Mario F, Bonvicini P, Naccarato R. Oxidative DNA damage accumulation in gastric carcinogenesis. Gastroenterology. 1998;42:351-356.

Fraga CG, Shigenaga MK, Park JW, Degan P, Ames BN. Oxidative damage to DNA during aging: 8-hydroxy-2’-deoxyguanosine in rat organ DNA and urine. Proceedings of the National Academy of Sciences of the UnitedStates of America. 1990;87:4533-4537.

Girgis S, Suh JR, Jolivet J, Stover PJ. 5-Formyltetrahydrofolate regulates homocysteine remethylation in human neuroblastoma. J Biol Chem. 1997;272:4729-4734.

Gregory III JF, Cuskelly GK, Shane B, Toch JP, Baumgartner TG, Stecpole P. Primed, constant infusion with [2H3] serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism. Am J Clin Nutr. 2000;72:1535-1541.

Grewal S, Morrison EE, Ponnambalam S, Walker JH. Nuclear localisation of cytosolic phospholipase A2-alpha in the EA.hy.926 human endothelial cell line is proliferation dependent and modulated by phosphorylation. J Cell Sci. 2002;115:4533-4543.

Gutsaeva DR, Suliman HB, Carraway MS, Demchenko IT, Piantadosi CA. Oxygen-induced mitochondrial biogenesis in the rat hippocampus. Neuroscience. 2006;137:493-504.

Harman D. The biologic clock: the mitochondria. J Am Geriatr Soc. 1972;20:145-147.

Hankey GJ, Eikelboom JW. Homocysteine and stroke. Curr Opin Neurol. 2001;14:95-102.

Heil SG, Van der Put NM, Waas ET, den Heijer M, Trijbels FJ, Blom HJ. Is mutated serine hydroxymethyltransferase (SHMT) involved in the etiology of neural tube defects? Mol Genet Metab. 2001;73:164-172.

Herbig K, Chiang EP, Lee LR, Hills J, Shane B, Stover PJ. Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses. J Biol Chem. 2002;277:38381-38389.

Ho PI, Ashline D, Dhitavat S, Ortiz D, Collins SC, Shea TB, Rogers E. Folate deprivation induces neurodegeneration: roles of oxidative stress and increased homocysteine. Neurobiol Dis. 2003;14:32-42.

Hoffbuhr KC, Davidson E, Filiano BA, Davidson M, Kennaway NG, King MP. A pathogenic 15-base pair deletion in mitochondrial DNA-encoded cytochrome c oxidase subunit III results in the absence of functional cytochrome c oxidase. J Biol Chem. 2000;275:13994-14003.

Hruszkewycz AM. Lipid peroxidation and mtDNA degeneration. A hypothesis. Mutat Res. 1992;275:243-248.

Hruszkewycz AM, Bergtold DS. Oxygen radicals, lipid peroxidation and DNA damage in mitochondria. Basic Life Sci. 1988;49:449-456.

Huang RFS. (1993) Interreaction between mitochondria and cytosolic folate-mediated one carbon metabolism. Ph. D. dissertation of the University of California at Berkely.

John LV, Walsh ML, Chen LB. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci USA. 1980;77:990-994.

Joshi R, Adhikari S, Patro BS, Chattopadhyay S, Mukherjee T. Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med. 2001;30:1390-1399.

Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2’-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutation Research. 1997;387:147-163.

Kang KW, Ryu JH, Kim SG. The essential role of phosphatidylinositol 3-kinase and of p38 mitogen-activated protein kinase activation in the antioxidant response element-mediated rGSTA2 induction by decreased glutathione in H4IIE hepatoma cells. Mol Pharmacol. 2000;58:1017-1025.

Kasai H, Nishimura S, Kurokawa Y, Hayashi Y. Oral administration of the renal carcinogen, potassium bromate, specifically produces 8-hydroxydeoxyguanosine in rat target organ DNA. Carcinogenesis. 1987;8:1959-1961.

Korczyn AD. Homocysteine, stroke, and dementia. Stroke. 2002;33:2343-2344.

Kowaltowski AJ, Vercesi AE. Mitochondrial damage induced by conditions of oxidative stress. Free Rad Biol Med. 1999;26:463–471.

Knott L, Hartridge T, Brown NL, Mansell JP, Sandy JR. Homocysteine oxidation and apoptosis: a potential cause of cleft palate. In Vitro Cell Dev Biol Anim. 2003;39:98-105.

Lee HC, Wei YH. Mitochondrial role in life and death of the cell. J Biomed Sci. 2000;7:2-15.

Leonard JV, Schapira AH. Mitochondrial respiratory chain disorders I: mitochondrial DNA defects. Lancet. 2000;355:299-304.

