‘Fukusensor:’ a genetically engineered plant for reporting DNA damage in response to gamma radiation

Transgenic plants can be designed to be ‘phytosensors’ for detection of environmental contaminants and pathogens. In this study, we describe the design and testing of a radiation phytosensor in the form of green fluorescence protein (GFP)‐transgenic Arabidopsis plant utilizing a DNA repair deficiency mutant background as a host. Mutant lines of Arabidopsis AtATM (At3g48190), which are hypersensitive to gamma irradiation, were used to generate stable GFP transgenic plants in which a gfp gene was under the control of a strong constitutive CaMV 35S promoter. Mutant and nonmutant genetic background transgenic plants were treated with 0, 1, 5, 10 and 100 Gy radiation doses, respectively, using a Co‐60 source. After 1 week, the GFP expression levels were drastically reduced in young leaves of mutant background plants (treated by 10 and 100 Gy), whereas there were scant visible differences in the fluorescence of the nonmutant background plants. These early results indicate that transgenic plants could serve in a relevant sensor system to report radiation dose and the biological effects to organisms in response to radionuclide contamination.     

The effects of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanization

Previously we have shown that growth of melanized fungi is stimulated by low levels of gamma radiation. The goal of this study was to examine the effects of visible light, UV light, and gamma radiation on the energy level (ATP concentration) in melanized Cryptococcus neoformans cells. Melanized C. neoformans cells as well as non-melanized controls were subjected to visible, UV or gamma radiation, and ATP was quantified by measuring the amount of light emitted by the ATP-dependent reaction of luciferase with luciferin. We found that all three forms of radiation led to a reduction in the ATP levels in melanized C. neoformans cells. This points to a universal melanin-related mechanism underlying observation of ATP decrease in irradiated melanized cells. In contrast, in non-melanized cells visible light led to increase in ATP levels; gamma radiation did not cause any changes while UV exposure resulted in some ATP decrease, however, much less pronounced than in melanized cells.     

Gamma radiolysis of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate

The applications of room-temperature ionic liquids in the nuclear fuel cycle and radiation chemistry depend on a comprehensive knowledge of their stability and chemical properties under radiation conditions. In this work, the effect of gamma radiation on pure ionic liquid [bmim][PF6] was investigated in detail. The radiolysis of [bmim][PF6] leads to an increase of UV-vis absorbance and a decrease of fluorescence intensity with increasing radiation dose. Raman spectra proved that gamma radiation induced significant chemical scission of the n-butyl group (e.g. C-H and C-C scission) and damage to the [PF6]- anion. When the irradiated [bmim][PF6] samples were cooled, two crystal structures were found to coexist, and they suffered a continuous destruction under irradiation; their dose dependence, however, was different.     

Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.     

Asymmetric somatic cell hybridization in plants

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.     

Potassium cyanate modification of the toxic and mutagenic effects of gamma radiation and benzo(a)pyrene

In experiments with CHO-AT3-2 cell culture, a study was made of the effect of potassium cyanate (KNCO) on the effect of gamma radiation and benzo(a)pyrene (BP) by the following tests: cell viability, induction of cells with micronuclei and fragmented nuclei and mutations by thymidine kinase (TK) and Na+/K+-ATPase loci. Some tests have revealed the increase in the effect of gamma radiation and BP produced by potassium cyanate. It is suggested that the sensitizing effects are related to repair system inhibition and/or changes in the cell chromatin structure produced by KNCO.     

Role of Ferulic Acid in the Amelioration of Ionizing Radiation Induced Inflammation: A Murine Model

Ionizing radiation is responsible for oxidative stress by generating reactive oxygen species (ROS), which alters the cellular redox potential. This change activates several redox sensitive enzymes which are crucial in activating signaling pathways at molecular level and can lead to oxidative stress induced inflammation. Therefore, the present study was intended to assess the anti-inflammatory role of ferulic acid (FA), a plant flavonoid, against radiation-induced oxidative stress with a novel mechanistic viewpoint. FA was administered (50 mg/kg body wt) to Swiss albino mice for five consecutive days prior to exposing them to a single dose of 10 Gy ⁶⁰Co γ-irradiation. The dose of FA was optimized from the survival experiment and 50 mg/kg body wt dose showed optimum effect. FA significantly ameliorated the radiation induced inflammatory response such as phosphorylation of IKKα/β and IκBα and consequent nuclear translocation of nuclear factor kappa B (NF-κB). FA also prevented the increase of cycloxygenase-2 (Cox-2) protein, inducible nitric oxide synthase-2 (iNOS-2) gene expression, lipid peroxidation in liver and the increase of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in serum. It was observed that exposure to radiation results in decreased activity of superoxide dismutase (SOD), catalase (CAT) and the pool of reduced glutathione (GSH) content. However, FA treatment prior to irradiation increased the activities of the same endogenous antioxidants. Thus, pretreatment with FA offers protection against gamma radiation induced inflammation.     

