Analysis of Common Deletion (CD) and a novel deletion of mitochondrial DNA induced by ionizing radiation

PURPOSE: In order to identify supportive evidence of radiation exposure to cells, we analyzed the relationship between exposure to ionizing radiation and the induction of deletions in mitochondrial DNA (mtDNA). MATERIALS AND METHODS: Using human hepatoblastoma cell line, HepG2 and its derivatives, HepG2-A, -89 and -400, established after long term exposure to X-ray, mtDNA deletions were analyzed by polymerase chain reaction (PCR) and real-time PCR after cells were subjected to radiation and genotoxic treatments. RESULTS: Common Deletion (CD), the most extensively studied deletion of mtDNA, was induced within 24 h after exposure to 5 Gray (Gy) of X-rays and was associated with replication of mtDNA. CD became undetectable several days after the exposure due to the death of cells containing mitochondria within which CD had been induced. Furthermore, we found a novel mtDNA deletion that consisted of a 4934 base-pair deletion (4934del) between nucleotide position 8435 and 13,368. A lower dose of ionizing radiation was required to induce the 4934del than for CD and this was independent of the quality of radiation used and was not induced by treatments with hydrogen peroxide (H(2)O(2)) and other genotoxic reagents including bleomycin. CONCLUSION: CD is induced by ionizing radiation, however, the amount of CD detected at a certain point in time after radiation exposure is dependent on the initial frequency of CD induced and the death rate of cells with mtDNA containing CD. The novel mtDNA deletion found in this study, therefore, will be used to determine whether cells were exposed to ionizing radiation.     

Mitochondrial DNA deletions in tissues of mice after ionizing radiation exposure

Purpose: The objective of the study was to estimate the presence of large mtDNA deletions in brain and spleen tissues of mice four months after exposure to 2 and 5?Gy.

Materials and methods: The male BALB/c mice underwent X-ray total-body acute radiation. Four months after irradiation, the mice were decapitated, and the samples of spleen and brain tissues were examined. A long-distance PCR was used to detect mtDNA deletions and their levels in samples of brain and spleen tissues.

Results: Four months after irradiation the levels of mtDNA deletions in the brain and spleen tissues were higher in animals exposed to 5?Gy than in animals at an irradiation dose of 2?Gy and in control mice. The levels of deletions in the mice brain tissues were higher 4 months than 1 month after X-ray exposure to both doses (2 and 5?Gy). In spleen tissues, a higher level of deletions was observed only at an irradiation dose of 5?Gy.

Conclusions: Our data have shown that ionizing radiation induces an increase of mtDNA copies with deletions in tissues of mice four months after the post-irradiation period. The level of deletions depends on the animal age, type of tissue, irradiation dose and length of the post-irradiation period.     

Telomere shortening and ionizing radiation: A possible role in vascular dysfunction?

Abstract

Purpose: In recent years, growing epidemiological evidence has linked ionizing radiation exposure to cardiovascular atherosclerotic disease. However, there are still major gaps in the knowledge of the molecular mechanisms of radiation-induced vascular disease, especially for low-dose levels. Telomeres, repetitive DNA sequences of (TTAGGG)_n located at the ends of eukaryotic chromosomes, play a role in regulating vascular aging, and shorter leukocyte telomere length has been demonstrated to predict cardiovascular disease and mortality. There is also evidence supporting the crucial role of telomeres in the formation of chromosome and chromatid aberrations induced by ionizing radiation.

Conclusions: The purpose of the present paper is to review the recent advances in the biological mechanisms determining telomere length erosion after ionizing radiation exposure as well as to examine the hypothesis that telomere shortening may be the crucial mediator leading to detrimental vascular effects after ionizing radiation exposure.     

P53 oncosuppressor influences selection of genomic imbalances in response to ionizing radiations in human osteosarcoma cell line SAOS-2

Purpose: To investigate the impact of TP53 (tumor protein 53, p53) on genomic stability of osteosarcoma (OS).

