Plasmid DNA strand breaks induced by laser irradiation of 193 nm wavelength

DNA of plasmid pBR322 irradiated with laser at a wavelength of 193 mm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type). The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks. The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks. A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks. The inactivation of the plasmid (in AB1157) is mainly determined by the number of directly formed laser-induced single-strand breaks, whereas the contribution of enzymatically produced single- and double-strand breaks is insignificant.     

Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents

The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells.     

In vitro repair of gaps in bacteriophage T7 DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.     

A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II-DNA complex

Amrubicin is a novel, completely synthetic 9-aminoanthracycline derivative. Amrubicin and its C-13 alcohol metabolite, amrubicinol, inhibited purified human DNA topoisomerase II (topo II). Compared with doxorubicin (DXR), amrubicin and amrubicinol induced extensive DNA-protein complex formation and double-strand DNA breaks in CCRF-CEM cells and KU-2 cells. In this study, we found that ICRF-193, a topo II catalytic inhibitor, antagonized both DNA-protein complex formation and double-strand DNA breaks induced by amrubicin and amrubicinol. Coordinately, cell growth inhibition induced by amrubicin and amrubicinol, but not that induced by DXR, was antagonized by ICRF-193. Taken together, these findings indicate that the cell growth-inhibitory effects of amrubicin and amrubicinol are due to DNA-protein complex formation followed by double-strand DNA breaks, which are mediated by topo II.     

Differential role of hydrogen peroxide and organic peroxides in augmenting asbestos-mediated DNA damage: implications for asbestos induced carcinogenesis

The incubation of asbestos with DNA in presence of peroxides augmented DNA damage several fold as compared to the damage caused by individual treatments. Asbestos in presence of hydrogen peroxide causes DNA double strand breaks, damage to its deoxyribose sugar moiety and enhanced DNA fidelity. However, only DNA double strand breaks and enhanced DNA fidelity could be recorded in presence of organic hydroperoxide/peroxide but no DNA sugar damage could be observed. Further, the extent of DNA damage could be correlated to the carcinogenic potential of asbestos fibre. Crocidolite, the most carcinogenic variety of asbestos, produces maximum damage to DNA in presence of both hydrogen peroxide and organic hydroperoxide/peroxide while chrysolite which is only a co-carcinogen produces significantly less DNA damage. The observed differences in DNA damage by hydrogen peroxide and organic hydroperoxide/peroxide have been ascribed to the differential reactivity of DNA with hydroxyl and alkoxy/aryloxy free radicals produced respectively from these inorganic and organic peroxides.     

Efficacy of novel antimicrobials against clinical isolates of opportunistic amebas

Using atomic force microscopy (AFM), we have investigated neutron-induced DNA double-strand breaks in plasmids in aqueous solution. AFM permits direct measurement of individual DNA molecules with an accuracy of a few nanometers. Furthermore, the analysis of the DNA fragment size distribution is non-parametric, whereas other methods are dependent on the model. Neutron irradiation of DNA results in the generation of many short fragments, an observation not made for damage induced by low-LET radiation. These data provide clear experimental evidence for the existence of clustered DNA double-strand breaks and demonstrate that short DNA fragments may be produced by such radiations in the absence of a nucleosomal DNA structure.     

Modelling DNA damage induced by different energy photons and tritium beta-particles

PURPOSE: To model the production of single- and double-strand breaks (ssb and dsb) in DNA by ionizing radiations. To compare the predicted effectiveness of different energy photon radiations and tritium beta-particles. MATERIALS AND METHODS: Modelling is carried out by Monte Carlo and includes consideration of direct energy depositions in DNA molecules, the production of species, their diffusion and interactions with each other and DNA. Computer-generated electron tracks in liquid water are used to model energy deposition and to derive the initial positions of chemical species. Atomistic representation of the DNA in B form with a first hydration shell is used. Photon radiations in the energy range 70keV-1MeV and tritium beta-particles are considered. RESULTS: A tentative increase for dsb yield has been predicted for 70 keV photons and tritium compared with 137Cs. This increase is more pronounced for complex dsb. Double-strand breaks are much more prone compared with ssb to combine with additional strand breaks and base damage, which contributes to break complexity. At least half of DNA breaks are hydroxyl radical mediated. CONCLUSIONS: The developed model makes predictions compatible with features of available experimental data. Break complexity has to be addressed in biophysical modelling when the relative effectiveness of radiations in DNA damage is studied. Obtained data strongly argue against the dominance of direct radiation action in DNA damage in the cellular environment predicted by some theoretical studies.     

