DNA-damage processing in a radiation-sensitive mouse cell line

The induction and repair of radiation-induced DNA damage was assessed in 3 mouse cell lines, including the parental L cell line, a radiation-sensitive, SL3-147 mutant cell line and the H5 revertant to radiation resistance. The yield of neither radiation-induced DNA single- nor double-strand breaks could explain the variable sensitivity of the 3 cell lines. Closure of DNA single-strand breaks proceeded at a similar rate in both the L and SL3-147 cell lines. Closure of DNA double-strand breaks however was significantly slower and less complete in the SL3-147 cell line than in either of the radiation-resistant cell lines. The results are consistent with the increased radiation sensitivity of the SL3-147 cell line resulting from a defect in their ability to repair radiation-induced DNA double-strand breaks.     

Dose- and time-dependent responses for micronucleus induction by X-rays and fast neutrons in gill cells of medaka (Oryzias latipes)

Medaka fish (Oryzias latipes) were exposed to various doses of X-rays or fast neutrons, and the frequency of micronucleated cells (MNCs) was measured in gills sampled at 12- or 24-hr intervals from 12 to 96 hr after exposure. The resulting time course of MNC frequency was biphasic, with a clear peak 24 hr after exposure, irrespective of the kind of radiation applied and the dose used. The half-life of MNCs induced in the gill tissues by the two exposures fluctuated around 28 hr, with no significant dose-dependent trend for either X-ray- or neutron-exposed fish. As assayed 24 hr after exposure, the MNC frequency increased linearly over the control level with increasing doses of both X-rays and fast neutrons. The relative biological effectiveness (RBE) of fast neutrons to X-rays for MNC induction was estimated to be 4.3 +/- 0.6. This value is close to the RBE value of 5.1 +/- 0.3 reported for fast neutron induction of somatic crossing-over mutations in Drosophila melanogaster that arise from recombination repair of DNA double-strand breaks. These results and other data support our conclusion that the medaka gill cell micronucleus assay is a reliable short-term test for detecting potential inducers of DNA double-strand breaks.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant,BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells show normal strand break repair kinetics.The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells.Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alterations in DNA strand breaks induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.     

Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line

DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate.     

Assessment of DNA strand breaks induced by bleomycin in barley by the comet assay

Comet assay was applied to study induction and repair of DNA damage produced by bleomycin in barley genome. Experimental conditions were adapted to achieve efficient detection of both DNA single- and double-strand breaks. Substantial increase of the parameter "% of DNA in tail" was observed coupled with almost linear dependence from bleomycin concentration, more pronounced for the induction of DNA double-strand breaks. Data obtained at different recovery periods displayed rapid restoration of breakage, revealing that efficient mechanisms for repair of strand discontinuities induced by bleomycin are functional in barley DNA loop domains.     

Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radioation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.     

Comparison of Monte Carlo calculated electron slowing-down spectra generated by 60Co gamma-rays,electrons, protons and light ions

When analysing the factors affecting the relative biological effectiveness (RBE) of different radiation qualities, it is essential to consider particularly the low-energy slowing-down electrons (around 100 eV to 1 keV) since they have the potential of inflicting severe damage to the DNA. We present a modified and extended version of the Monte Carlo code PENELOPE that enables scoring of slowing-down spectra. mean local energy imparted spectra and average intra-track nearest-neighbour energy deposition distances of the secondary electrons generated by different radiation qualities, such as electrons, photons, protons and light ions in general. The resulting spectra show that the low-linear energy transfer (LET) beams, 60Co gamma-rays and electrons with initial energies of 0.1 MeV and higher, have as expected approximately the same electron slowing-down fluence per unit dose in the biologically important low-energy interval. Consistent with the general behaviour of the RBE of low-energy electrons, protons and light ions, the low-energy electron slowing-down fluence per unit dose is larger than for low-LET beams, and it increases with decreasing initial projectile energy.     

DNA damage and aging

The hypothesis discussed here is that a major component of aging in metazoans is oxidative damage to nuclear DNA. Such a viewpoint would be consistent with the fact that all of the thus far identified premature aging syndromes in mammals involve mutations in nuclear proteins. Several of these nuclear proteins are enzymes that are related to DNA metabolism or DNA repair. Among the single- and double-stranded DNA damage repair pathways present in eukaryotes, only one pathway often fails to restore the full information content of the genome and typically would result in a deletion of a few base pairs. This pathway is called nonhomologous DNA end joining (NHEJ) and it is a major pathway for the repair of double-strand DNA breaks. Repetitive DNA content may determine the extent to which any organism can use this pathway, and therefore, may dictate a key factor in the balance between oxidation and organismal lifespan.     