Induction and assessment of persistent radioresistance in murine leukocytes in vivo

The aim of the present study was to investigate whether weekly exposure to gamma rays causes a persistent increase in the number of radioresistant leukocytes in mice in vivo. Using the comet assay, 1 Gy radiation exposure decreased the percentage of leukocytes with less than 5% DNA in the tail (<5% DNAT), and we propose that radioresistance induction might increase the number of cells with <5% DNAT after radiation exposure. We exposed mice to 1 Gy gamma rays weekly for four weeks or 2 Gy per week for nine weeks. We observed a significant increase in cells with <5% DNAT after the third week and up to nine weeks of exposure. We exposed animals to gradually increasing radiation doses and finally challenged the lymphocytes with 1 Gy radiation both in vivo and in vitro. We observed increased radioresistance in vitro, providing evidence that a cellular process is involved. However, more radioresistance was observed in vivo than in vitro, suggesting a physiological effect. Cells challenged in vitro were maintained on ice during and after exposure, which likely caused a reduction in DNA repair. Radioresistance induction likely arose from mutation selection in stem cells because leukocytes are unable to proliferate in peripheral blood.     

Effect of ionizing radiation on the DNA damage response in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The DNA damage response (DDR) is induced by various DNA damaging factors and maintains genome stability in all organisms. The Chlamydomonas reinhardtii genome contains putative homologous genes involved in DDR; however, little is known about the functions and responses of these genes to DNA damage. In this study, DDR by gamma radiation was determined in C. reinhardtii. Irradiation with 80, and 200 Gy gamma radiation caused death in approximately 47 and 97 % of C. reinhardtii cells, respectively. The absolute lethality of cells was at 300 Gy. The rate of DNA breaks was also determined using comet assays after exposure to different doses of gamma radiation. Irradiation with 80 and 400 Gy resulted in 17 and 34 % of nuclear degradation in C. reinhardtii cells, respectively. To identify the major DDR pathway of C. reinhardtii induced by gamma radiation, 24 putative DDR genes were selected from the Joint Genome Institute (JGI) database. Gamma radiation significantly affected expression of 15 genes among these. Therefore, these genes displaying expressional changes by gamma radiation are involved in DDR, which indicate that C. reinhardtii may possess a fundamental conserved DDR pathway with higher plants. Furthermore, radiation responsive proteins were identified by proteomic analysis, which are involved in metabolisms of carbohydrate, energy, and photosynthesis. This is the first report to describe the responses of DDR homologous genes to gamma radiation and to identify gamma radiation-responsive proteins in C. reinhardtii. Our data should provide molecular insights into gamma radiation responses including DNA damage in green algae.     

Coccomyxa actinabiotis sp. nov. (Trebouxiophyceae,Chlorophyta), a new green microalga living in the spent fuel cooling pool of a nuclear reactor

Life can thrive in extreme environments where inhospitable conditions prevail. Organisms which resist, for example, acidity, pressure, low or high temperature, have been found in harsh environments. Most of them are bacteria and archaea. The bacterium Deinococcus radiodurans is considered to be a champion among all living organisms, surviving extreme ionizing radiation levels. We have discovered a new extremophile eukaryotic organism that possesses a resistance to ionizing radiations similar to that of D. radiodurans. This microorganism, an autotrophic freshwater green microalga, lives in a peculiar environment, namely the cooling pool of a nuclear reactor containing spent nuclear fuels, where it is continuously submitted to nutritive, metallic, and radiative stress. We investigated its morphology and its ultrastructure by light, fluorescence and electron microscopy as well as its biochemical properties. Its resistance to UV and gamma radiation was assessed. When submitted to different dose rates of the order of some tens of mGy · h^?1 to several thousands of Gy · h^?1, the microalga revealed to be able to survive intense gamma‐rays irradiation, up to 2,000 times the dose lethal to human. The nuclear genome region spanning the genes for small subunit ribosomal RNA‐Internal Transcribed Spacer (ITS) 1‐5.8S rRNA‐ITS2‐28S rRNA (beginning) was sequenced (4,065 bp). The phylogenetic position of the microalga was inferred from the 18S rRNA gene. All the revealed characteristics make the alga a new species of the genus Coccomyxa in the class Trebouxiophyceae, which we name Coccomyxa actinabiotis sp. nov.     

