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 flanking the beta-globin gene. Simian virus 40-transformed fibroblasts treated with 200 microM 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 ROS-associated diseases.

Figures

Figure 1

Kinetics of H2O2 depletion from the culture medium. SV40-transformed fibroblast monolayers (1 × 106 cells) were incubated at 37°C in 3 ml of serum-free MEM supplemented with either 50 μM (▴), 100 μM (▪), or 200 μM (•) H2O2. The H2O2 (200 μM) content in the medium in the absence of cells is represented by (▾). Medium aliquots were removed and the amount of H2O2 remaining was determined by spectrophotometric analysis and a standard curve of known H2O2 concentrations. Data are expressed as the mean ± SEM (n = 2 cell experiments in duplicate for each time point; error bars were omitted because the SEM were <1%). First-order rate constants of H2O2 depletion were 0.067 min−1 (▴), 0.047 min−1 (▪), and 0.045 min−1 (•) for 50, 100, and 200 μM, respectively.

Figure 2

H2O2 dose response in human fibroblast cells. Human fibroblast cells were exposed to increasing concentrations of H2O2, in duplicate plates for each dose, for 1 hr at 37°C, and total cellular DNA was isolated. Control cultures were incubated in serum-free medium alone. (A) Representative autoradiogram depicting the decrease in amplification of the 17.7-kb β-globin fragment (Upper) and the 16.2-kb mitochondrial fragment (Lower). (B) The amount of radioactivity associated with each amplification product relative to the nondamaged controls was determined by PhosphorImager (Molecular Dynamics) analysis and is plotted as a function of H2O2 concentration: (▪), 16.2-kb mitochondrial fragment; (□), 17.7-kb β-globin fragment. (C) The decrease in relative amplification from B was then converted to lesion frequency using the Poisson equation as described. The data are expressed as the mean ± SEM from a minimum of two biological experiments in which 3–5 PCRs were performed per experiment. Statistically significant differences in the lesion frequency for both fragments at each dose were calculated using the unpaired Student’s t test (100 μM, P = 0.05; 200 μM, P = 0.004; 400 μM, P = 0.0001). (D) Mitochondrial function assayed by MTT reduction. Fibroblast cells were plated in 60-mm dishes and exposed to increasing concentrations of H2O2 for 60 min at 37°C. Following H2O2 exposure, the cells were rinsed with PBS and incubated for 60 min with conditioned medium containing 2.0 μg/ml MTT. Afterward the medium was removed, the cells lysed, and the absorbance measured at 570 nm (•). MTT reduction was determined with a standard curve and normalized to nontreated controls and is reported as a fraction of control. The data are expressed as the mean ± SD for triplicate plates (error bars were omitted because the SD were <1%).

Figure 3

DNA repair activity in mtDNA and nuclear DNA. Fibroblast cells were exposed to 200 μM H2O2 for either 15 (A) or 60 (B) min as described in Fig. 2 and either harvested immediately or allowed to recover in conditioned medium for the indicated times. QPCR was performed for both the mitochondrial (open bars) and β-globin fragments (hatched bars). The data are expressed as the mean ± SD (n = 3).

Figure 4

Effect of H2O2 on mitochondrial function and cell growth. Fibroblast cells were treated with 200 μM H2O2 for either 15 or 60 min as described in Fig. 2 and then allowed to recover in conditioned medium for the indicated times. (A) Mitochondrial function for control cells (open bars) or cells treated with H2O2 for 15 min (hatched bars) or 60 min (filled bars) was determined after incubating the cells with 2.0 μg/ml MTT for 60 min at 37°C during the final hour of the indicated recovery time. The data are expressed as the mean ± SEM (n = 2 biological experiments, where each point was performed in duplicate) relative to nontreated controls at time zero. (B) The total number of cells was determined, following a 15-min (▪) or 60-min (▴) exposure to H2O2, by manual counting at 12, 24, and 36 hr posttreatment, and treated cells were compared with nontreated cells (•). These measurements were performed simultaneously with the MTT assay (A). The data are expressed as the mean ± SEM (n = 2 biological experiments). (C) Increased expression of WAF1/CIP1 in response to H2O2. WAF1/CIP1 levels were analyzed in control cells (lanes a, d, and g), and cells were exposed to 200 μM H2O2 for either 15 min (lanes b, e, and h) or 60 min (lanes c, f, and i) at 12 hr (lanes a–c), 24 h (lanes d–f), and 36 h (lanes g–i) of recovery.

Figure 5

Schematic representation of the role of mitochondria in the generation of ROS and oxidative stress. The mitochondrion is responsible for oxidative phosphorylation, the biochemical pathway that generates ATP via the respiratory chain. During this process, 1–2% of the oxygen that is consumed is released as ROS, which can damage mtDNA and subsequently be repaired. However, under ROS-stressed conditions, the generation of ROS leads to persistent mtDNA damage. The propagation of mitochondrial damage through the generation of secondary ROS could lead to a decline in oxidative phosphorylation and of mitochondrial physiology. In response to changes in mitochondrial physiology, stress response genes are activated and cells undergo growth arrest, which may be followed by apoptosis.