Contrast nephrotoxicity: predictive value of urinary enzyme markers in a rat model

Asbestos, proven to be carcinogenic in humans and animals, is reported to have no genotoxic effect. Asbestos workers have an increased risk of lung cancer, mesothelioma, and other tumours. Earlier findings showed that crocidolite can induce DNA strand breaks in cultured rat embryo cells as assessed by nick translation. We investigated DNA double-strand breaks in white blood cells (WBC) of ten workers occupationally exposed to asbestos. According to our results, obtained with neutral filter elution, individuals who had been exposed to asbestos fibres showed two to four times more DNA double-strand breaks (dsb) in white blood cells than ten non-exposed persons. The induced DNA fragments are of about 250 kb (compared to chromosomal DNA of Saccharomyces cerevisiae standard marker). Using additionally the chromosomal DNA protective method of agarose-plugs, DNA fragments in the range of 200 to 1000 kb have been found in the white blood cells of the same ten workers occupationally exposed to asbestos. In the white blood cells of non-exposed subjects no DNA fragments could be detected with this method. Compared to 51 non-exposed persons, elevated anti-ds DNA antibody concentrations were found in ten workers occupationally exposed to asbestos. The fact that workers occupationally exposed to asbestos have distinctly more double-strand breaks and anti-ds DNA antibodies could mean that an increased incidence of DNA-fragments may be an important indicator in the chronic effect of asbestos-associated carcinogenesis. Apparently, the chronic effects of asbestos observed here do not seem to be identical with that of previously reported acute in vitro effects.     

Calcitonin versus etidronate for the treatment of postmenopausal osteoporosis: a meta-analysis of published clinical trials

DNA double-strand breaks are very genotoxic lesions that can result in chromosome aberrations. The current view is that DNA double-strand breaks are repaired most efficiently through homologous recombination in yeast and simple end-joining in mammalian cells. However, recent experiments reveal that both repair pathways are conserved from yeast to mammals, including humans. The challenge ahead is to put the different pieces of the jigsaw together into coherent mechanisms for both pathways and to determine their relative contributions to ionizing-radiation resistance and to the prevention of genetic instability and carcinogenesis.     

DNA damage in folate deficiency

Folate deficiency significantly increases uracil content and chromosome breaks (as measured by micronucleated cells) in human leukocyte DNA. Folate supplementation reduces both the uracil content of DNA and the frequency of micronucleated cells, indicating that uracil misincorporation may play a causative role in folate deficiency-induced chromosome breaks. A calculation is presented to explain how the levels of uracil found in DNA could cause chromosome breaks. Based on this calculation, the frequency of uracil repair events that might result in double-strand DNA breaks increases by 1752-fold. These results are consistent with clinical and epidemiological evidence linking folate deficiency to DNA damage and cancer.     

bcl-2 protein inhibits etoposide-induced apoptosis through its effects on events subsequent to topoisomerase II-induced DNA strand breaks and their repair

Previous studies have shown that bcl-2 overexpression can inhibit apoptosis induced by DNA-damaging agents widely used in cancer chemotherapy, including X-irradiation, alkylating agents (hydroperoxycyclophosphamide, etc.), and topoisomerase II inhibitors (etoposide, etc.). However, little is known about the mechanism by which bcl-2 overexpression inhibits apoptosis triggered by these agents. In this study, we examined whether bcl-2 overexpression could have effects on etoposide-induced DNA damage and its repair. For these experiments, we developed CH31 clones (mouse B-cells) stably transfected with human bcl-2 sense plasmids and compared these clones with a parental CH31 clone or CH31 clones with antisense plasmids. Overexpression of bcl-2 protein inhibited etoposide-induced apoptosis and cytotoxicity. However, there was no or little difference in the production and repair of DNA-protein cross-links, DNA single-strand breaks, and double-strand beaks among a parental CH31 clone and CH31 clones with human bcl-2 sense or antisense plasmids. These findings indicate that (a) apoptosis or cytotoxicity induced by etoposide can be separated into early events (formation of double-strand breaks, DNA single-strand breaks, and double-strand breaks) and later events (secondary DNA fragmentation or cell death) and (b) bcl-2 inhibits apoptosis and cytotoxicity induced by etoposide at some steps between these events.     