Effect of Irradiation on Microorganisms in a Nuclear Reactor

The effects of irradiation in the JRR-1 (Japan Research Reactor No. 1, a homogeneous light water nuclear reactor; max. power, 50 KW) on microorganisms such as bacterial and fungal spores and yeast cells were investigated in comparison with those of ⁶⁰Co gamma radiation. As far as the lethal effect was concerned the dose rate of radiation in the experimental hole No. 16 of the JRR-1 was equivalent to 3.0×l0⁶~3.4×l0⁶ r/hr with ⁶⁰Co gamma radiation, and a ratio of the neutron effect to the gamma radiation effect on microorganisms in this hole was estimated to be approximately 3~5.4. The results different from those with gamma radiation were obtained in experiments such as post-NaCl treatment and spore germination. The considerable contribution of fast neutrons to the total biological effect of neutrons, in comparison with the thermal neutron effect, could be presumed from the microbiological experiments with the help of physical and chemical data. Morphological changes in post-irradiation growth were observed by means of phase contrast microscopy. No specific aftereffect was found.     

Exoemission instrument and technology to explore gamma radiation influence on bones

This article aims at developing a new technology and instrument to explore the effect of gamma radiation on bone material. Exoelectron emission (EEE) phenomenon underlies a new electron spectroscopy to explore alteration of the electronic structurally dependence properties of bone material. The development of EEE technology for exploring the gamma radiation effects on bones is of high importance. Moreover, the influence of gamma radiation with different energies on the bone structure is discussed. It was found that the changes in EEE vary with radiation energy and radiation dose and that the response is non-linear.     

Perspectives of Radioactive Contamination in Nuclear War

The degrees of risk associated with the medical, industrial and military employment of nuclear energy are compared. The nature of radioactive contamination of areas and of persons resulting from the explosion of nuclear weapons, particularly the relationship between the radiation exposure and the amount of physical debris, is examined.Some theoretical examples are compared quantitatively. It is concluded that the amount of radio-activity that may be carried on the contaminated person involves a minor health hazard from gamma radiation, compared to the irradiation arising from contaminated areas.     

DNA radiation damage and asymmetric somatic hybridization: Is UV a potential substitute or supplement to ionising radiation in fusion experiments?

Recent reports have revealed that the asymmetric nature of the nuclear genome of somatic hybrids, produced following the irradiation of one of the parents with X- or gamma rays, is generally much less than had been anticipated. As a consequence, we have begun to investigate whether UV radiation might be used as an alternative or indeed a supplement to the presently-used ionising radiation techniques in such experiments. Cell culture studies have revealed that UV radiation induces the desired physiological effects in sugar beet ( Beta vulgaris ) protoplasts, namely, a prevention of cell division without immediate cytotoxicity. Preliminary studies using denaturing and pulsed field gel electrophoresis have shown that UV can also induce substantial physical fragmentation of DNA. When using the same techniques, less breakdown was observed following gamma radiation. All results were highly reproducible. Such results augur well for the potential use of UV in asymmetric somatic cell fusion experiments.     

On-chip detection of protein glycosylation based on energy transfer between nanoparticles

We describe a chip-based method to detect protein glycosylation based on the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs). Our assay system relies on modulations in the energy transfer between the nanoparticles on a surface. The photoluminescence (PL) of lectin-coated QDs (energy donor) immobilized on a glass slide is quenched by carbohydrate-coated AuNPs (energy acceptor), and the presence of the glycoprotein causes the increase of the PL of QDs. As a proof-of-concept, Concanavalin A-coated QDs (ConA-QDs) and dextran-coated AuNPs (Dex-AuNPs) were used to detect the mannosylated proteins. As a result, the PL intensity of QDs was found to be linearly correlated with the concentration and the number of glycan moiety of the glycoprotein. We anticipated that our simple assay system will find applications for the analysis of glycoproteins with high selectivity and sensitivity in a high-throughput manner.     

