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PubMed1981

Modification of the action of misonidazole. III. The effect of diamide on toxicity and radiosensitization.

Diamide, a compound which depletes cells of reduced glutathione, decreases toxicity of misonidazole to hypoxic cells, as measured by single-strand break (SSB) production, cell inactivation, and uptake of radioactive misonidazole. As a radiosensitizer, the effect of diamide in cell pellets is additive to that of misonidazole. Individually, both misonidazole and diamide enhance SSB production in DNA of cells irradiated under hypoxia in dilute suspension, but a combination of the two drugs does not appear to produce a greater number of SSB than misonidazole alone.

Animals↗

PubMed1982

The effects of 9-beta-D-arabinofuranosyladenine on the repair of DNA strand breaks in X-irradiated Ehrlich ascites tumour cells.

The effects of the DNA synthesis inhibitor 9-beta-D-arabinofuranosyladenine (beta-ara A), a nucleoside analogue of desoxyadenine, on repair of DNA single and double strand breaks (ssb and dsb) were investigated in X-irradiated Ehrlich ascites tumour cells. Repair of ssb was followed using the unwinding method, and repair of dsb was measured with both the unwinding and the neutral sucrose centrifugation methods. Repair of ssb was inhibited in the presence of beta-ara A; however, even at high concentrations some repair took place. It is suggested that this proportion of the breaks (about 30 per cent) are joined by polynucleotide ligase, and do not require insertion of nucleotides. Dsb repair was strongly inhibited by beta-ara A, the inhibition being complete at high concentrations. It seems likely therefore that dsb repair has an absolute requirement for DNA polymerization. When cells were treated with beta-ara A (200 mumols/1, 2 hours) after irradiation dsb repair was inhibited; however, when the drug was washed away, repair of dsb returned. At 6 hours more breaks were found to have persisted in beta-ara A treated cells than in the untreated controls. Cells treated after X-irradiation with beta-ara A for 7 hours at 120 mumols/1 in conditioned medium and afterwards in fresh medium free of beta-ara A for 24 hours showed a higher number of residual dsb than control cells. It is suggested that these residual dsb may be relevant to the increased killing effect caused by adding beta-ara A to X-irradiated cultures.

Animals↗

PubMed1985

Glutathione requirement for the rejoining of radiation-induced DNA breaks in misonidazole-treated cells.

The role of glutathione (GSH) in the rejoining of radiation-induced single-strand DNA breaks (ssb) was studied in human fibroblast cultures sensitized to radiation by a 30 min treatment with 1 mM misonidazole (MISO). Hypoxically irradiated cells, deficient in GSH, either inherently, or due to a 16 h incubation with 1 mM buthionine sulphoximine (BSO), rejoined the breaks after MISO treatment at a lower rate and to a lesser extent than did GSH-proficient cells. Without MISO treatment, the hypoxically induced ssb were rejoined in the GSH-deficient cells as effectively as in the proficient cells. It is concluded that a large proportion of the breaks which arise after hypoxic irradiation in the presence of MISO are of a different type to those which arise in the absence of the drug, and require a particular GSH-dependent, enzymatic repair system. This requirement for rejoining in hypoxically irradiated, MISO-treated cells is similar to that seen earlier in MISO-untreated, oxically irradiated cells, and suggests that the ssb induced by radiation in the presence of MISO or oxygen are of a similar nature.

Buthionine Sulfoximine↗

PubMed1986

The influence of oxygen on the induction of radiation damage in DNA in mammalian cells after sensitization by intracellular glutathione depletion.

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and non-protein-bound SH (NPSH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10-20 per cent for the three types of radiation damage. Also under severely hypoxic conditions (0.01 microM oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0-30 per cent). At somewhat higher concentrations of oxygen (0.5-10 microM) however, the sensitization amounted to about 90 per cent for the induction of ssb and dsb and about 50 per cent for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen.

Animals↗

PubMed1987

Hydroxyl radical-induced strand break formation of poly(U) in anoxic solution. Effect of dithiothreitol and tetranitromethane.

