Bidirectional function of shenghe powder on repair of radiation-induced DNA damage in glioma and astrocyte

The study assessed the effect of Chinese herbs of Shenghe Powder (SHP) on the repair capacity of gamma-radiation-induced DNA damage in rat glioma cells (C6) compared with normal human astrocytes (NHA). C6 and NHA Cells treated with SHP and irradiated with 2Gy of gamma radiation. Cells growth inhibition were analysed by MTT assay, DNA damage and repair were evaluated using phosphorylated histone H2AX (γH2AX) at the appointed time. Apoptosis was observed by flow cytometry, and the expression of DNA-dependent protein kinase (DNA-PK) and surviving proteins were assessed by Western blot analysis. SHP depressed the radiation-induced DNA double-strand break and enhanced the DNA repair capacity in NHA, which correlated with promotion of DNA-PK phosphorylation. In contrast, SHP enhanced radiosensitivity of C6 cells, the pre-treatment with SHP resulted in reduced numbers of γH2AX foci in irradiated C6 cells, and decreased the expression of DNA-PK and survivn(P<0.005). It significant effect on inhibition of C6 cell proliferation and induced C6 cells apoptosis in a time-depdendent manner than radiation alone (P<0.001). SHP showed a novel bidirectional function to improve the radioresistance of NHA and enhanced radiosensitivity of C6 cells. This implies that SHP can protect the NHA from radiant damage and enhanced the sensitivity of C6 cells to radiation, which could be attributed to the alteration of survivin DNA-PK in DNA repair processes.     

Kinetics of gamma-H2AX focus formation upon treatment of cells with UV light and alkylating agents

Histone H2AX is rapidly phosphorylated in response to DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). Here we show that DNA damage induced by alkylating agents [methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)] and ultraviolet light (UV-C) leads to a dose and time dependent accumulation of phosphorylated H2AX (gamma-H2AX). Time course experiments revealed that the number of gamma-H2AX foci reached peak levels 8 hr after MMS or MNNG treatment and declined to almost control values within 24 hr after exposure. Upon UV-C treatment, a biphasic response was observed with a maximum 12 hr after treatment. In 43-3B cells deficient in nucleotide excision repair (NER) the number of gamma-H2AX foci increased steadily. gamma-H2AX foci were preferentially formed in BrdU labeled cells. In proliferation compromised cells, the gamma-H2AX level was significantly reduced, indicating that most of the gamma-H2AX foci induced by UV-C and alkylating agent treatments were replication dependent. The data are in line with the view that DNA damage induced by UV-C light and simple alkylating agents, leads to the formation of DSBs during DNA replication giving rise to H2AX phosphorylation. In replicating NER defective cells, DSBs accumulate due to nonrepaired primary DNA lesions that produce a high level of DSBs during replication. The data support that gamma-H2AX foci are a useful marker of DSBs that are induced by S-phase dependent genotoxins during replication.     

Gamma-H2AX immunofluorescence for the detection of tissue-specific genotoxicity in vivo

The phosphorylation of histone H2AX in Serine 139 (gamma-H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma-H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma-H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma-H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma-H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma-H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma-H2AX staining allows the detection of tissue-specific effects in situ. Moreover, since gamma-H2AX staining can be performed on formalin-fixed and paraffin-embedded tissue sections generated during repeated-dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals. Environ. Mol. Mutagen. 60:4–16, 2019. © 2018 Wiley Periodicals, Inc.     

Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks

Nonhomologous end-joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PK catalytic subunit (DNA-PKcs), is activated by DNA in vitro and is required for NHEJ. We report that DNA-PKcs is autophosphorylated at Thr2609 in vivo in a Ku-dependent manner in response to ionizing radiation. Phosphorylated DNA-PKcs colocalizes with both gamma-H2AX and 53BP1 after DNA damage. Mutation of Thr2609 to Ala leads to radiation sensitivity and impaired DSB rejoining. These findings establish that Ku-dependent phosphorylation of DNA-PKcs at Thr2609 is required for the repair of DSBs by NHEJ.     