Effect of polyamine-induced compaction and aggregation of DNA on the formation of radiation-induced strand breaks: quantitative models for cellular radiation damage

The yield of DNA single-strand breaks, G(SSB), upon gamma irradiation of SV40 DNA and SV40 minichromosomes in aqueous solution under aerobic conditions was determined at physiological ionic strength in the presence of various potential radioprotective agents. Putrescine (PUT), spermidine (SPD), glutathione, trans-4,5-dihydroxy-1,2-dithiane, 2-mercaptoethyl disulfide and cystamine, all at 0.1-10 mM, spermine (SPM, 0.1-1 mM) and WR-33278 (WRSSWR, 0.1-2 mM) lowered G(SSB) of SV40 DNA. These results were expected from the ability of these agents to scavenge OH radical in the bulk solution. However, SPD, above 10 mM, and SPM and WRSSWR, each above 2 mM, produced dramatic radioprotection attributed to polyamine-induced compaction and aggregation of the DNA (PICA effect). The DNA of SV40 minichromosomes was inherently less radiosensitive and was subject to a PICA effect at lower polyamine concentrations, i.e. approximately 5 mM SPD, approximately 0.6 mM SPM and approximately 0.5 mM WRSSWR. The PICA effect decreased G(SSB) for SV40 DNA and minichromosomes by one to two orders of magnitude, depending upon the scavenging capacity of the medium. The final yields were similar for SV40 DNA and minichromosomes and were comparable to the corresponding yield determined for cells. Results for the yield of double-strand breaks indicated that the yield of double-strand breaks, G(DSB), for DNA and minichromosomes is subject to a PICA effect by SPM and SPD comparable to that measured for G(SSB). Values of G(SSB) for SV40 DNA and minichromosomes subjected to the PICA effect were well approximated by calculations based upon a 30-nm cylinder assumed to model their condensed states. The results indicate that a major fraction of the formation of SSBs in condensed DNA and minichromosomes results from nonscavengeable radical intermediates. Minichromosomes subjected to the PICA effect of 2 mM SPM were protected against formation of radiation-induced SSBs 1.5-fold by 20 mM DTT but 5-fold by 10 mM DTT plus 10 mM WR-1065 relative to 2 mM SPM alone. Thus WR-1065 is capable of providing marked protection of compacted and aggregated minichromosomes, a protection ascribed to the chemical repair of DNA radicals by WR-1065.     

Thyrotropin receptor expression in adrenal, kidney, and thymus

Inflammatory conditions characterized by neutrophil activation are associated with a variety of chronic diseases. Reactive oxygen species are produced by activated neutrophils and produce DNA damage which may lead to tissue damage. Previous studies have shown that activated murine neutrophils induce DNA strand breaks in a target plasmacytoma cell, RIMPC 2394. We studied the effect of a water soluble nitroxide anti-oxidant, Tempol, on murine neutrophil induction of DNA strand breaks in this system. Murine neutrophils were isolated from the peritoneal cavity of BALB/cAn mice after an i.p. injection of pristane oil. Neutrophils were activated by the phorbol ester PMA and co-incubated with RIMPC 2394 cells. Control alkaline elution studies revealed progressive DNA strand breaks in RIMPC cells with time. The addition of Tempol to the incubation mixture prevented DNA damage in a dose dependent fashion. Five mM Tempol provided complete protection. Tempol protection against DNA strand breaks was similar for both stimulated neutrophils and exogenously added hydrogen peroxide. Measurement of hydrogen peroxide produced by stimulated neutrophils demonstrated that Tempol did not decrease hydrogen peroxide concentration. Oxidation of reduced metals, thereby interfering with the production of hydroxyl radical, is the most likely mechanism of nitroxide protection, although superoxide dismutase (SOD) like activity and scavenging of carbon-based free radicals may also account for a portion of the observed protection. The anti-oxidant activity of Tempol inhibited DNA damage by activated neutrophils. The nitroxides as a class of compounds may have a role in the investigation and modification of inflammatory conditions.     

