Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress

Abstract

A significant amount of reactive oxygen species (ROS) is generated during mitochondrial oxidative phosphorylation. Several studies have suggested that mtDNA may accumulate more oxidative DNA damage relative to nuclear DNA. This study used quantitative PCR to examine the formation and repair of hydrogen peroxide-induced DNA damage in a 16.2-kb mitochondrial fragment and a 17.7-kb fragment f lanking the ␤-globin gene. Simian virus 40-transformed fibroblasts treated with 200 M hydrogen peroxide for 15 or 60 min exhibited 3-fold more damage to the mitochondrial genome compared with the nuclear fragment. Following a 60-min treatment, damage to the nuclear fragment was completely repaired within 1.5 hr, whereas no DNA repair in the mitochondrion was observed. Mitochondrial function, as assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, also showed a sharp decline. These cells displayed arrested-cell growth, large increases in p21 protein levels, and morphological changes consistent with apoptosis. In contrast, when hydrogen peroxide treatments were limited to 15 min, mtDNA damage was repaired with similar kinetics as the nuclear fragment, mitochondrial function was restored, and cells resumed division within 12 hr. These results indicate that mtDNA is a critical cellular target for ROS. A model is presented in which chronic ROS exposure, found in several degenerative diseases associated with aging, leads to decreased mitochondrial function, increased mitochondrial-generated ROS, and persistent mitochondrial DNA damage. Thus persistent mitochondrial DNA damage may serve as a useful biomarker for ROSassociated diseases.

Increased salivary level of 8-hydroxydeoxyguanosine is a marker of premature oxidative mitochondrial DNA damage in gingival tissue of patients with periodontitis

“…Human mtDNA is naked and is located in the vicinity of mitochondrial inner membranes, where abundant ROS and free radicals are continually generated. It is not protected by histones and replicates faster than nuclear DNA, without proofreading or an efficient DNA repair systems (Yakes and van Houten 1997). In the last decade a number of pathogenic mutations of mtDNA, such as large-scale deletions and point mutations, have been established as responsible for or associated with several distinct human diseases (Wallace et al 1995).…”

Reduced Flavins Promote Oxidative DNA Damage in Non-respiringEscherichia coli by Delivering Electrons to Intracellular Free Iron

“…Measurement of Polymerase-blocking DNA Lesions-DNA damage can be quantified by the ability of DNA lesions to block PCR amplification of the DNA template (35). Cells were grown to A 600 ϭ 0.1-0.2, and they were then treated with H 2 O 2 and/or KCN as described above.…”

Chronic inflammation and oxidative stress in human carcinogenesis

“…42 Fragments of mitochondrial DNA have been found to be inserted into nuclear DNA, suggesting a possible mechanism of oncogene activation. Mitochondrial DNA (mtDNA), which is bound to the inner mitochondria membrane, is particularly liable to oxidative damage because of the lack of histone proteins, the presence of incomplete repairing mechanisms [53][54][55] and its proximity to the mitochondrial electron transport chain. Damage to mtDNA causes mitochondrial respiratory chain dysfunction, which in turn increases the production of hydroxyl radicals which are the major source of oxidative damage for DNA.…”