Hepatitis C virus NS3/4A protein interacts with ATM, impairs DNA repair and enhances sensitivity to ionizing radiation

Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Nonstructural protein 3 (NS3) of HCV possesses serine protease, nucleoside triphosphatase, and helicase activities, while NS4A functions as a cofactor for the NS3 serine protease. Here, we show that HCV NS3/4A interacts with the ATM (ataxia-telangiectasia mutated), a cellular protein essential for cellular response to irradiation. The expression of NS3/4A caused cytoplasmic translocation of either endogenous or exogenous ATM and delayed dephosphorylation of the phosphorylated ATM and gamma-H2AX following ionizing irradiation. As a result, the irradiation-induced gamma-H2AX foci persisted longer in the NS3/4A-expressing cells. Furthermore, these cells showed increased comet tail moment in single-cell electrophoresis assay, indicating increased double-strand DNA breaks. The cells harboring an HCV replicon also exhibited cytoplasmic localization of ATM and increased sensitivity to irradiation. These results demonstrate that NS3/4A impairs the efficiency of DNA repair by interacting with ATM and renders the cells more sensitive to DNA damage. This effect may contribute to HCV oncogenesis.     

Short-term culture and gammaH2AX flow cytometry determine differences in individual radiosensitivity in human peripheral T lymphocytes

Histone H2AX, a subfamily of histone H2A, is phosphorylated and forms proteinaceous repair foci at the sites of DNA double-strand breaks in response to genotoxic insults, such as ionizing radiation. This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro. Irradiation of short-term ( approximately 7 days) cultured T lymphocytes exhibited significant interindividual, but not interexperimental, differences in the cellular content of gammaH2AX 6 hr after 4 Gy of X-irradiation in three independent experiments using peripheral blood lymphocytes from six healthy donors. However, these differences were not as marked in uncultured lymphocytes, or lymphocytes that were cultured for a prolonged period ( approximately 13 days). The variation of gammaH2AX focus formation in lymphocytes of individuals was reproducible, with differences reaching about 1.5-fold following 7 days of culture. Therefore, the FCM-based gammaH2AX measurement appeared to reflect both the temporal course and the amount of DNA damage within the irradiated lymphocytes. Further, we confirmed that the differences in residual lymphocyte subsets were not involved in individual radiosensitivity. These results suggest that the FCM-based gammaH2AX assay using cultured T lymphocytes might be useful for the rapid and reliable assessment of individual radiation sensitivity involved in DNA damage repair.     

Radiation-induced double strand breaks and subsequent apoptotic DNA fragmentation in human peripheral blood mononuclear cells

In case of accidental radiation exposure or a nuclear incident, physical dosimetry is not always complete. Therefore, it is important to develop tools that allow dose estimates and determination that are based on biological markers of radiation exposure. Exposure to ionizing radiation triggers a large-scale activation of specific DNA signaling and repair mechanisms. This includes the phosphorylation of γH2AX in the vicinity of a double-strand break (DSB). A DNA DSB is a cytotoxic form of DNA damage, and if not correctly repaired can initiate genomic instability, chromosome aberrations, mutations or apoptosis. Measurements of DNA DSBs and their subsequent repair after in?vitro irradiation has been suggested to be of potential use to monitor cellular responses. The bone marrow and the blood are known to be the most radiosensitive tissues of the human body and can therefore be of particular importance to find radiation-induced biological markers. In the present study, changes in H2AX phosphorylation and apoptosis of irradiated human peripheral blood mononuclear cells (PBMCs) were analyzed. Freshly isolated PBMCs from healthy donors were irradiated with X-rays (0.1, 0.25, 0.5, 1, 2 and 4?Gy). The phosphorylation of γH2AX was measured at different time points (0, 0.25, 1, 2, 4, 6 and 24?h) after irradiation. We detected a linear dose-dependency of γH2AX phosphorylation measured by γH2AX foci scoring using immunofluorescence microscopy as well as by γH2AX fluorescence detection using flow cytometry. Apoptosis was detected by measuring DNA fragmentation at different time points (0, 24, 48, 72, 96?h) after X-irradiation using DNA ladder gel electrophoresis. The apoptotic DNA fragmentation increased in a dose-dependent manner. In conclusion, DNA DSBs and subsequent apoptotic DNA fragmentation monitoring have potential as biomarkers for assessing human exposure in radiation biodosimetry.     

