Mitochondrial DNA damage as a biomarker of oxidative stress

One of the most ubiquitous DNA damaging agents is oxidative stress, an endogenous DNA damaging agent generated by normal cellular respiration. During the formation of ATP, electrons flow down the electron chain complexes, which generate a proton-gradient that is harvested by complex V, ATPase synthase. The final acceptor of electrons is molecular oxygen which is reduced in a four-electron addition to water. However reactive oxygen products of one- and two-electron additions, superoxide and hydrogen peroxide, respectively, are generated in the mitochondria. It has been estimated that as much as 1-2% of all the oxygen we consume is released as reactive oxygen species.

Interestingly, we found that hydrogen peroxide induced three times more damage to mitochondrial DNA (mtDNA) than nuclear targets in human fibroblasts.9 This damage was repaired within 1.5 h after a 15-min treatment with 200 mM hydrogen peroxide. After 3 h of repair, amplification of both genomes increased over the control levels. This unexpected result probably represents an induction of DNA repair enzymes by the oxidative stress such that their action reduces DNA damage to levels lower than the basal levels.1011 Strangely, a 60-min treatment with 200 mM hydrogen peroxide lead to persistent mitochondrial DNA damage even though the nuclear damage was repaired within 1.5 h (Figure 11.3). What is the basis of this persistent mtDNA damage? For a long time, it was believed that mitochondria lack efficient repair, but work in many laboratories12 during the last decade have clearly demonstrated that mitochondria have some but not all the repair pathways associated with nucleus. For example, mitochondria are deficient in the removal of UV-induced pyrimidine dimers or cisplatin adducts, lesions which are repaired by NER (mentioned above). However, base excision repair enzymes, which are primarily responsible for the removal of oxidative lesions, have been isolated from mitochondria.12 Glu-

Mitochondrial Gene Panel

Figure 11.2 Repair kinetics of UV-induced damage in human cells. SV-40 transformed fibroblasts (Panel A) or human lymphoblastoid cells (Panel B) were treated with 10 J/m2 of UVC and allowed various periods of time for repair. The DNA was extracted and subjected to quantitative PCR. Repair was examined in two gene regions, nonexpressed beta-globin region (closed symbols) and the expressed HPRT gene (open symbols). (From Van Houten, B., Cheng, S., and Chen, Y., Mut. Res., 460, 81-94, 2000. With permission.)

Figure 11.2 Repair kinetics of UV-induced damage in human cells. SV-40 transformed fibroblasts (Panel A) or human lymphoblastoid cells (Panel B) were treated with 10 J/m2 of UVC and allowed various periods of time for repair. The DNA was extracted and subjected to quantitative PCR. Repair was examined in two gene regions, nonexpressed beta-globin region (closed symbols) and the expressed HPRT gene (open symbols). (From Van Houten, B., Cheng, S., and Chen, Y., Mut. Res., 460, 81-94, 2000. With permission.)

cose oxidase (GO), a steady generator of more physiological concentrations of hydrogen peroxide, resulted in large amounts of mtDNA damage with little or no nuclear damage.13 Human fibroblasts treated with 6 milliunits GO/ml of media generated about 10 mM hydrogen peroxide during a 15-min treatment and produced approximately 1.0 lesion/mitochondrial genome. This mtDNA damage was repaired within 4 h after the treatment.13 This finding of increased mtDNA damage after an oxidant has been repeated in a large number of cell types from both human and rodent cells.14-16 It is nuclear genome mitochondrial genome n

GDNE

H202

Figure 11.3 Oxidative damage to mitochondrial and nuclear DNA. Yeast wild type strain ale 1000 (1x107 cells/mL) were inoculated into YPD broth. Eight hours after inoculation, cells were harvested and resuspended in PBS. The cells were treated for 30 min with either 1mM of 1-chloro-2,4dinitrobenzene (CDNB) or 20 mM of hydrogen peroxide alone, or a combined exposure in which CDNB was administered first followed by 30 min to H2O2. Lesions (per 10 Kb), observed in both nuclear and mitochondrial genomes, are plotted as a function of treatments (data of mitochondria normalized for mitochondria copy number).

interesting to note that the antiapoptotic protein, Bcl-2, while not capable of blocking ROS-induced mitochondrial damage, lead to increased rates of mtDNA repair.15

Even yeast cells suffer significantly more mtDNA damage than nuclear damage following oxidant stress.7 Oxidative stress was induced in yeast by treatment with hydrogen peroxide or of 1-chloro-2,4dinitrobenzene (CNDB), a glutathione reactive compound. CNDB leads to the loss of glutathione, an important co-factor of glutathione peroxidase, the primary hydrogen peroxide detoxifying enzyme in mitochondria. It is interesting to note that depletion of glutathione leads to an increase damage in mtDNA as compared to a nuclear gene. Hydrogen peroxide produced approximately three times more damage in the mitochondrial DNA than a nuclear gene. Furthermore combination of CNDB and hydrogen peroxide leads to synergistic amounts of damage in both genomes.

These observations have lead to two working hypotheses: increased divalent metal ions in mitochondria produce more mtDNA damage and damaged mitochondria can generate ROS and lead to a vicious cycle of damage, which even in the presence of an active DNA repair system results in persistent mtDNA damage. These hypotheses provide a paradigm for the molecular aspects of many chronic diseases associated with aging, such as Alzheimer's disease and Parkinson's disease discussed in more detail below.

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