The role of mitochondrial folate metabolism to protect against oxidant stress-induced mitochondrial DNA injuries, enhanced mitochondrial biogenesis and mitochondrial dysfunction

• Main Authors: Ye iling, 葉宜玲
• Other Authors: Rwei-Fen S. Huang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/00245278057855886134

碩士 === 輔仁大學 === 營養科學系 === 95 === 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.