Li GM, Presnell SR, Gu L. Folate deficiency, mismatch repair -dependent apoptosis, and human disease. J Nutr Biochem. 2003;14:568-575.

Lin MT, Simon DK, Ahn CH, Kim LM, Beal MF. High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer's disease brain. Hum Mol Genet. 2002;15:133-145.

Linnane AW, Marzuki S, Ozawa T, Tanaka M. Mitochondrial DNA mutations as an important contributor to aging and degenerative diseases. Lancet. 1989;1:642-645.

Ljubicic V, Adhihetty PJ, Hood DA. Role of UCP3 in state 4 respiration during contractile activity-induced mitochondrial biogenesis. J Appl Physiol. 2004;97:976-983.

Maniura-Weber K, Goffart S, Garstka HL, Montoya J, Wiesner RJ. Transient overexpression of mitochondrial transcription factor A (TFAM) is sufficient to stimulate mitochondrial DNA transcription, but not sufficient to increase mtDNA copy number in cultured cells. Nucleic Acids Res. 2004;32:6015-6027.

Mattson MP, Shea TB. Folate and homocysteine metabolism in neural plasticity and neurodegenerative disorders. Trends Neurosci. 2003;26:137-146.

McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol. 1969;56:111-128.

Mirabella M, Giovanni SD, Silvestri G, Tonali P, Servidei S. Apoptosis in mitochondrial encephalomyopathies with mitochondrial DNA mutations: a potential pathogenic mechanism. Brain. 2000;123:93-104.

Miranda S, Foncea R, Guerrero J, Leighton F. Oxidative stress and upregulation of mitochondrial biogenesis genes in mitochondrial DNA-depleted HeLa cells. Biochem Biophys Res Commun. 1999;258:44-49.

Mohamed SA, Wesch D, Blumenthal A, Bruse P, Windler K, Ernst M, Kabelitz D, Oehmichen M, Meissner C. Detection of the 4977 bp deletion of mitochondrial DNA in different human blood cells. Experi. Gerontol. 2004;39:181-188.

Narkewicz MR, Sauls SD, Tjoa SS, Teng C, Fennessey PV. Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells. Biochem J. 1996;313:991-996.

Nisoli E, Clementi E, Paolucci C, Cozzi V, Tonello C, Sciorati C, Bracale R, Valerio A, Francolini M, Moncada S, Carruba MO. Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide. Science. 2003;299:896-899.

Perez-de-Arce K, Foncea R, Leighton F. Reactive oxygen species mediates homocysteine-induced mitochondrial biogenesis in human endothelial cells: modulation by antioxidants. Biochem Biophys Res Commun. 2005;338:1103-1109.

Pfendner W, Pizer LI. The metabolism of serine and glycine in mutant lines of Chinese hamster ovary cells. Arch Biochem Biophys. 1980;200:503-512.

Piantadosi CA, Suliman HB. Mitochondrial transcription factor A induction by redox activation of nuclear respiratory factor 1. J Biol Chem. 2006;281:324-333.

Porteous WK, James AM, Sheard PW, Porteous CM, Packer MA, Hyslop SJ, Melton JV, Pang CY, Wei YH, Murphy MP. Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion. Eur J Biochem. 1998;257:192-201.

Racek J, Rusnakova H, Trefil L, Siala KK. The influence of folate and antioxidants on homocysteine levels and oxidative stress in patients with hyperlipidemia and hyperhomocysteinemia. Physiol Res. 2005;54:87-95.

Rezk BM, Haenen GR, van der Vijgh WJ, Bast A. Tetrahydrofolate and 5-methyltetrahydrofolate are folates with high antioxidant activity. Identification of the antioxidant pharmacophore. FEBS Lett. 2003;555:601-605.

Ricci JE, Gottlieb RA Green DR. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J Cell Biol. 2003;160:65-75.

Richter C, Park JW, Ames BN. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Natl Acad Sci U S A. 1988;85:6465-6467.

Richter C, Gogvadze V, Laffranchi R, Schlapbach R, Schweizer M, Suter M, Walter P, Yaffee M. Oxidants in mitochondria: from physiology to diseases. Biochim Biophys Acta. 1995;1271:67-74.

Salet C, Moreno G. Photodynamic action increases leakage of the mitochondrial electron transport chain. Int J Radiat Biol. 1995;67:477-480.

Scarpulla RC. Nuclear activators and coactivators in mammalian mitochondrial biogenesis. Biochim Biophys Acta. 2002;1576:1-14.

Scarpulla RC. Nuclear control of respiratory gene expression in mammalian cells. J Cell Biochem. 2006;97:673-683.

