Quantitation of the oxidatively induced DNA lesion 8-oxo-dG, and its repair, in mammalian mitochondrial DNA

Mitochondrial DNA (mtDNA) has been reported to contain high levels of oxidative DNA damage, which increase with age. Using photoactivated methylene blue, 8-oxo-dG was induced both in DNA in vitro and in cultured cells. This technique was used, first, to provide a substrate for use in establishing quantitative conditions for the enzymatic/Southern blot assay. This assay is used to determine the level of 8-oxo-dG in a DNA sample, using the bacterial repair enzyme, formamidopyrimidine DNA glycosylase (Fpg). Enzyme:substrate ratios and digestion times were optimized so that specific cutting reached completion and non-specific cutting was minimized. Second, the relative role of repair and degradation in the removal of 8-oxo-dG from mtDNA was studied. Removal of damage induced in cultured (WI38) fetal lung fibroblasts was determined to be due to true repair rather than replication, cell loss, or degradation of damaged genomes. Repair was similar in both DNA strands of the frequently transcribed ribosomal region of the mitochondrial genome and in both strands of the non-ribosomal region. Repair does not correlate with the expected rate of transcription. Third, the reported age related increase in 8-oxo-dG in mtDNA could be due to an age related decrease in mtDNA repair capacity. Oxidative damage was induced in cultured skin fibroblasts from young and old human donors. In these cells, damage removal was accompanied by mtDNA degradation. Neither process was correlated with age or with the cell's pre-treatment replicative capacity. Fourth, the levels of ten different oxidatively induced lesions were analyzed by gas chromatography/mass spectrometry (GC/MS) in both mitochondrial and nuclear rat liver DNA as a function of age. No age effects were observed, and contrary to previous reports using the same technique, nuclear levels of damage were slightly higher than mitochondrial levels. Finally, quantitation of 8-oxo-dG in DNA using GC/MS, high performance liquid chromatography with electrochemical detection (HPLC/ECD), and the Fpg/Southern blot assay was compared. HPLC/ECD and the Fpg/Southern blot assay were equivalent at physiological levels of damage. Using the Fpg/Southern blot assay, damage was found to be higher in mtDNA than nuclear DNA only when the mitochondria were isolated prior to DNA purification.