Inhibitory effect of ascorbic acid post-treatment on radiation-induced chromosomal damage in human lymphocytes in vitro

In the present study, the effect of exposure to ascorbic acid (vitamin C) after gamma-ray-induced chromosomal damage in cultured human lymphocytes was examined to explore the mechanism by which this antioxidant vitamin protects irradiated cells Non-irradiated lymphocytes were exposed to increasing concentrations of ascorbic acid (1-100 micro g/ml) and DNA damage was estimated using chromosomal aberration analysis and the comet assay. The results showed that ascorbic acid did not influence the frequency of chromosomal aberrations in non-irradiated cells, except at the highest concentration (20 micro g/ml), which induced breakage-type chromosomal aberrations. Vitamin C at the concentration of 50 micro g/ml caused DNA damage detected by the comet assay. A significant (34%) decrease in the frequency of chromosomal aberrations was observed in lymphocytes exposed to gamma-radiation and then cultured in the presence of ascorbic acid (1 micro g/ml). The removal of DNA breaks in cells exposed to 2 Gy of gamma-radiation was accelerated in the presence of ascorbic acid as determined by the comet assay, suggesting that it may stimulate DNA repair processes.     

Elevated DNA damage in a mouse model of oxidative stress: impacts of ionizing radiation and a protective dietary supplement

Transgenic growth hormone (Tg) mice express elevated free radical processes and a progeroid syndrome of accelerated ageing. We examined bone marrow cells of Tg mice and their normal (Nr) siblings for three markers of DNA damage and assessed the impact of free radical stress using ionizing radiation. We also evaluated the radiation protection afforded by a dietary supplement that we previously demonstrated to extend longevity and reduce cognitive ageing of Nr and Tg mice. Spectral karyotyping revealed few spontaneous chromosomal aberrations in Nr or Tg. Tg mice, however, had significantly greater constitutive levels of both gammaH2AX and 8-hydroxy-deoxyguanosine (8-OHdG) compared to Nr. When exposed to a 2-Gy whole-body dose of ionizing radiation, both Nr and Tg mice showed significant increases in DNA damage. Compared to Nr mice, irradiated Tg mice had dramatically higher levels of gammaH2AX foci and double the levels of chromosomal aberrations. In unirradiated mice, the dietary supplement significantly reduced constitutive gammaH2AX and 8-OHdG in both Nr and Tg mice (normalizing both gammaH2AX and 8-OHdG in Tg), with little difference in gammaH2AX and 8-OHdG over constitutive levels. Induced chromosomal aberrations were also reduced, and in Nr mice, virtually absent. Remarkably, supplemented mice expressed 6-fold lower levels of radiation-induced chromosomal aberrations compared to unsupplemented Nr or Tg mice. Based on our data, the dietary supplement appeared to scavenge free radicals before they could cause damage. This study validates Tg mice as an exemplary model of oxidative stress and radiation hypersensitivity and documents unprecedented radioprotection by a dietary supplement comprised of ingredients available to the general public.     

MCPH1在电离辐射诱导食管癌细胞DNA损伤通路中的作用

目的:研究MCPH1在电离辐射诱导食管癌细胞DNA损伤通路中的作用。方法:应用已构建的沉默MDC1的食管癌ECA109细胞株接受8 Gy电离辐射后1 h,检测相关因子核内斑点形成情况。构建沉默MCPH1的食管癌ECA109细胞株,检测此细胞株接受同样照射条件后相关因子核内斑点的形成情况。结果:成功构建沉默MCPH1的食管癌ECA109细胞;电离辐射使MDC1、MCPH1与γ-H2AX蛋白相互作用。沉默MDC1不影响γ-H2AX和MCPH1核内斑点的形成;沉默MCPH1使电离辐射导致的MDC1核内斑点减少,不影响γ-H2AX核内斑点的形成。结论:MCPH1在电离辐射诱导的DNA损伤通路中可能位于H2AX下游、MDC1上游,可以调控MDC1核内斑点形成。     

