Mitochondrial complex II dysfunction can contribute significantly to genomic instability after exposure to ionizing radiation

Abstract

Ionizing radiation induces chronic metabolic oxidative stress and a mutator phenotype in hamster fibroblasts that is mediated by H(2)O(2), but the intracellular source of H(2)O(2) is not well defined. To determine the role of mitochondria in the radiation-induced mutator phenotype, end points of mitochondrial function were determined in unstable (CS-9 and LS-12) and stable (114) hamster fibroblast cell lines derived from GM10115 cells exposed to 10 Gy X rays. Cell lines isolated after irradiation demonstrated a 20-40% loss of mitochondrial membrane potential and an increase in mitochondrial content compared to the parental cell line GM10115. Surprisingly, no differences were observed in steady-state levels of ATP (P > 0.05). Unstable clones demonstrated increased oxygen consumption (two- to threefold; CS-9) and/or increased mitochondrial electron transport chain (ETC) complex II activity (twofold; LS-12). Using Western blot analysis and Blue Native gel electrophoresis, a significant increase in complex II subunit B protein levels was observed in LS-12 cells. Furthermore, immunoprecipitation assays revealed evidence of abnormal complex II assembly in LS-12 cells. Treatment of LS-12 cells with an inhibitor of ETC complex II (thenoyltrifluoroacetone) resulted in significant decreases in the steady-state levels of H(2)O(2) and a 50% reduction in mutation frequency as well as a 16% reduction in CAD gene amplification frequency. These data show that radiation-induced genomic instability was accompanied by evidence of mitochondrial dysfunction leading to increased steady-state levels of H(2)O(2) that contributed to increased mutation frequency and gene amplification. These results support the hypothesis that mitochondrial dysfunction originating from complex II can contribute to radiation-induced genomic instability by increasing steady-state levels of reactive oxygen species.

FIG. 1

Genomically unstable cells show a depolarized mitochondrial membrane potential, an increase in mitochondrial content and no difference in ATP levels. Panel A: Mitochondrial membrane potential as measured by JC-1. The error bars represent ±1 SD from three independent experiments. * P < 0.05 compared to wild-type cells. Panel B: Mitochondrial content as shown by Mitotracker green staining showed modest differences among clones. Error bars represent ±1 SD from three separate dishes. * P < 0.05 compared to wild-type cells. Panel C: ATP production measured by the luciferase assay. The error bars represent ±1 SD from three separate experiments. ATP levels were not significantly different.

FIG. 2

Genomically unstable clones show high oxygen consumption and high complex II levels. Panel A: Oxygen consumption. The error bars represent ±1 SD from five independent experiments. CS-9 cells were significantly different from GM10115 cells and stable clone 114. * P < 0.001 compared to wild-type cells, ¥ P < 0.001 compared to 114 cells. Panel B: Complex II activity normalized to mitochondrial protein. * P < 0.05 compared to wild-type cells, ¥ P < 0.05 compared to 114 cells. Panel C: Western blots of whole cell lysate using 100 μg protein and SDHB antibody. Quantification shown by numerical data was achieved by normalizing SDHB density to actin density. Panel D: Coomassie blue staining of a Blue Native gel (lower panel) and a Western blot of a parallel gel probed with SDHB antibody.

FIG. 3

Model by which disruption of complex II assembly may participate in radiation-induced genomic instability.

FIG. 4

Genomically unstable clone LS-12 shows improper assembly of complex II. Panel A: Western blot showing levels of immunoprecipitated SDHB after transfer on a nitrocellulose membrane. Unstable clone LS-12 shows the presence of SDHB in the soluble fraction. Panel B: Immunoprecipitation of SDHA proteins carried out on 150 μg isolated mitochondria using antibody against SDHA and Sepharose protein A beads. Samples were separated on a 4%–20% gradient gel, which was stained with Coomassie blue.

FIG. 5

TTFA treatment reduces steady-state levels of H₂O₂ and the mutation frequency of LS-12 clone. Panel A: H₂O₂ per 10⁶cells. Error bars are ±1 SD for three separate experiments (n = 4–9). * P < 0.05 compared to GM10115 cells, £ P < 0.05 compared to LS-12 cells. Panel B: HPRT mutation frequency. Error bars are ±1 SD for three separate experiments (n = 6–9). £ P < 0.05 compared to LS-12 cells.