Materials and methods: In first instance, we expressed in OS cell line SAOS-2 (lacking p53) a wild type (wt) p53 construct, whose protein undergoes nuclear import and activation in response to ionizing radiations (IR). Thereafter, we investigated genomic imbalances (amplifications and deletions at genes or DNA regions most frequently altered in human cancers) associated with radio-resistance relative to p53 expression by mean of an array-based comparative genomic hybridization (aCGH) strategy. Finally we investigated a putative marker of radio-induced oxidative stress, a 4,977 bp deletion at mitochondrial (mt) DNA usually referred to as ‘common’ deletion, by mean of a polimerase chain reaction (PCR) strategy.

Results: In radio-resistant subclones generated from wt p53-transfected SAOS-2 cells DNA deletions were remarkably reduced and the accumulation of ‘common’ deletion at mtDNA (that may let the persistence of oxidative damage by precluding detoxification from reactive oxygen species [ROS]) completely abrogated.

Conclusions: The results of our study confirm that wt p53 has a role in protection of OS cell DNA integrity. Multiple mechanisms involved in p53 safeguard of genomic integrity and prevention of deletion outcome are discussed.     

Role of ROS-mediated autophagy in radiation-induced bystander effect of hepatoma cells

Abstract

Purpose: Autophagy plays a crucial role in cellular response to ionizing radiation, but it is unclear whether autophagy can modulate radiation-induced bystander effect (RIBE). Here, we investigated the relationship between bystander damage and autophagy in human hepatoma cells of HepG2.

Materials and methods: HepG2 cells were treated with conditioned medium (CM) collected from 3 Gy γ-rays irradiated hepatoma HepG2 cells for 4, 12, or 24 h, followed by the measurement of micronuclei (MN), intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and protein expressions of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 in the bystander HepG2 cells. In some experiments, the bystander HepG2 cells were respectively transfected with LC3 small interfering RNA (siRNA), Beclin-1 siRNA or treated with 1% dimethyl sulfoxide (DMSO).

Results: Additional MN and mitochondrial dysfunction coupled with ROS were induced in the bystander cells. The expressions of protein markers of autophagy, LC3-II/LC3-I and Beclin-1, increased in the bystander cells. The inductions of bystander MN and overexpressions of LC3 and Beclin-1 were significantly diminished by DMSO. However, when the bystander cells were transfected with LC3 siRNA or Beclin-1 siRNA, the yield of bystander MN was significantly enhanced.

Conclusion: The elevated ROS have bi-functions in balancing the bystander effects. One is to cause MN and the other is to induce protective autophagy.     

Effect of low-dose X-ray irradiation on micronucleus formation in human embryo,newborn and child cells

Purpose: It is well known that a high-dose of ionizing radiation is sufficient to break DNA strands, which leads to elevated genotoxic risks; however, the risks associated with low doses of ionizing radiation remain unclear. In addition, there is little data about the effect of low-dose ionizing radiation on human-derived embryo, newborn and child cells. We investigated the frequency of micronucleus (MN) formation in these cells to understand the genotoxic effects of ionizing radiation.

Materials and methods: We irradiated the cells with X-rays from 0.02–2?Gy at a rate of 0.0635?Gy/min. After irradiation, we investigated the effect of low-dose X-ray irradiation on cellular viability and frequency of MN formation.

Results: Increases in MN formation were largely dose-dependent; however, there were no differences between controls and doses lower than 0.2?Gy, except in KMST-6 human transformed embryo cells.

Conclusion: We could not detect an obvious effect of low-dose X-ray irradiation at doses lower than 0.1?Gy. The embryonic cells were more sensitive to X-ray irradiation than newborn and child cells. The threshold for X-ray-induced MN formation appears to be in the range of 0.05–0.1?Gy in cultured human embryo, newborn and child cells.     

Ionizing radiation impacts photochemical quantum yield and oxygen evolution activity of Photosystem II in photosynthetic microorganisms

Purpose: Long-term space exploration requires biological life support systems capable of coping with the deleterious space environment. The use of oxygenic photosynthetic microorganisms represents an intriguing topic in this context, mainly from the point of view of food and O₂ production. The aim of the present study was to assess the effects of space ionizing radiation exposure on the photosynthetic activity of various microorganisms.