Kinetics of gamma-H2AX induction and removal in bone marrow and testicular cells of mice after X-ray irradiation

Male germ cells have been shown to differ in their DNA damage response (DDR) with respect to somatic cells. In addition, DDR pathways are modulated along spermatogenesis, accompanying profound chromatin modifications. Histone H2AX phosphorylation is a fundamental step of DDR. Few data are available on the long-term kinetics of phosphorylated H2AX (γ-H2AX) after in vivo irradiation. We have investigated, by microscopic and flow cytometric immunochemistry, γ-H2AX induction and removal in testicular cells of irradiated mice, in comparison with bone marrow cells. In unirradiated testicular cells, much higher levels of γ-H2AX were measured by flow cytometry with respect to bone marrow cells. Irradiation induced a redistribution of γ-H2AX into discrete foci detectable by microscopy. In irradiated bone marrow, the percentage of labelled cells peaked at 1 h and rapidly declined, in agreement with data on in vitro cell lines. In contrast, spermatocytes and round spermatids showed persistent labelling until 48 h. During this time, in spermatids, topological changes were observed in γ-H2AX foci from a pattern of many uncountable dots to a pattern of few large spots. Observations of testicular sections confirmed this trend in the reduction of foci number in spite of substantially invariable percentages of labelled cells in the analysed timeframe. To assess whether γ-H2AX persistence in testicular cells was due to unrepaired DNA breaks, we performed comet assay and immunofluorescence analysis of Mdc1, a marker of DDR different from γ-H2AX. Comet assay showed that most breaks were repaired within 2 h. Forty-eight hours after irradiation, contrary to γ-H2AX foci that remained detectable in 80% of initially labelled cells, Mdc1 foci were observed in only 20-30% of cells. These data suggest that, at long times after irradiation, mechanisms additional to impairment of DNA break repair may account for the long persistence of γ-H2AX foci in male germ cells.     

Sesamol ameliorates radiation induced DNA damage in hematopoietic system of whole body γ‐irradiated mice

Ionizing radiation exposure is harmful and at high doses can lead to acute hematopoietic radiation syndrome. Therefore, agents that can protect hematopoietic system are important for development of radioprotector. Sesamol is a potential molecule for development of radioprotector due to its strong free radical scavenging and antioxidant properties. In the present study, sesamol was evaluated for its role in DNA damage and repair in hematopoietic system of γ‐irradiated CB57BL/6 mice and compared with amifostine. C57BL/6 male mice were administered with sesamol 20 mg/kg (i.p.) followed by 2 Gy whole body irradiation (WBI) at 30 min. Mice were sacrificed at 0.5, 3, 24 h postirradiation; bone marrow, splenocytes, and peripheral blood lymphocytes were isolated to measure DNA damages and repair using alkaline comet,γ‐H2AXand micronucleus assays. An increase in % of tail DNA was observed in all organs of WBI mice. Whereas in pre‐administered sesamol reduced %DNA in tail (P ≤ 0.05). Sesamol has also reduced formation of radiation induced γ‐H2AX foci after 0.5 h in these organs and further lowered to respective control values at 24 h of WBI. Similar reduction of % DNA in tail and γ‐H2AX foci were observed with amifostine (P ≤ 0.05). Analysis of mnPCE frequency at 24 h has revealed similar extent of protection by sesamol and amifostine. Interestingly, both sesamol and amifostine, alone and with radiation, also increased the granulocytes count significantly compared to the control (P ≤ 0.05). These findings suggest that sesamol has strong potential to protect hematopoietic system by lowering radiation induced DNA damages and can prevent acute hematopoietic syndrome in mice. Environ. Mol. Mutagen. 59:79–90, 2018. © 2017 Wiley Periodicals, Inc.     

