Severe mitochondrial damage associated with low-dose radiation sensitivity in ATM- and NBS1-deficient cells

Low-dose radiation risks remain unclear owing to a lack of sufficient studies. We previously reported that low-dose, long-term fractionated radiation (FR) with 0.01 or 0.05 Gy/fraction for 31 d inflicts oxidative stress in human fibroblasts due to excess levels of mitochondrial reactive oxygen species (ROS). To identify the small effects of low-dose radiation, we investigated how mitochondria respond to low-dose radiation in radiosensitive human ataxia telangiectasia mutated (ATM)- and Nijmegen breakage syndrome (NBS)1-deficient cell lines compared with corresponding cell lines expressing ATM and NBS1. Consistent with previous results in normal fibroblasts, low-dose, long-term FR increased mitochondrial mass and caused accumulation of mitochondrial ROS in ATM- and NBS1-complemented cell lines. Excess mitochondrial ROS resulted in mitochondrial damage that was in turn recognized by Parkin, leading to mitochondrial autophagy (mitophagy). In contrast, ATM- and NBS1-deficient cells showed defective induction of mitophagy after low-dose, long-term FR, leading to accumulation of abnormal mitochondria; this was determined by mitochondrial fragmentation and decreased mitochondrial membrane potential. Consequently, apoptosis was induced in ATM- and NBS1-deficient cells after low-dose, long-term FR. Antioxidant N-acetyl-L-cysteine was effective as a radioprotective agent against mitochondrial damage induced by low-dose, long-term FR among all cell lines, including radiosensitive cell lines. In conclusion, we demonstrated that mitochondria are target organelles of low-dose radiation. Mitochondrial response influences radiation sensitivity in human cells. Our findings provide new insights into cancer risk estimation associated with low-dose radiation exposure.</p

Possible relationship between mitochondrial changes and oxidative stress under low dose-rate irradiation

Objectives: High dose-rate ionizing radiation (IR) causes severe DSB damage, as well as reactive oxygen species (ROS) accumulation and oxidative stress. However, it is unknown what biological processes are affected by low dose-rate IR; therefore, the molecular relationships between mitochondria changes and oxidative stress in human normal cells was investigated after low dose-rate IR. Methods: We compared several cellular response between high and low dose-rate irradiation using cell survival assay, ROS/RNS assay, immunofluorescence and western blot analysis. Results: Reduced DSB damage and increased levels of ROS, with subsequent oxidative stress responses, were observed in normal cells after low dose-rate IR. Low dose-rate IR caused several mitochondrial changes, including morphology mass, and mitochondrial membrane potential, suggesting that mitochondrial damage was caused. Although damaged mitochondria were removed by mitophagy to stop ROS leakage, the mitophagy-regulatory factor, PINK1, was reduced following low dose-rate IR. Although mitochondrial dynamics (fission/fusion events) are important for the proper mitophagy process, some mitochondrial fusion factors decreased following low dose-rate IR. Discussion: The dysfunction of mitophagy pathway under low dose-rate IR increased ROS and the subsequent activation of the oxidative stress response.</p

IR induces microcephaly in mice.

<p>(A) Mouse embryos at E13.5 were exposed in utero to IR of 1 or 2 Gy, and then embryonic brains were sampled at the indicated days. (B) Embryonic brains were weighed and average weights were calculated using at least 3 embryos. Error bars indicate the standard error. (C) Mouse embryos at E13.5 were exposed to 1 or 2 Gy, and then the brains of newborn mice (P0.5) were cryosectioned and stained with antibodies against Brn1 (green) and Foxp2 (red), used as markers of cerebral cortex layers II–IV and layer VI, respectively. Scale bar: 100 μm.</p

Apoptotic cell death in the mouse embryonic cerebral cortex after IR exposure.