Materials and methods: Ground-based irradiation experiments were performed using fast neutrons and gamma rays on microorganisms maintained at various light conditions. A stratospheric balloon and a European Space Agency (ESA) flight facility were used to deliver organisms to space at the altitude of 38 and 300 km, respectively. During the balloon flight, the fluorescence activity of the organisms was real-time monitored by means of a special biosensor.

Results: The quantum yield of Photosystem II (PSII), measured directly in flight, varied among the microorganisms depending on the light conditions. Darkness and irradiation of cells at 120 and 180 μmol m^?2 s^?1 enhanced the radiation-induced inhibition of photosynthetic activity, while exposure to weaker light irradiance of 20 and 70 μmol m^?2 s^?1 protected the cells against damage. Cell permanence in space reduced the photosynthetic growth while the oxygen evolution capacity of the cells after the flight was enhanced.

Conclusions: A potential role of PSII in capturing and utilizing ionizing radiation energy is postulated.     

The magnitude and time-dependence of the apoptotic response of normal and malignant cells subjected to ionizing radiation versus hyperthermia

Purpose:?The purpose of the study was to examine the optimal time of exposure and dose of heat and ionizing radiation that results in the killing of human cancer cells in vitro via apoptosis vs. necrosis.

Materials and methods:?Human mammary carcinoma, colorectal carcinoma and normal bovine capillary endothelial (BCE) cell lines were subjected to 20 Gy ionizing radiation and 6, 12, 24, and 72 h later assessed for apoptosis using detection of apoptotic bodies and caspase assays. Necrosis was detected by loss of cells from the surface and lactate dehydrogenase (LDH) release. The colorectal carcinoma cells were subjected to hyperthermia using temperatures ranging from 39 – 44°C for 5, 15 or 45 min. exposures and at varying times post-treatment, apoptosis and necrosis were measured.

Results:?In response to ionizing radiation, none of the cells underwent necrosis and some cell types apoptosed 24 and 72 h posttreatment. The colorectal cancer cells exhibited a steady increase of apoptosis at 6, 12, and 24 h. When these cells were exposed to 40°C for 5 min, caspases increased within 6 h and a significant fraction (50%) of cells apoptosed. If the time of exposure to 40°C was increased to 15 or 45 min, 80% and 100% of the dying cells apoptosed, respectively. A temperature of 39°C did not cause cell death even after 45 min exposures. If heat was elevated to 42 or 44°C, increased necrosis was observed with a corresponding decrease in apoptosis.

Conclusions:?These studies reveal time and temperature dependent in vitro cell responses to ionizing radiation and water-bath hyperthermia.     

Evaluation of the genotoxic effects of chronic low-dose ionizing radiation exposure on nuclear medicine workers

IntroductionNuclear medicine workers are occupationally exposed to chronic ionizing radiation. It is known that ionizing radiation may have damaging effects on chromosomes. In the present study, we investigated the genotoxic effects of ionizing radiation on nuclear medicine workers. We used two different indicators of genotoxicity methods: sister chromatid exchange (SCE) and micronucleus (MN).MethodsThe present research was carried out using 21 nuclear medicine workers (11 females and 10 males) during two periods: during normal working conditions and after a 1-month vacation. The radiation dose varied from 1.20 to 48.56 mSv, which accumulated during the occupational exposure time between two vacations. Peripheral blood samples were taken from each subject for two distinct lymphocyte cultures (SCE and MN) in each period.ResultsIn nearly all subjects, SCE values increased significantly during radiation exposure compared to the postvacation period (P<.05). Similarly, MN frequencies in most of the subjects increased significantly during radiation exposure compared to the postvacation period (P<.05).ConclusionsThis study revealed that both SCE and MN frequencies in most of the subjects were significantly higher during exposure to ionizing radiation than after a 1-month vacation period. However, this genotoxic effect was reversible in most of the subjects.     