Preferential localization of γH2AX foci in euchromatin of retina rod cells after DNA damage induction

DNA damage may lead to cell transformation, senescence, or death. Histone H2AX phosphorylation, immunodetected as γH2AX foci, is an early response to DNA damage persisting even after DNA repair. In cycling mammalian cells with canonical nuclear architecture, i.e., central euchromatin and peripheral heterochromatin, γH2AX foci map preferentially to euchromatin. Mice retina rods are G0 cells displaying an inverted nuclear architecture 28 days after birth (P28). Rod nuclei exhibit one or two central constitutive heterochromatin chromocenters encircled by facultative heterochromatin. Euchromatin resides at the nuclear periphery, extending to the equator in cells with two chromocenters. To assess the impact of chromatin relocation in the localization of DNA damage, γH2AX and TUNEL foci induced ex vivo by radiomimetic bleomycin were mapped in H3K4me3 immunolabeled P28 rod nuclei. A preferential localization of γH2AX foci in euchromatin was detected together with foci clustering. Besides, a decay of H3K4me3 signal at γH2AX foci sites was observed. TUNEL and γH2AX foci exhibited similar localization patterns in BLM-treated rod cells thus excluding curtailed access of anti-γH2AX antibodies to heterochromatin. Lack of γH2AX foci in rod chromocenters appears to be unrelated to the occurrence of mid-range foci movements. Foci clusters may arise through DNA double-strand break proximity, local non-directional chromatin movements or chromatin relaxation. H3K4me3 signal reduction at γH2AX foci could stem from local chromatin decondensation or downregulation of histone H4 methylation. The observed topology of DNA damage in retina-differentiated rods indicates that euchromatin is damage-prone, regardless of the canonical or inverted nuclear architecture of mammalian cells.     

53BP1 contributes to survival of cells irradiated with X-ray during G1 without Ku70 or Artemis

Ionizing radiation (IR) induces a variety of DNA lesions. The most significant lesion is a DNA double-strand break (DSB), which is repaired by homologous recombination or nonhomologous end joining (NHEJ) pathway. Since we previously demonstrated that IR-responsive protein 53BP1 specifically enhances activity of DNA ligase IV, a DNA ligase required for NHEJ, we investigated responses of 53BP1-deficient chicken DT40 cells to IR. 53BP1-deficient cells showed increased sensitivity to X-rays during G1 phase. Although intra-S and G2/M checkpoints were intact, the frequency of isochromatid-type chromosomal aberrations was elevated after irradiation in 53BP1-deficient cells. Furthermore, the disappearance of X-ray-induced gamma-H2AX foci, a marker of DNA DSBs, was prolonged in 53BP1-deficient cells. Thus, the elevated X-ray sensitivity in G1 phase cells was attributable to repair defect for IR-induced DNA-damage. Epistasis analysis revealed that 53BP1 plays a role in a pathway distinct from the Ku-dependent and Artemis-dependent NHEJ pathways, but requires DNA ligase IV. Strikingly, disruption of the 53BP1 gene together with inhibition of phosphatidylinositol 3-kinase family by wortmannin completely abolished colony formation by cells irradiated during G1 phase. These results demonstrate that the 53BP1-dependent repair pathway is important for survival of cells irradiated with IR during the G1 phase of the cell cycle.     

Extremely Low Frequency Magnetic Fields Do Not Induce DNA Damage in Human Lens Epithelial Cells In Vitro