Sciacco M, Bonilla E, Schon EA, DiMauro S, Moraes CT. Distribution of wild-type and common deletion forms of mtDNA in normal and respiration-deficient muscle fibers from patients with mitochondrial myopathy. Hum Mol Genet. 1994;3:13-19.

Senior AE. ATP synthesis by oxidative phosphorylation. Physiol Rev. 1988;68:177-231.

Shigenaga MK, Gimeno CJ, Ames BN. Urinary 8-hydroxy-2’ -deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proceedings of the National Academy of Sciences of the United States of America. 1989;86:9697-9701.

Shibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation damaged base 8-oxodG. Nature. 1991;349:431-434.

Shigenaga MK, Ames BN. Assays for 8-hydroxy-2’- deoxyguanosine: as a biological marker of in vivo oxidative DNA damage. Free Radical Biology and Medicine. 1991;10:202-226.

Shoffner JM 4th, Wallace DC. Oxidative phosphorylation diseases. Disorders of two genomes. Adv Hum Genet. 1990;19:267-330.

Skibola CF, Smith MT, Hubbard A, Shane B, Roberts AC, Law GR, Rollinson S, Roman E, Cartwright RA, Morgan GJ. Polymorphisms in the thymidylate synthase and serine hydroxymethyltransferase genes and risk of adult acute lymphocytic leukemia. Blood. 2002;99:3786-3791.

Stanger O, Weger M. Interactions of homocysteine, nitric oxide, folate and radicals in the progressively damaged endothelium. Clin Chem Lab Med. 2003;41:1444-1454.

Stover P, Schirch V. 5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase. J Biol Chem. 1991;266:1543-1550.

Suliman HB, Carraway MS, Welty-Wolf KE, Whorton AR, Piantadosi CA.　 Lipopolysaccharide stimulates mitochondrial biogenesis via activation of nuclear respiratory factor-1. J Biol Chem. 2003;278:41510-41518.

Taanman JW. The mitochondrial genome: structure, transcription, translation and replication. Biochim Biophys Acta. 1999;1410:103-123.

Taylor RT, Hanna ML. Folate-dependent enzymes in cultured Chinese hamster ovary cells: impaired mitochondrial serine hydroxymethyltransferase activity in two additional glycine--auxotroph complementation classes. Arch Biochem Biophys. 1982;217:609-623.

Thomas AH, Suarez G, Cabrerizo FM, Martino R, Capparelli AL. Study of the photolysis of folic acid and 6-formylpterin in acid aqueous solutions. J Photochem Photobiol. 2000;135:147-154.

Titus SA, Moran RG. Retrovirally mediated complementation of the glyB phenotype. Cloning of a human gene encoding the carrier for entry of folates into mitochondria. J Biol Chem. 2000;275:36811-36817.

Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J. 1980;191:421-427.

Twiddy D, Brown DG, Adrain C, Jukes R, Martin SJ, Cohen GM, MacFarlane M, Cain K. Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex. J Biol Chem. 2004;279:19665-19682.

Wallace DC, Ye J, Necklemann SN, Singh G, Webster KA, Greenberg BD. Sequence analysis of cDNAs for the human and bovine ATP synthase beta subunit: mitochondrial DNA genes sustain seventeen times more mutations. Curr Genet. 1987;12:81-90.

Wallace DC. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science. 1992;256:628-632.

Wang H, Fliegel L, Cass CE, Penn AM, Michalak M, Weiner JH, Lemire BD. Quantification of mitochondrial DNA in heteroplasmic fibroblasts with competitive PCR. Biotechniques. 1994;17:76 - 78, 80, 82.

Wang YM, Guo W, Zhang XF, Li Y, Wang N, Ge H, Wei LZ, Wen DG, Zhang JH. Correlations between serine hydroxymethyltransferase1 C1420T polymorphisms and susceptibilities to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma. Ai Zheng. 2006;25:281-286.

Watherhouse NJ. The cellular energy crisis: mitochondria and cell death. Med Sci Sports Exerc. 2002;35:105-110.

Wei YH, Lee HC Oxidative stress, mitochondrial DNA mutation, and impairment of antioxidant enzymes in aging. Exp Biol Med. 2002;227:671-682.

Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Scarpulla RC, Spiegelman BM. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell. 1999;98:115-124.

Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci U S A. 1997;94:514-549.

Yoshida T, Azuma H, Aihara K, Fujimura M, Akaike M, Mitsui T, Matsumoto T. Vascular smooth muscle cell proliferation is dependent upon upregulation of mitochondrial transcription factor A (mtTFA) expression in injured rat carotid artery. Atherosclerosis. 2005;178:39-47.

Zhang J, Block ER, Patel JM. Down-regulation of mitochondrial cytochrome c oxidase in senescent porcine pulmonary artery endothelial cells. Mech Ageing and Dev. 2002;123:1363-1374.