Radiation induced oxidative stress: I. Studies in Ehrlich solid tumor in mice

Understanding the response of tumors to ionizing radiation might potentially lead to improvement in tumor control and patient morbidity. Since the antioxidant status is likely to be linked to radioresponse, its modulation needs to be examined. Therefore, Swiss albino male mice (7–8 weeks old) with Ehrlich solid tumors were irradiated with different doses of gamma rays (0–9 Gy) at a dose rate of 0.0153 Gy/s; and enzymes involved in antioxidant functions were determined in the tumors. Radiation effects in terms of oxidative damage, LDH, nitric oxide and DNA fragmentation were also examined.In tumors, the specific activity of SOD was increased with dose but declined 6 Gy onwards. GST, DTD and GSH showed an almost progressive increase. These enhanced activities might have resulted from the increased protein expression. This possibility was supported by the Western Blot analysis for GST protein. These changes might be closely linked to the radiation-induced oxidative stress as reflected by the enhanced levels of peroxidative damage, DNA fragmentation, LDH activity and nitric oxide levels. These findings may have relevance to radiation therapy of cancer as the elevated antioxidant status of irradiated tumors is likely to limit the effectiveness of radiation dose and adversely affect the therapeutic gain.     

In vivo radioprotection by 5-androstenediol: stimulation of the innate immune system

We showed previously that 5-androstenediol stimulates myelopoiesis, increases the numbers of circulating neutrophils and platelets, and enhances resistance to infection in gamma-irradiated mice. We have extended those studies to include monocytes, natural killer (NK) cells, eosinophils and basophils, and we have measured the activation marker CD11b using flow cytometry. Androstenediol (160 mg/kg) was administered subcutaneously to female B6D2F1 mice 24 h before whole-body gamma irradiation. Androstenediol treatments increased the blood levels of neutrophils, monocytes and NK cells in unirradiated animals; decreased the numbers of circulating eosinophils; and ameliorated radiation-induced decreases in neutrophils, monocytes, NK cells, erythrocytes and platelets. The androstenediol treatments had no significant effect on the numbers of circulating B cells or T cells. CD11b labeling intensity on monocytes was decreased slightly after androstenediol treatment. In contrast, radiation or androstenediol alone caused increases in CD11b labeling intensity on NK cells. Androstenediol and radiation combined caused a marked increase in NK cell CD11b. The results indicate that androstenediol increases the numbers of the three major cell types of the innate immune system (neutrophils, monocytes and NK cells), that androstenediol-induced changes in blood elements in irradiated animals persist for at least several weeks, and that there is a significant positive interaction between radiation and administration of androstenediol in the activation of NK cells.     

External gamma radiation and mortality from cardiovascular diseases in the German WISMUT uranium miners cohort study, 1946–2008

It is currently unclear whether exposure of the heart and vascular system, at lifetime accumulated dose levels relevant to the general public (<500 mGy), is associated with an increased risk of cardiovascular disease. Therefore, data from the German WISMUT cohort of uranium miners were investigated for evidence of a relationship between external gamma radiation and death from cardiovascular diseases. The cohort comprises 58,982 former employees of the Wismut company. There were 9,039 recorded deaths from cardiovascular diseases during the follow-up period from 1946 to 2008. Exposures to external gamma radiation were estimated using a detailed job-exposure matrix. The exposures were based on expert ratings for the period 1946–1954 and measurements thereafter. The excess relative risk (ERR) per unit of cumulative gamma dose was obtained with internal Poisson regression using a linear ERR model with baseline stratification by age and calendar year. The mean cumulative gamma dose was 47 mSv for exposed miners (86 %), with a maximum of 909 mSv. No evidence for an increase in risk with increasing cumulative dose was found for mortality from all cardiovascular diseases (ERR/Sv = ?0.13; 95 % confidence interval (CI): ?0.38; 0.12) and ischemic heart diseases (n = 4,613; ERR/Sv = ?0.03; 95 % CI: ?0.38, 0.32). However, a statistically insignificant increase (n = 2,073; ERR/Sv = 0.44; 95 % CI: ?0.16, 1.04) for mortality from cerebrovascular diseases was observed. Data on smoking, diabetes, and overweight are available for subgroups of the cohort, indicating no major correlation with cumulative gamma radiation. Confounding by these factors or other risk factors, however, cannot be excluded. In conclusion, the results provide weak evidence for an increased risk of death due to gamma radiation only for cerebrovascular diseases.