Non‐ionizing radiations, e.g., radiofrequency electromagnetic fields, could induce DNA damage and oxidative stress in human lens epithelial cells (LECs) which can be early events in cataractogenesis. Extremely low frequency magnetic fields (ELF MF) as another common form of man‐made electromagnetic fields has been considered as suspected human carcinogen by International Agency for Research on Cancer (IARC) and become a focus that people play more and more attentions to. This study aimed to determine whether ELF MF can induce DNA damage in cultured human LECs at a relatively low intensity. Human LECs were exposed or sham‐exposed to a 50 Hz ELF MF which produced by a well‐designed exposure system at the intensity of 0.4 mT. DNA damage in human LECs was examined by the phosphorylated form of histone variant H2AX (γH2AX) foci formation assay and further explored with western blot, flow cytometry, and alkaline comet assay. Immunofluorescence analysis showed that 0.4 mT ELF MF did not significantly increase γH2AX foci formation in human LECs after 2, 6, 12, 24, or 48 hr exposure. No significant differences had been detected in γH2AX expression level between the ELF MF‐ and sham‐exposure groups, while no obvious chromosomal DNA fragmentation was detected by alkaline comet assay after ELF MF exposure. The results indicate an absence of genotoxicity in ELF MF‐exposed human epithelial cells and do not support the hypothesis that environmental ELF MF might be causally led to genomic instability via chromosomal damage response processes. Neither short nor long term continuous exposure to 50 Hz ELF MF at 0.4 mT could induce DNA damage in human lens epithelial cells in vitro. Anat Rec, 299:688–697, 2016. © 2016 Wiley Periodicals, Inc.     

Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54(-/-)Ku80(-/-) mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54(-/-)Ku80(-/-) mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54(-/-)Ku80(-/-) animals accumulated foci of gamma-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.     

γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G‐003M on foci generation and persistence. G‐003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co⁶⁰ gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G‐003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G‐003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11‐15 in terminally ill animals. G‐003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re‐appearance of foci is a strong indicator of imminent death in animals, and that G‐003M provides protection against radiation. Environ. Mol. Mutagen. 57:455–468, 2016. © 2016 Wiley Periodicals, Inc.     

Susceptibility of peripheral lymphocytes of brain tumour patients to in vitro radiation-induced DNA damage,a preliminary study

OBJECTIVE: The present investigation aimed to study the susceptibility of lymphocytes collected from brain tumour patients to radiation-induced DNA damage under in vitro conditions. METHODS: The peripheral lymphocytes collected from brain tumour patients were exposed to 2-Gy gamma radiation. Susceptibility of lymphocytes to radiation-induced DNA damage and their repair ability was assessed by alkaline comet assay. RESULTS: Lymphocytes of patients with benign and malignant tumour had a significantly higher (p < 0.001) baseline DNA damage compared to lymphocytes from normal subjects. A significant increase (p < 0.001) in DNA damage was observed immediately after irradiation of lymphocytes from healthy subjects and brain tumour patients. However, at 1 h after irradiation the level of DNA damage dropped significantly (p < 0.001) compared to that of immediately after irradiation of respective groups. DISCUSSION: In conclusion, the lymphocytes of brain tumour patients possess a higher level of basal DNA damage and exhibit a higher susceptibility to a clastogenic agent like radiation.     

High-energy pulse-electron-beam-induced molecular and cellular damage in Saccharomyces cerevisiae

The high-energy pulse electron beam (HEPE) is a new method for mutation breeding. Previously, we demonstrated that HEPE radiation improved thermotolerance and ethanol production of Saccharomyces cerevisiae. To investigate the influence of HEPE on yeast molecular and cellular damage, cells were separately treated with HEPE radiation at different doses (0, 200, 400, 600, 800, 1000, 1200 and 1400 Gy). Based on results obtained, protein leakage and diffusion of intracellular nucleotide and propidium iodide (PI) uptake assays showed that HEPE clearly enhanced the cell membrane permeability of yeast depending on the dose of exposure. Yeast cells treated with HEPE radiation had significantly elevated levels of DNA instability, as detected by the chromosome spreading assay. These results correlated well with the measurement of increased levels of chromosomal aberrations and apoptosis. Intracellular reactive oxygen species (ROS) and caspase 3 activity were also measured in HEPE-applied yeast cells. Caspase 3 appeared to be involved in HEPE-induced apoptosis. Use of dihydroethidium staining and confocal laser scanning microscopy (CLSM) showed increased levels of intracellular ROS as a consequence of augmented pulsing. Moreover, yeast cells retained some photoreactivation capacity when the dose of HEPE exposure was less than 600 Gy. Thereafter, the level of damage was too serious to repair. Thus, photoreactivation had a repair effect upon HEPE radiation-induced damage. The results of our studies provide a possible explanation for the molecular and cellular effects of HEPE radiation upon S. cerevisiae.