Adaptive response to DNA and chromosomal damage induced by X-rays in human blood lymphocytes

Nucleoid sedimentation, single-cell gel electrophoresis (comet assay) and premature chromosome condensation (PCC) technique were utilized to estimate the involvement of DNA strand breaks and chromosomal damage in radio-adaptive response of stimulated human lymphocytes. Conditioning of cells with 0.02 Gy X-rays rendered them more resistant to single- and double-strand DNA breaks produced by 1 Gy challenging treatment as revealed by the sedimentation behaviour of the nucleoids and the comet assay. Nucleoid sedimentation also demonstrated that adaptive reaction towards X-ray-induced DNA damage is favoured in the presence of oxygen. A concomitant decrease in the amount of interphase chromosomal breaks visualized by PCC under the same experimental conditions was observed. Data indicate that adaptation of human lymphocytes to X-rays is tightly linked to the reduced susceptibility towards generation of DNA and chromosomal breaks. It is proposed that the very persistence of DNA strand discontinuities might serve as a triggering signal for the adaptation of human lymphocytes against ionizing radiation exposure.     

λ-Radiation-induced irreparable damage to DNA of HeLa cells

Exposure of HeLa cells to λ-radiation at 0.1 Gy and then at 5 Gy reduces their ability to repair double-strand DNA breaks to a greater extent than irradiation with a single dose of 5 Gy. Modifying effects of 0.1 Gy on double-strand DNA breaks and on cell survival are observed after irradiation during logarithmic but not stationary phase of growth. Primary λ-induced irreparable double-strand breaks correlates with cell survival regardless the irradiation regime. It is suggested that such a damage is primarily responsible for reproductive death of HeLa cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 7, pp. 53–56, July, 1997     

Genome instability in the context of chromatin structure and fragile sites

Genomes are exposed to various external stimuli that induce DNA damage in the form of single- or double-stranded DNA breaks. Fragile sites in the human genome are sensitive to genotoxic stress and, when not appropriately repaired, are responsible for chromosomal aberrations, including the gene amplifications observed in a variety of tumors. Moreover, when DNA lesions from different chromosomes are in close proximity and not repaired, the probability of chromosome translocations is greatly increased. These events can be induced by ionizing radiation that, in a majority of cells, induces a G2/M cell cycle arrest and is characterized by the repositioning of many tumor-related genes closer to the nuclear interior. On the basis of this knowledge, we review functional and structural aspects of chromosomal rearrangements and the DNA repair machinery.     

The Apicomplexan AP2 family: integral factors regulating Plasmodium development

Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to replace missing nucleotides and rejoin broken strands. Little is known about DNA repair in Plasmodium falciparum, including the roles of PNKP in repairing parasite DNA. We identified a P. falciparum gene encoding a protein with 24% homology to human PNKP and thus suggestive of a putative PNKP. In this study, the PNKP gene of P. falciparum strain K1 (PfPNKP) was successfully cloned and expressed in E. coli as a GST-PfPNKP recombinant protein. MALDI-TOF/TOF analysis of the protein confirmed the identity of PfPNKP. Assays for enzymatic activity were carried out with a variety of single- and double-stranded substrates. Although 3'-phosphatase activity was detected, PfPNKP was observed to dephosphorylate single-stranded substrates or double-stranded substrates with a short 3'-single-stranded overhang, but not double-stranded substrates that mimicked single-strand breaks. We hypothesize that unlike human PNKP, PfPNKP may not be involved in single-strand break repair, since alternative terminal processing mechanisms can substitute for PfPNKP, and that PfPNKP DNA repair actions may be confined to overhanging termini of double-strand breaks.     