DNA strand breakage by bivalent metal ions and ionizing radiation

Purpose: To investigate mechanisms of DNA breakage via the interaction of bivalent metal ion, thiol reducing agent and ionizing radiation, in ?OH scavenging abilities comparable to those in cells.

Materials and methods: We measured the effects of 10 min exposure to 200 μM Fe²⁺ vs. Fe³⁺ on the induction of single (SSB) and double (DSB) strand breaks in unirradiated and oxically irradiated SV40 DNA, in aqueous solution containing 75 or 750 mM glycerol and/or 5 mM glutathione (GSH).

Results: Fe²⁺ or GSH alone produced little DNA damage. However, their combination produced a dramatic increase in the production of both SSB and DSB. Experiments with ferric ion suggest that it produces DNA damage only after partial reduction to ferrous by GSH. Induction efficiencies for SSB in the presence of Fe²⁺/GSH showed additivity of the effects of radiation alone with those from Fe²⁺/GSH. However, the corresponding induction efficiencies for DSB demonstrated a 2.5-fold enhancement.

Conclusions: Our results are consistent with a model in which reduced bivalent metal ions plus thiols, in the presence of O₂, produce DSB in DNA primarily via local clusters of hydroxyl radicals arising from site specific Fenton reactions. The synergism observed between DSB production by Fe/GSH and by ionizing radiation, also believed to occur via local clusters of hydroxyl radicals, is consistent with this model. Our results suggest that both normally present intracellular iron and ionizing radiation may be important sources of oxidative stress in cells.     

Memory CD4 T-cell subsets discriminated by CD43 expression level in A-bomb survivors

Purpose:?Our previous study showed that radiation exposure reduced the diversity of repertoires of memory thymus-derived cells (T cells) with cluster of differentiation (CD)- 4 among atomic-bomb (A-bomb) survivors. To evaluate the maintenance of T-cell memory within A-bomb survivors 60 years after radiation exposure, we examined functionally distinct memory CD4 T-cell subsets in the peripheral blood lymphocytes of the survivors.

Methods:?Three functionally different subsets of memory CD4 T cells were identified by differential CD43 expression levels and measured using flow cytometry. These subsets consist of functionally mature memory cells, cells weakly responsive to antigenic stimulation, and those cells functionally anergic and prone to spontaneous apoptosis.

Results:?The percentages of these subsets within the peripheral blood CD4 T-cell pool all significantly increased with age. Percentages of functionally weak and anergic subsets were also found to increase with radiation dose, fitting to a log linear model. Within the memory CD4 T-cell pool, however, there was an inverse association between radiation dose and the percentage of functionally mature memory cells.

Conclusion:?These results suggest that the steady state of T cell memory, which is regulated by cell activation and/or cell survival processes in subsets, may have been perturbed by prior radiation exposure among A-bomb survivors.     

Anti‐erbB receptor strategy as a gene therapeutic intervention to improve radiotherapy in malignant human tumours

Purpose: We explored and quantified the therapeutic potential of using dominant‐negative EGFR transduction with replication‐incompetent adenovirus (Ad‐EGFR‐CD533 or Ad‐CD533) as a genetic approach for radiosensitization in different carcinoma and malignant glioma cell lines in vitro and in established tumour xenografts in vivo.

Material and methods: The cell lines MDA‐MB‐231, A‐431, U‐373 MG, U‐87 MG and T47D were used. The ErbB expression profiles were quantified by Western blotting. MAPK immune complex assay measured MAPK activity with or without EGFR‐CD533 expression after ionizing radiation. Radiosensitization was determined and quantified in vitro by colony‐formation assays, in vivo by use of an ex vivo‐in vitro colony‐formation assay after intratumoral infusion of the adenoviral vectors expressing EGFR‐CD533 or the control LacZ.

Results: Western blotting demonstrated widely varied expression levels of the ErbB receptors in the tested cell lines. Expression of EGFR‐CD533 effectively blocked the radiation‐induced activation of MAPK, leading to significant radiosensitization in vitro and in vivo.

