Association of Imbalance of Free Radical Scavenging Enzymes with Mitochondrial DNA Mutations in Fibroblasts of Elderly Subjects and Patients with CPEO Syndrome

• Main Authors: Ching-You Lu, 盧青佑
• Other Authors: Yau-Huei Wei
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/28323173505249952059

博士 === 國立陽明大學 === 生物化學研究所 === 87 === The cellular respiratory functions decline in aging human tissues and affected tissues of patients with mitochondrial diseases. This is accompanied by an increase of reactive oxygen species (ROS) production in mitochondria via the increased electron leak of the respiratory chain. As a result, an ever-increasing amount of ROS may escape from the defense systems and cause oxidative damage to biomolecules. To investigate the relationship between free radical scavenging enzymes and oxidative damage, a total of 76 normal subjects of different ages were recruited in this study. I found an age-dependent increase of 8-OHdG content and proportion of 4,977 bp mitochondrial DNA (mtDNA) deletion in skin tissues from subjects above the age of sixty years. Moreover, I also found an age-dependent increase of lipid peroxides in cultured fibroblasts of the subjects above the age of sixty years. To evaluate the antioxidant defense system from individuals of different ages, I measured the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione peroxidase (GPx). The activities of Cu,Zn-SOD, CAT and GPx were found to decrease with age, and the activity of Mn-SOD was increased with age before sixty years but was decreased thereafter. This indicated that elevated oxidative stress caused by an imbalance between the production and removal of ROS occurred in skin fibroblasts after sixty years of age. Taken together, I suggest that the functional decline of free radical scavenging enzymes and the elevation of oxidative stress may play an important role in eliciting oxidative damage and mutation of mtDNA during the human aging process. On the other hand, I investigated the alterations of age-dependent mitochondrial gene expression associated with mtDNA deletions. Total RNAs were isolated and analyzed by Northern hybridization. There were no significant differences of the mtRNAs among the skin fibroblasts from subjects of different ages. Moreover, I measured the activities of respiratory enzymes NCCR, SCCR and CCO in fibroblasts of subjects of different ages. There was an age-dependent decrease in the activities of the three enzyme complexes. More interestingly, I found a 75-year-old woman who had no mitochondrial disease but harbored 60% of the 4,977 bp deleted mtDNA in the fibroblasts established from her face skin. This deletion caused dramatic alteration in the mRNA levels and the activities of respiratory enzymes of mitochondria. In the second part of this study, skin and muscle fibroblasts were cultured from nine patients with chronic progressive external ophthalmoplegia (CPEO) syndrome for studying the relationship between mtDNA mutation and free radical scavenging enzymes. The skin and muscle fibroblasts from these CPEO patients carried different mtDNA mutations at different levels. The fibroblasts from patients had the common 4,977 bp mtDNA deletion, a 4,366 bp mtDNA, a 6,190 bp mtDNA deletion, a ~10 kb mtDNA deletion and the A3243G mutation in mitochondrial tRNALeu(UUR) gene. To examine whether these mtDNA mutations are associated with a defect or imbalance in the free radical scavenging enzymes. I assayed the enzyme activities of Mn-SOD, catalase and GPx of the fibroblasts. I found that the skin and muscle fibroblasts from the nine CPEO patients all had higher enzyme activity and mRNA level of Mn-SOD but those of catalase and GPx were not increased or even decreased. These results indicated an imbalance between the H2O2 generation and removal systems in the fibroblasts of the patients with CPEO syndrome. The ratios of activities of Mn-SOD/catalase in muscle fibroblasts (between 3.39 and 8.36) were about 1.4 to 3.5 fold higher than those of the control subjects. The ratios in skin fibroblasts of patients CPEO4, CPEO6, and CPEO8 were also significantly different from those of the control subjects. But the imbalance was much more pronounced in muscle fibroblasts. This tissue-specific imbalance of free radical scavengers was confirmed by analysis of the corresponding mRNA levels. The average mRNA ratio of Mn-SOD/catalase in fibroblasts of the nine CPEO patients was about 1.21 and that was 0.44 for control skin fibroblasts. By contrast, the average Mn-SOD/catalase mRNA ratio was 1.58-6.29 in muscle fibroblasts of these patients, which was much higher than those of the control fibroblasts (0.79). These results are consistent with the clinical observation that muscle is the main affected tissue of patients with CPEO syndrome. I suggest that the imbalance between free radical scavenging enzymes play an important role in the pathogenesis and age-dependent progression of the CPEO syndrome.