Protective Role of Glutathione Synthesis on Radiation-Induced DNA Damage in Rabbit Brain

1. Radiotherapy has attracted increasing interest in recent years. It is known that ionizing radiation induces oxygen radical injury, whereas oxidative stress by the radiation can cause cellular responses to defense cellular injury. In this study, the metabolism of antioxidants in response to ionizing radiation to the brain was studied in the brain using experimental rabbits.2. Ionizing radiation to the hemicerebrum caused an increase in the levels of glutathione (GSH) and the activity of a GSH synthesizing enzyme, -glutamylcysteine synthetase (-GCS), and Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Ionizing radiation also induced DNA-damage estimated by the formation of 8-hydroxydeoxyguanosine. These changes were dependent on the radiation dose.3. Previous intrathecal-administration of buthionine sulfoximine (100 M), a specific inhibitor of -GCS, increased DNA damage by radiation in the radiated hemicerebrum. That of S-methyl GSH, on the other hand, resulted in a significant reduction of DNA damage by radiation.4. These results suggest that synthesis of GSH and Cu,Zn-SOD is responsive to ionizing radiation and this induction of antioxidants may play a role in reducing tissue damage in radiotherapy.     

Dds20 operates in Cds1-independent mechanism of tolerance to UV-induced DNA damage in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> cells

Repair of DNA double-stranded breaks caused by ionizing radiation or cellular metabolization, homologous recombination, is an evolutionary conserved process controlled by RAD52 group genes. Genes of recombinational repair also play a leading role in the response to DNA damage caused by UV light. Cells with deletion in gene dds20 of recombinational repair were shown to manifest hypersensitivity to the action of UV light at lowered incubation temperature. Epistatic analysis revealed that dds20 ⁺ is not a member of the NER and UVER gene groups responsible for the repair of DNA damage induced by UV light. The Dds protein has functions in the Cds1-independent mechanism of UV damage tolerance of DNA.     

Protective effects of melatonin against oxidation of guanine bases in DNA and decreased microsomal membrane fluidity in rat liver induced by whole body ionizing radiation

The aim of the study was to examine the potential protective effect of melatonin against whole body ionizing radiation (800 cGy). Changes in 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, an index of DNA damage, and alterations in membrane fluidity (the inverse of membrane rigidity) and lipid peroxidation in microsomal membranes, as indices of damage to lipid and protein molecules in membranes, were estimated. Measurements were made in rat liver, 12 h after their exposure to radiation. To test the potential protective effects of melatonin, the indole was injected (i.p. 50 mg/kg b.w.) at 120, 90, 60 and 30 min prior to radiation exposure. Both 8-OH-dG levels and microsomal membrane rigidity increased significantly 12 h after radiation exposure. Melatonin completely counteracted the effects of ionizing radiation. Changes in 8-OH-dG levels and membrane fluidity are early sensitive parameters of DNA and microsomal membrane damage, respectively, induced by ionizing radiation and our findings document the protective effects of melatonin against ionizing radiation.     

Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.     

Clustered DNA damage on subcellular level: effect of scavengers

Clustered DNA damages are induced by ionizing radiation, particularly of high linear energy transfer (LET). Compared to isolated DNA damage sites, their biological effects can be more severe. We investigated a clustered DNA damage induced by high LET radiation (C 290 MeV u^?1 and Fe 500 MeV u^?1) in pBR322 plasmid DNA. The plasmid is dissolved in pure water or in aqueous solution of one of the three scavengers (coumarin-3-carboxylic acid, dimethylsulfoxide, and glycylglycine). The yield of double strand breaks (DSB) induced in the DNA plasmid-scavenger system by heavy ion radiation was found to decrease with increasing scavenging capacity due to reaction with hydroxyl radical, linearly with high correlation coefficients. The yield of non-DSB clusters was found to occur twice as much as the DSB. Their decrease with increasing scavenging capacity had lower linear correlation coefficients. This indicates that the yield of non-DSB clusters depends on more factors, which are likely connected to the chemical properties of individual scavengers.     

Preserving Genome Integrity: The DdrA Protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.     