Conclusions: The radiation‐induced ErbB activation can be effectively modulated by a gene therapeutic approach of over‐expressing EGFR‐CD533 leading to tumour cell radiosensitization after single and repeated radiation exposures both in vitro and in vivo.     

Morphological and molecular alterations at the junctional complex in irradiated human colon adenocarcinoma cells,Caco-2

Purpose: Ionizing radiation is one of the main modalities used in the treatment of colorectal cancer. Despite a number of epigenetic or non-targeted effects of radiation exposure that have been described, the effect of radiation on cell-cell adhesion in the epithelium has been less studied. We report morphological and molecular alterations induced by ionizing radiation at the junctional complex level of human colon cancer Caco-2 cells.

Materials and methods: Cells were irradiated with doses of 2, 5 or 10 Gy and the effects on the junctional complex were monitored for different times after irradiation. Alterations of tight and adherens junction components were observed by measuring the transepithelial electrical resistance, by immunofluorescence and immunoblotting and electron microscopy analyses.

Results: Ionizing radiation caused alterations in the junctional complex, as evidenced by: (a) a decrease in the transepithelial electrical resistance, (b) alterations in the pattern of the distribution of junctional proteins as observed for E-cadherin, occludin, and zonula occludens 1 (ZO-1), but with minor changes in claudin-1 localization, and (c) wide spaces between opposed cells. These effects were dose and time-dependent since minor doses of irradiation caused a reversible effect on E-cadherin distribution and transepithelial electrical resistance.

Conclusions: The results obtained show that ionizing radiation caused redistribution of the main junctional proteins E-cadherin, occludin and ZO-1 with minor changes for claudin-1, leading to disassembly of the junctional complex and loss of its functionality in Caco-2 cells. The molecular mechanisms responsible for these events need further elucidation.     

Effect of hypothermia on radiation-induced micronuclei and delay of cell cycle progression in TK6 cells

Abstract

Purpose: Low temperature (hypothermia) during irradiation leads to a reduced frequency of micronuclei in TK6 cells and it has been suggested that perturbation of cell cycle progression is responsible for this effect. The aim of the study was to test this hypothesis.

Materials and methods: Human lymphoblastoid TK6 cells were treated by a combination of hypothermia (0.8°C) and ionizing radiation in varying order (hypothermia before, during or after irradiation) and micronuclei were scored. Growth assay and two-dimensional flow cytometry was used to analyze cell cycle kinetics following irradiated of cells at 0.8°C or 37.0°C.

Results: The temperature effect was observed at the level of micronuclei regardless of whether cells were cooled during or immediately before or after the radiation exposure. No indication of cell cycle perturbation by combined exposure to hypothermia and radiation could be detected.

Conclusions: The protective effect of hypothermia observed at the level of cytogenetic damage was not due to a modulation of cell cycle progression. A possible alternative mechanism and experiments to test it are discussed.     

Mutagenic effect of low energy neutrons on human chromosome 11

Purpose: The shape of the dose–effect curve for neutrons, i.e. the question as to whether the curve is linear or supralinear in the low‐dose region, is still not clear. Therefore, the mutagenic effect of very low doses of low‐energy neutrons was determined.

Materials and methods: Human–hamster hybrid A_L cells contain human chromosome 11, which expresses the membrane protein CD59. This membrane protein can be detected immunologically and quantified by flow cytometry. The A_L cells were irradiated with neutrons of 0.565, 2.5 or 14.8?MeV and the results were compared with those after 200?kVp X‐rays. Before irradiation, cells spontaneously mutated in the CD59 gene were removed by magnetic cell sorting (MACS).

Results: The relative biological effectiveness (RBE) for CD59 mutation induction was 19.8 (±2.7) for 0.565?MeV, 10.2 (±1.9) for 2.5?MeV, and 10.2 (±1.6) for 14.8?MeV neutrons. Linear mutation responses were obtained with all radiations except for 14.8?MeV neutrons where a supralinear curve may be a better fit. The deletion spectrum of mutated cell clones showed 29?Mbp deletions on average after irradiation with 0.069?Gy of 0.565?MeV neutrons. This scale of deletions is similar to that after 3?Gy 100?kV X‐rays (=34?Mbp). For 50% cell survival, the RBE of the neutrons was 11 compared with 200?kV X‐rays.