Damage induced by hydroxyl radicals generated in the hydration layer of gamma-irradiated frozen aqueous solution of DNA

Aqueous DNA solutions with or without the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) were exposed to gamma-rays at 77 K. After thawing the solutions, three experiments were carried out to confirm the generation of OH radicals in the hydration layer of DNA and to examine whether they act as an inducer of DNA strand breaks and base alterations. Observation with the ESR-spin trapping method showed ESR signals from PBN-OH adducts in the solution containing PBN and DNA, but there were few signals in the solution containing PBN alone, suggesting that reactive OH radicals were produced in the hydration layer of gamma-irradiated DNA and were effectively scavenged by PBN, and that unreactive OH radicals were produced in the free water layer of gamma-irradiated DNA. Agarose gel electrophoresis of DNA proved that PBN had no effect on the formation of strand breaks, whereas examination with the high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method showed that PBN suppressed the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). From these results it was concluded that OH radicals generated in the hydration layer of gamma-irradiated DNA did not induce DNA strand breaks but induced base alterations.     

Differences in weight gain in relation to race, gender, age and education in young adults: the CARDIA Study. Coronary Artery Risk Development in Young Adults

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, to O2 and H2O2, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may be injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage phi X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H2O2. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.     

Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase.     

Activation of nuclear factor kappa B inflammatory bowel disease

Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing double-strand DNA breaks. V(D)J recombination is a double-strand DNA breakage and rejoining process that relies on NHEJ for the joining steps. Here we show that the targeted disruption of both DNA ligase IV alleles in a human pre-B cell line renders the cells sensitive to ionizing radiation and ablates V(D)J recombination. This phenotype can only be reversed by complementation with DNA ligase IV but not by expression of either of the remaining two ligases, DNA ligase I or III. Hence, DNA ligase IV is the activity responsible for the ligation step in NHEJ and in V(D)J recombination.     

Comparison of strand breaks in plasmid DNA after positional changes of Auger electron-emitting iodine-125

To elucidate the kinetics of the induction of DNA strand breaks by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA after the decay of 125I (1) in proximity to DNA after minor-groove binding (125I-iodoHoechst 33342, 125IH) and (2) at a distance from DNA (125I-iodoantipyrine, 125IAP). Iodine-125 bound to the minor groove in DNA or free in solution is equally effective per decay in producing single-strand breaks (SSBs), while 125I bound to the minor groove is 6.7-fold more efficient than 125I free in solution in producing double-strand breaks (DSBs) (1.08 +/- 0.13 compared to 0.16 +/- 0.01 DSB/decay). Consequently, SSB to DSB ratios for 125IAP and gamma radiation (20.7 +/- 2.9 and 43.8 +/- 1.5, respectively) are greater than that for 125IH (2.9 +/- 0.4). Finally, the decay of 125IH leads to fragmentation of plasmid DNA beyond SSBs and DSBs.     

Radiolysis of DNA in the presence of a protein studied by HPL-gel chromatography

The influence of bovine serum albumin (BSA) on the radiolysis of double-stranded DNA was studied by measuring the loss of highly polymerized DNA with HPL-gel chromatography. The scavenger capacity of BSA for OH.-radicals kBSA [BSA] was kept constant. at 7.8 x 10(5) s-1, when DNA (0.1 mg/ml) was irradiated under different gas conditions (air, N2 and N2O), at pH 7 and 5 and with different ionic conditions. The resulting protein radicals react with DNA producing DNA protein crosslinks and DNA double-strand breaks. The yield and the kind of DNA damage depend on the nature of the protein radicals and their association with DNA. High phosphate concentration prevents the association of BSA with DNA and causes a reduction of the protection by BSA against double-strand break-age of DNA. Radiolysis in the presence of BSA in perchlorate solution leads to more strand breakage and less protein crosslinking than in phosphate solution because perchlorate is more chaotropic than phosphate. Changing the pH from 7 to 5 increases the protection by BSA against DNA strand breakage.