Suppression of E. multilocularis Hydatid Cysts after Ionizing Radiation Exposure

Background

Heavy-ion therapy has an advantage over conventional radiotherapy due to its superb biological effectiveness and dose conformity in cancer therapy. It could be a potential alternate approach for hydatid cyst treatment. However, there is no information currently available on the cellular and molecular basis for heavy-ion irradiation induced cell death in cystic echinococcosis.

Methododology/Principal Findings

LD50 was scored by protoscolex death. Cellular and ultrastructural changes within the parasite were studied by light and electron microscopy, mitochondrial DNA (mtDNA) damage and copy number were measured by QPCR, and apoptosis was determined by caspase 3 expression and caspase 3 activity. Ionizing radiation induced sparse cytoplasm, disorganized and clumped organelles, large vacuoles and devoid of villi. The initial mtDNA damage caused by ionizing radiation increased in a dose-dependent manner. The kinetic of DNA repair was slower after carbon-ion radiation than that after X-rays radiation. High dose carbon-ion radiation caused irreversible mtDNA degradation. Cysts apoptosis was pronounced after radiation. Carbon-ion radiation was more effective to suppress hydatid cysts than X-rays.

Conclusions

These studies provide a framework to the evaluation of attenuation effect of heavy-ion radiation on cystic echinococcosis in vitro. Carbon-ion radiation is more effective to suppress E. multilocularis than X-rays.     

The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO(3)). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.     

DNA strand breaks in mammalian tissues induced by methylarsenics

DNA damage induced by administration of dimethylarsinic acid (DMAA) to rats and mice was investigated. At 12 h after administration of DMAA, DNA single-strand breaks were induced markedly in lung. The majority of dimethylarsine, one of the main metabolites, in the expired air was excreted within 6–18 h after administration of DMAA to rats. In vitro experiments using nuclei isolated from lung of mice indicated that DNA strand breaks were caused by dimethylarsine. Furthermore, the strand breaks after exposure to dimethylarsine were reduced in the presence of catalase and/or superoxide dismutase. These results strongly suggest that the strand breaks are induced not by dimethylarsine itself but by active oxygen, e.g., O ₂ ^? and ·OH, produced both by dimethylarsine and molecular oxygen. When DNA was exposed to dimethylarsine, thiobarbituric acid (TBA)-reactive intermediates andcis-thymine glycol were produced. Dimethylarsine appears to induce DNA damage by the mechanism similar to the damage produced by ionizing radiation.     

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.     

Repair of γ-Ray-induced Thymine Damage in <Emphasis Type="Italic">Micrococcus radiodurans</Emphasis>

MOST attempts to correlate the biological effect of ionizing radiation with the damage introduced into DNA have focused on radiation-induced strand breakage. Little is known about the extent of the destruction of the heterocyclic bases in DNA after exposure of cell cultures to ionizing radiation, probably because of the lack of reliable methods for the detecting and auantifying of such damage. We have developed a sensitive radiochemical procedure for the detection and determination of the principal radiolysis product(s) of thymine, 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine (Ia) and/or 5,6-dihydroxy-5,6-dihydrothymine (Ib)^1–3. (Although Ia is the major radiolysis product of thymine formed in the presence of oxygen, several related dihydrothymine derivatives are also obtained (R. Teoule and J. Cadet, personal communication and ref. 4).) By this method we have studied thymine damage in the DNA of Micrococcus radiodurans caused by exposure to γ-rays of cobalt-60. It seems that Ia and/or Ib are important products formed in DNA by γ-rays in vivo and that these products are removed from the DNA during incubation after irradiation.     