<p>(A) Embryonic brains were sampled at 24 h after 1 or 2 Gy IR exposure and stained with antibodies against cleaved-caspase-3 (apoptosis marker) and βIII-tubulin (Tuj1; neuron marker). (B) and (C) Cleaved-caspase-3-positive cells after 1 or 2 Gy exposure were quantified. Error bars indicate the standard error. (D) Embryonic brains were sampled at 24 h after 1 or 2 Gy exposure and stained with a Tbr2 antibody (neural-progenitor marker), and then the Tbr2-positive cells were quantified. At least 3 independent mouse embryos were used for each experiment. V: ventricle; VZ: ventricular zone; SVZ: subventricular zone. The border of VZ and SVZ was delineated according to the method by Pulvers JN., et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158236#pone.0158236.ref012" target="_blank">12</a>]. Scale bar: 100 μm.</p

Randomized controlled trial of a water-soluble formulation of lutein in humans

Lutein is poorly absorbed owing to their high hydrophobicity and crystallinity. This double-blind crossover trial involved eight healthy males who were administrated capsules containing either a lutein water-soluble formulation or a lutein oil suspension for 8 days. In the formulation group, plasma and erythrocytes lutein concentrations and baseline-corrected AUC were two-fold higher than those in the oil suspension group.</p

Cell proliferation in the mouse embryonic cerebral cortex after IR exposure.

<p>(A) Mouse embryos at E13.5 were irradiated with 1 or 2 Gy, and 24 h later, brains were sampled and stained with an antibody against Ser10-phosphorylated histone H3 (pH3; red, mitotic-cell marker) and the Tuj1 antibody (green, neuron marker), and counterstained with DAPI (white). Apical and basal mitotic cells are indicated by white and yellow arrows, respectively. (B) and (C) All pH3-positive cells in the apical and basal surface were quantified separately after irradiation with 1 Gy (B) or 2 Gy (C). Black and white bars indicate the apical and the basal surface, respectively. At least 3 independent mouse embryos were used for each experiment. Error bars represent the standard error. V: ventricle; VZ: ventricular zone; SVZ: subventricular zone. Scale bar: 100 μm.</p

Abnormal cytokinesis in the mouse embryonic cerebral cortex after IR exposure.

<p>(A) Mouse embryos at E13.5 were irradiated with 1 or 2 Gy, and 48 h later, brains were sampled and stained with antibodies against Ser10-phosphorylated histone H3 (pH3; red in merged image) and γ-tubulin (green in merged image), used as markers of mitotic cells and centrosomes. Cells were counterstained with DAPI (blue in merged image). Quantification of (B) multipolar cell division, (C) lagging chromosomes, and (D) centrosome clustering among pH3-positive cells; at least 100 pH3-positive cells were counted. Error bars represent the standard error. Scale bar: 10 μm.</p

Disruption of the apical layer in the mouse embryonic cerebral cortex after IR exposure.

<p>Mouse embryos at E13.5 were irradiated with 1 or 2 Gy and the brains were sampled 24 h later and stained with antibodies against (A) ZO1 (green) and Sox2 (blue), used as markers of adherence junctions and neural progenitors, respectively; and (B) γ-tubulin (green), used as a spindle marker. VZ: ventricular zone; SVZ: subventricular zone. Scale bar: 100 μm.</p

Nucleolin contributes to ATM-dependent DNA damage responses.

<p>(A and B) U2OS cells were transfected by nucleolin siRNA or negative control siRNA, and after 2 days these cells were irradiated by 5 Gy of γ-ray. After 30 minutes, their cells were fixed and immuno-staining was performed using indicated antibodies. Percentage of phospho-ATM or 53BP1 foci-positive cell at indicated times after 2 Gy of irradiation was shown in (B). Open column: control, closed column: nucleolin siRNA. (C) 48BR cells were transfected by nucelolin siRNA. After 2 days, these cells were irradiated by 5 Gy of γ-ray and were harvested at indicated times after IR and analyzed by Western blot using indicated antibodies.</p