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Updated: Oct 16, 2026
Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals
Published on: May 19, 2023
Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells
1Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei, Taiwan 112, Republic of China.
Abstract:
Mitochondrial respiratory function is impaired in the target tissues of patients with mitochondrial diseases and declines with age in various human tissues. It is generally accepted that respiratory-chain defects result in enhanced production of reactive oxygen species and free radicals in mitochondria. Recently, we have demonstrated that the copy number of mitochondrial DNA (mtDNA) is increased in the lung tissues of elderly human subjects. The mtDNA copy number was suggested to be increased by a feedback mechanism that compensates for defects in mitochondria harbouring mutated mtDNA and a defective respiratory system. However, the detailed mechanism remains unclear. In this study, we treated a human lung fibroblast cell line, MRC-5, with H(2)O(2) at concentrations of 90-360 microM. After the treatment for 24-72 h, we found that cells were arrested at G(0) and G(1) phases but that mitochondrial mass and mtDNA content were significantly increased in a concentration- and time-dependent manner. Moreover, the oxidative stress induced by buthionine sulphoximine was also found to cause an increase in mitochondrial mass of the treated cells. Increased uptake of a vital mitochondrial dye Rhodamine 123 and enhanced tetrazolium [MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] reduction revealed that the mitochondria increased by H(2)O(2) treatment were functional. In addition, the increase in the mitochondrial mass was also observed in cell-cycle-arrested cells induced by mimosine, lovastatin and genistein. Taken together, these findings suggest that the increase in mitochondrial mass and mtDNA content are the early molecular events of human cells in response to endogenous or exogenous oxidative stress through cell-cycle arrest.
Insights
Oxidative stress triggers an increase in mitochondrial mass and mitochondrial DNA (mtDNA) content in human cells. This response occurs alongside cell-cycle arrest, suggesting a protective mechanism against cellular damage.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Oxidative Stress Research
Background:
- Mitochondrial dysfunction and impaired respiratory function are hallmarks of aging and mitochondrial diseases.
- Increased reactive oxygen species (ROS) production is linked to respiratory chain defects.
- Previous studies indicated increased mitochondrial DNA (mtDNA) copy number in aged lung tissue, possibly as a compensatory mechanism.
Purpose of the Study:
- To investigate the cellular response to oxidative stress, specifically focusing on changes in mitochondrial mass and mtDNA content.
- To elucidate the mechanism behind increased mtDNA copy number in response to cellular damage.
- To determine if functional mitochondria increase under oxidative stress conditions.
Main Methods:
- Treatment of human lung fibroblast cell line (MRC-5) with hydrogen peroxide (H2O2) and buthionine sulfoximine to induce oxidative stress.
- Cell cycle analysis to assess cell cycle arrest at G0 and G1 phases.
- Quantification of mitochondrial mass and mtDNA content.
- Assessment of mitochondrial function using Rhodamine 123 uptake and MTT reduction assays.
- Induction of cell cycle arrest using mimosine, lovastatin, and genistein to observe mitochondrial changes.
Main Results:
- Hydrogen peroxide treatment led to significant, dose- and time-dependent increases in mitochondrial mass and mtDNA content.
- Cells treated with H2O2 exhibited arrest at the G0 and G1 phases of the cell cycle.
- Oxidative stress induced by buthionine sulfoximine also resulted in increased mitochondrial mass.
- The increased mitochondria were functional, as evidenced by enhanced Rhodamine 123 uptake and MTT reduction.
- Cell cycle arrest induced by various agents (mimosine, lovastatin, genistein) also correlated with increased mitochondrial mass.
Conclusions:
- Increased mitochondrial mass and mtDNA content are early molecular events in human cells responding to oxidative stress.
- Cell cycle arrest appears to be a key component of the cellular response to oxidative stress, potentially involving mitochondrial biogenesis.
- These findings provide insights into cellular adaptation mechanisms under conditions of oxidative damage.
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