The effect of mild hypothermia (34 degrees C) and mild hyperthermia (39 degrees C) on DNA damage, repair and aging of human diploid fibroblasts

We used mild hypothermia (34 degrees C) and mild hyperthermia (39 degrees C) to examine aging at the cellular level in relation to DNA damage and repair. With the filter elution technique we monitored spontaneous single-strand breaks (SSBs) and double-strand breaks (DSBs) in DNA during in vitro aging at 34 degrees C, 37 degrees C and 39 degrees C of normal human diploid fibroblasts (HDF). DNA repair was assessed after ionizing and non-ionizing (ultraviolet) radiation of HDF at different population doubling levels (PDLs): the former was assayed by filter elution and the latter by unscheduled DNA synthesis. Survival was assessed by trypan blue dye exclusion and colony formation. Cells at 37 degrees C achieve a higher cumulative PDL (67 +/- 6) than cells at 39 degrees C (60 +/- 5) or at 34 degrees C (55 +/- 6). The level of spontaneous SSBs and DSBs, and radiosensitivity of DNA to either 6 Gy or 100 Gy gamma rays, do not change with in vitro age at any of the three temperatures. Repair of SSBs (induced by 6 Gy) and DSBs (induced by 100 Gy) does not change with in vitro age: rejoining is 86-104% complete by 60 min repair and generally does not differ across temperatures. Response to non-ionizing radiation (254 nm, 75, 150, 300 ergs/mm2) does not change with in vitro age at 37 degrees C or 39 degrees C, whereas excision repair increases with age at 34 degrees C even though cell survival does not. The results do not support the rate of living theory of aging (Pearl, R., The Rate of Living, University of London Press, London, 1928) as applied to temperature effects on HDF aging in vitro (as measured by proliferative lifespan) and on their response to radiation-induced DNA damage.     

Cell cycle-dependent repair of double-strand DNA breaks in a γ-ray-sensitive Chinese hamster cell

A Chinese hamster cell mutant has been isolated which is extremely sensitive to killing by -irradiation in the G₁, and early S phases of the cell cycle (LD₅₀ of 20 vs. 250 rads for parent), but which has nearly normal resistance in late S. The mutant cell is able to repair single-stranded DNA breaks introduced by -radiation. However, in comparison to its parental cell, the mutant is deficient in the repair of double-stranded DNA breaks produced by -irradiation during the sensitive G₁-early S period, while in the resistant late S period, the repair is nearly the same for both cell types. This correlation between -ray sensitivity and repair strongly suggests that an inability to repair double-strand DNA breaks in G₁ is the basis for the hypersensitivity of the mutant to killing by -rays in this phase of the cell cycle. It also provides direct evidence in mammalian cells that the ability to repair double-strand DNA breaks induced by ionizing radiation is an important biochemical function in cell survival and supports the hypothesis that unrepaired double-strand breaks are a major lethal lesion in mammalian cells. A plausible explanation for the appearance of the cell cycle phenotype of the mutant is that in normal cells there are at least two pathways for the repair of double-strand breaks, one of which functions primarily in late S phase, and the other, either throughout the cell cycle or only in the G₁ and early S phases.     

Evidence to support the existence of efficient DNA double-strand break rejoining in a radiosensitive mutant of V79-4 following irradiation with 250 kVp X-rays or neutrons

The repair of ionising-radiation-induced DNA double-strand break type damage was measured by Kohn neutral elution in an X-ray-sensitive mutant of V79-4, irsl. This was done in order to investigate further the likelihood that irsl carries a defect which leads to error-prone repair of DNA damage, and not simply a reduced ability to rejoin DNA double-strand breaks.The mutant displayed an equal increase in sensitivity to the lethal of neutrons, as compared to X-rays. Both irsl and V79-4 showed an increased sensitivity to the killing effects of neutrons of around 2 at 10% survival. irsl also showed an exponential survival after either X-rays or neutrons.The induction of DNA double-strand breaks was measured in both cell lines over a dose range of 10–40 Gy using Kohn neutral filter elution. Induction of breaks by X-rays in irsl seemed to increase slightly with dose, relative to induction in V79-4, so that at 40 Gy 1.5 times more DNA double-strand breaks were measured in irsl cells than in V79-4. Neutron irradiation resulted in a more similar level of induction in either strain after 10–40 Gy. This difference in induction of damage may be due to a different cell-cycle composition in either cell line.The rejoining of X-ray induced double-strand breaks showed a very similar pattern (on a percentage rejoined basis) in both cell lines, although from the induction data at 40 Gy, the dose at which rejoining was measured, fewer breaks were rejoined in V79-4 but also fewer breaks remained unsealed. Neutron-induced breaks, however, were rejoined more efficiently in irsl again on a percentage basis, but also in absolute terms since similar induction was seen after 40 Gy. This data, together with the differences seen in the rejoining of X-ray compared to neutron induced breaks, may indirectly support the proposal that irsl is a misrepair mutant.     

