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Mitochondrial GPx1 decreases induced but not basal oxidative damage to mtDNA in T47D cells
J Legault1, C Carrier, P Petrov
1Unit of Health and Environment, CHUL Research Center and Laval University, Québec, Canada.
Abstract:
The production of oxyradicals by mitochondria (mt) is a source of oxidative damage to mtDNA such as 8-oxo-dG lesions that may lead to mutations and mitochondrial dysfunction. The potential protection of mtDNA by glutathione peroxidase-1 (GPx1) was investigated in GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants. GPx activity and GPx1-like antigen concentration in mitochondria were respectively at least 100-fold and 20- to 25-fold higher in GPx2 than Hygro-3 cells. In spite of this large difference in peroxide-scavenging capacity, the basal 8-oxo-dG frequency in mtDNA, assessed by carefully controlled postlabeling assay, was strikingly similar in both cell lines. In contrast, in response to menadione-mediated oxidative stress, induction of 8-oxo-dG and DNA strand breaks was much lower in the GPx1-proficient mitochondria (e.g., +14% 8-oxo-dG versus +54% in Hygro-3 after 1-h exposure to 25 microM menadione, P < 0.05). Our data indicate that the mitochondrial glutathione/GPx1 system protected mtDNA against damage induced by oxidative stress, but did not prevent basal oxidative damage to mtDNA, which, surprisingly, appeared independent of GPx1 status in the T47D model.
Insights
Mitochondrial glutathione peroxidase-1 (GPx1) protects mitochondrial DNA (mtDNA) from oxidative stress but does not prevent basal mtDNA damage. This suggests GPx1 is crucial for mitigating induced oxidative damage in mitochondria.
Area of Science:
- Mitochondrial biology
- Oxidative stress research
- DNA damage and repair
Background:
- Mitochondria produce oxyradicals, causing oxidative damage to mitochondrial DNA (mtDNA), leading to mutations and dysfunction.
- Glutathione peroxidase-1 (GPx1) is a key antioxidant enzyme potentially protecting mtDNA from oxidative damage.
Purpose of the Study:
- To investigate the protective role of GPx1 against oxidative damage in human breast T47D cell mitochondria.
- To compare mtDNA damage in GPx1-proficient and GPx1-deficient cells under basal and induced oxidative stress conditions.
Main Methods:
- Utilized GPx1-proficient (GPx-2) and GPx1-deficient (Hygro-3) human breast T47D cell transfectants.
- Quantified GPx activity and GPx1 antigen concentration in mitochondria.
- Assessed 8-oxo-dG frequency in mtDNA using a postlabeling assay.
- Induced oxidative stress using menadione.
Main Results:
- Mitochondria in GPx-2 cells showed significantly higher GPx activity and GPx1 levels than Hygro-3 cells.
- Basal 8-oxo-dG frequency in mtDNA was similar in both cell lines, irrespective of GPx1 status.
- Menadione-induced oxidative stress resulted in significantly lower 8-oxo-dG and DNA strand breaks in GPx1-proficient mitochondria compared to deficient ones.
Conclusions:
- The mitochondrial glutathione/GPx1 system effectively protects mtDNA against induced oxidative stress.
- Basal oxidative damage to mtDNA appears independent of GPx1 status in the T47D cell model.
- GPx1 plays a critical role in mitigating, but not preventing, oxidative damage to mtDNA.