Conclusion: Neutrons of low energies (0.565 or 2.5?MeV) produce a linear dose–response for mutation in the tested dose range of 0.015–0.15?Gy. The neutron curve of 14.8?MeV can be approximated by a curvilinear or linear function.     

The effects of chronic gamma irradiation on oxidative stress response and the expression of anthocyanin biosynthesis-related genes in wheat (Triticum aestivum)

Abstract

Purpose: To investigate the mechanisms of adaptation and tolerance to ionizing radiation using chronic radiation in wheat.

Materials and methods: We exposed wheat plants to chronic gamma irradiation (50 Gy) for 2, 4, and 6 weeks and measured various biological parameters.

Results: Plant height was reduced by exposure to gamma irradiation; this effect increased with increasing exposure time. Photosynthetic pigment levels decreased with increasing exposure time, while anthocyanin levels significantly increased after exposure to gamma rays. The activities of antioxidant enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], and peroxidase [POD]) and malondialdehyde (MDA) levels increased with increasing duration of exposure to gamma irradiation. Electron spin resonance (ESR) signals were strongly detected in wheat that was gamma-irradiated for two weeks and then gradually decreased with increasing exposure time. The expression of anthocyanin biosynthesis genes (flavanone 3-hydroxylase [F3H], dihydroflavonol reductase [DFR], anthocyanin reductase [ANS], and UDPG-flavonoid glucosyl transferase [UFGT]) and sugar contents increased after exposure to gamma rays.

Conclusions: This suggests that exposure to ionizing radiation according to increase of exposure time has led to efficient induction of anthocyanin and antioxidant enzyme activities. This study indicates that reactive oxygen species (ROS) is eliminated by biosynthesis of anthocyanin and antioxidant enzymes. This study helps elucidate the biological effects of various durations of low-dose exposure to chronic gamma radiation in wheat plants.     

Altered mitochondrial function and genome frequency post exposure to γ-radiation and bystander factors

Purpose:?To further evaluate irregular mitochondrial function and mitochondrial genome damage induced by direct γ-irradiation and bystander factors in human keratinocyte (HPV-G) epithelial cells and hamster ovarian fibroblast (CHO-K1) cells. This is as a follow-up to our recent reports of γ-irradiation-induced loss of mitochondrial function and mitochondrial DNA (mtDNA) damage.

Materials and methods:?Mitochondrial function was evaluated post direct radiation and irradiated cell conditioned medium (ICCM) by determining: Activity of the individual complexes of oxidative phosphorylation (OxPhos); mtDNA-encoded protein synthesis; and mitochondrial genome frequency and mtDNA damage.

Results:?Mitochondria show a loss of OxPhos enzyme function as early as 4?h post treatment with recovery observed 12–96?h in some but not all complexes demonstrating a non-uniform sensitivity to γ-radiation. We also identified irregular mtDNA-directed protein synthesis. Long range Polymerase Chain Reaction (PCR) analysis identified mitochondrial genome damage and real-time PCR identified increases in mitochondrial genome frequency.

Conclusions:?The study reaffirms the sensitive nature of mitochondria to both low-level direct radiation exposure and radiation-induced bystander factor mediated damage. Furthermore, we report for the first time, the loss of function in the enzymes of OxPhos post exposure to bystander factors and identify altered mtDNA-directed protein synthesis post both direct radiation and bystander factors.     

DNA Damage Does Not Appear to be a Trigger for Thermotolerance in Mammalian Cells

Summary

The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morrison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. We therefore used three alternative assay which are indicators of the subsequent development of thermotolerance. These were; (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after ionizing radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA stand breaks caused by the radiation. Our data therefore suggest that, in mammalian cells, the type of DNA damage caused by ionizing radiation is not the trigger for the induction of thermotolerance.