Specificity of protein-RNA and protein-protein interaction upon assembly of TMV in vivo and vitro.

Decay of ³²P incorporated into the DNA of bacteriophage T4D inactivates the phage and produces single- and double-strand breaks in phage DNA, all with single-hit kinetics. The lethal efficiency for ³²P-labeled phage stored in buffer at 4° was 0.10, and double-strand breaks in the DNA are formed at the same rate. When the storage medium is supplemented with 2.8% (w/v) AET (2-aminoethyl isothiouronium bromide hydrogen bromide, a free-radical trap), double-strand breaks and lethal damages occur at the rate of 0.06 per ³²P decay. This suggests that double-strand breaks are the lethal damages. Single-strand breaks accumulate at the rate of one per ³²P decay for ³²P-labeled phage stored in buffer at 4°. The lethal efficiency of ³³P for phage stored in buffer is about 65% that of ³²P. The protective effect of AET is nearly as great for ³³P-labeled T4D as it is for ³²P-labeled T4D. For ³²P, between 35 and 89% of the lethality is due to recoil. Not more than 10% of the lethality is due to radiation effects, and the remainder (if any) is due to transmutation. For ³³P, recoil accounts for less than half (probably no more than 5%) of the lethality. Radiation can account for nearly half of the lethality. Transumutation could account for all of the lethality and probably accounts for over half.     

Monte Carlo simulation of DNA damage induction by x-rays and selected radioisotopes

To better assess the potential biological consequences of diagnostic x-rays and selected gamma-emitting radioisotopes used in brachytherapy, we used the PENELOPE Monte Carlo radiation transport code to estimate the spectrum of initial electrons produced by photons in single cells and in an irradiation geometry similar to those used in cell culture experiments. We then combined estimates of the initial spectrum of electrons from PENELOPE with DNA damage yields for monoenergetic electrons from the fast Monte Carlo damage simulation (MCDS). The predicted absolute yields (Gbp(-1) Gy(-1)) and RBE values for single-strand break (SSB) and double-strand break (DSB) induction by 220 kVp x-rays are within 1% of the results from detailed track-structure simulations (Friedland et al 1999 Radiat. Environ. Biophys. 38 39). The measured RBE for DSB induction reported by Kühne et al (2005 Radiat. Res. 164 669) for gamma-rays from (60)Co and for 29 kVp x-rays with a 50 microm Rh (mammography) filter are in excellent agreement (1.15 versus 1.16). DSB yields predicted by the MCDS also agree to within 7% with the absolute DSB yields reported by de Lara et al (2001 Radiat. Res. 155 440) and Botchway et al (1997 Radiat. Res. 148 317) for the irradiation of V79 cells by low energy (<2 keV) characteristic x-rays. The predicted RBE for DSB induction by gamma-rays from bare (169)Yb and (131)Cs to (60)Co are 1.06 and 1.14, respectively. Tabulated RBE values for the single-cell and monolayer cell culture geometries differ by at most 15%. The proposed methodology is computationally efficient and may also be useful for the prediction of damage yields for mixtures of other types of charged particles, such as those found in proton therapy, space applications or internal dosimetry.     

A description of the damaged DNA produced during tissue injury

Warm ischemia produces DNA damage which is characterized by both single- and double-strand breaks with 5'-PO4 and 3'-OH ends. In contrast, cold ischemia produces mostly single-strand breaks for the first 60 hr and then, abruptly, double-strand damage is produced. Cold ischemia produces both 5'-OH and 5'-PO4 termini, but 5'-OH ends do not appear until after 24 hr of storage. Cold ischemia, also produces 3'-PO4 ends but we have not found any 3'-OH termini. Mechanisms for DNA degradation during warm and cold ischemia are presented to account for these results. It is suggested that damage to the genetic code may be prevented if degradation of the DNA can be confined to the production of the easily repairable 5'-PO4/3'-OH opposed ends.