Changes in DNA conformation induced by gamma irradiation in the presence of copper

Gamma irradiation of DNA solutions containing copper causes changes in DNA conformation in oligonucleotides and in natural and synthetic DNAs. Diagnostic for these conformational changes is a ubiquitous 187-nm peak in the circular dichroism (CD) difference spectrum that has been predicted for a transformation from a right-handed to a left-handed helical DNA conformation. Changes in CD are correlated with changes in the UV spectrum. Reduction of DNA-bound Cu(II) to Cu(I) with ascorbic acid produces similar changes in CD spectra. These changes can be produced by the peroxy radical anion (O2*-) and the OH radical in the presence of copper. O2*- is approximately twice as efficient as *OH in initiating these changes in natural DNA. The changes in DNA conformation induced by ionizing radiation are remarkable in that they are dependent on the copper-ion concentration in a highly nonlinear manner at low copper concentrations and are not observed in the absence of copper ions. Possible implications of our results for radiobiological and oxidative damage in the cell nucleus are discussed.     

The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX

Background

Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated.

Methods

Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting.

Results

In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2.

Conclusions

Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1.

General significance

Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.     

Methodologies for predicting the expected combined stochastic radiobiological effects of different ionizing radiations and some applications

A methodology for predicting the expected combined stochastic radiobiological effects of sequential exposure to different ionizing radiations is used to arrive at a methodology for predicting the radiobiological effects of simultaneous exposure. Both methodologies require developing additive-damage dose-effect models. Additive-damage dose-effect models are derived assuming (a) each radiation comprised by the combined exposure produces initial damage called critical damage that could lead to the radiobiological effect of interest; (b) doses of different radiations that lead to the same level of radiobiological effect (or risk) can be viewed as producing the same amount of critical damage and being indistinguishable as far as the effects of subsequently administered radiation. Derived dose-effect functions that describe the risk per individual, conditional on radiation dose, are called risk functions. The methodologies allow the use of known radiation-specific risk functions to derive risk functions for combined effects of different radiations. The risk functions for combined exposure to different radiations are called global risk functions. For sequential exposures to different ionizing radiations, the global risk functions derived depend on how individual radiation doses are ordered. Global risk functions can also differ for sequential and simultaneous exposure. The methodologies are used to account for some previously unexplained radiobiological effects of combined exposure to high and low linear-energy-transfer radiations.     

The Caenorhabditis elegans Werner Syndrome Protein Functions Upstream of ATR and ATM in Response to DNA Replication Inhibition and Double-Strand DNA Breaks

WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability. Relatively little is known as to how WRN-1 functions in DNA repair and DNA damage signaling. Here, we take advantage of the genetic and cytological approaches in C. elegans to dissect the epistatic relationship of WRN-1 in various DNA damage checkpoint pathways. We found that WRN-1 is required for CHK1 phosphorylation induced by DNA replication inhibition, but not by UV radiation. Furthermore, WRN-1 influences the RPA-1 focus formation, suggesting that WRN-1 functions in the same step or upstream of RPA-1 in the DNA replication checkpoint pathway. In response to ionizing radiation, RPA-1 focus formation and nuclear localization of ATM depend on WRN-1 and MRE-11. We conclude that C. elegans WRN-1 participates in the initial stages of checkpoint activation induced by DNA replication inhibition and ionizing radiation. These functions of WRN-1 in upstream DNA damage signaling are likely to be conserved, but might be cryptic in human systems due to functional redundancy.     

Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases

Background

Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G₁/S and G₂/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation.

Results

Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined.

Conclusion

Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of enhanced BER activities was found in irradiated cells arrested in G2 phase.     

Nativity of the macromolecule of isologous DNA as a condition for its reparative effect on radiation damage

The effect of isologous DNA on the course of postirradiation reparation of meristematic cells ofVicia faba primary roots was studied in detail. A considerable interest was devoted to determinations of fundamental qualitative and quantitative conditions of the above effect of isologous DNA. Main criteria of the effect were both mitotic activity of irradiated cellular population and dynamics of chromosome aberrations induced by radiation. One set of experiments compared the course of reparation as occurred in regard to applied dose of ionizing radiation in native isologous DNA, DNA denaturated by heat and degraded by DNAase, and post-irradiation reparation of induced damages was favorably affected by native isologous DNA only. Another set of results evaluated the dependence of positive reparative effect of native isologous DNA on the length of the molecule demonstrating that in the process of reparation the presence of a complete DNA macromolecule was not essential. The last experimental group was focused on observations on the dependence of the rate of native isologous DNA effect on concentration of applied solution of the macromolecule.