The role of dithiothreitol (DTT) and tetranitromethane (TNM) on the yields of radiation-induced strand break formation in polyuridylic acid (poly(U] was studied in anoxic aqueous solutions at neutral pH by low-angle laser light-scattering. From G (single-strand breaks) as a function of DTT concentration it follows that two different processes lead to OH radical-induced single-strand break (ssb) formation. Only one of the two processes, which accounts for 80 per cent of the ssb formation, is inhibited by DTT, the other one, 20 per cent, is not inhibited. The 'repair' process is attributed to H-donation to the C-6-yl radical of the uracil moiety. The C-6-yl radical is produced by OH addition to the C-5 position of the uracil moiety. It follows that the sugar radicals, in contrast to earlier suggestions, do not seem to be repaired by DTT at the low concentrations used. The strand break formation not inhibited by DTT is induced by radicals other than the uracil-6-yl radical, e.g. the uracil-5-yl or the OH radicals reacting with the sugar moiety. The strong reduction of G(ssb) from 2.3 to 0.2 on addition of TNM is also discussed.

Chemical Phenomena↗

PubMed1987

Determination of G-values for single and double strand break induction in plasmid DNA using agarose gel electrophoresis and a curve-fitting procedure.

Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine the yield of single strand breaks (ssb) and double strand breaks (dsb) as a consequence of X-irradiation. One ssb transforms DNA of the CC form to the nicked circular form (NC), whereas one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments. DNA (30-800 micrograms/ml) was irradiated in air-saturated sodium phosphate buffer. The different forms of DNA were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered by using a curve-fitting procedure. From the quantitative changes of each conformation D37 values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots. The following yields were determined: Gssb 3.4 X 10E-8 molJ-1, and Gdsb 3.3 X 10E-10 molJ-1. Gdsb refers only to those dsb produced directly. Yields are related to strand breaks without further treatment by heat or alkali.

DNA↗

PubMed1988

Hydroxyl radical-induced strand break formation in single-stranded polynucleotides and single-stranded DNA in aqueous solution as measured by light scattering and by conductivity.

Combining conductivity measurements and molecular weight determination by means of low-angle laser light scattering, we have found for the polyribonucleotides (polyuridylic acid (poly(U], polyadenylic acid (poly(A], polycytidylic acid (poly(C] and polyguanylic acid (poly(G] and for single-stranded DNA (ssDNA) that, on average, 8.5 counterions per single-strand break (ssb) are liberated under salt-free conditions. This relationship allows us to estimate, from conductivity measurements alone, G-values of single-strand break formation (G(ssb] for the polydeoxyribonucleotides (polydeoxyriboadenylic acid (poly(dA], polydeoxyribocytidylic acid (poly(dC], polydeoxyribothymidylic acid (poly(dT], polydeoxyribouridylic acid (poly(dU] and polydeoxyriboguanylic acid (poly(dG]. The following G(ssb) values (units of mumol J-1) have been obtained for anoxic conditions: poly(dA), 0.23; poly(dC), 0.14; poly(dT), 0.06; poly(dU), 0.046 and poly(dG), 0.009. Time-resolved conductivity measurements in pulse radiolysis enable us to measure the rate of strand break formation. The rate has been found to be similar for poly(dA) and ssDNA over a range of pH values. Poly(dC) and poly(dU) exhibit conductivity increase components with half-lives similar to those of poly(dA) and ssDNA at corresponding pH values. The implications of these results are discussed.

DNA Damage↗

PubMed1989

Cell killing and DNA damage in Chinese hamster V79 cells treated with hydrogen peroxide.

Hydrogen peroxide induces lesions in cells similar to those from ionizing radiation, by a Fenton-type production of hydroxyl radicals. At 4 degrees C significant levels of DAN single-strand breaks (ssb) could be measured using the alkaline elution technique, after a 20-min incubation with 10(-5) mol dm-3 H2O2. Only at higher concentrations of H2O2 (greater than 10(-4) mol dm-3) where the levels of ssb measured corresponded to that induced by more than 18 Gy of X-rays, was any significant cell killing detected in a clonogenic assay. Cell killing was observed to coincide with the measurement of significant levels of DNA double-strand breaks (dsb) using the filter elution technique at pH 9.6. This suggests that dsb and not ssb are important as regards hydroxyl-radical-induced cell kill, as found for ionizing radiation. The correlation of induced dsb with lethal events showed that the predicted lethal effect of the H2O2-induced dsb was approximately 5 times less than that of X-ray-induced dsb. This is compared with data previously obtained which showed differences in the lethality of dsb with the quality of radiation (Prise et al. 1987).

Animals↗

PubMed1989

Post-ultraviolet DNA synthesis in the absence of repair: role of the single-strand DNA-binding protein.

Post-ultraviolet DNA synthesis kinetics were investigated in the Escherichia coli uvrA recA strain and its isogenic counterpart, overproducing single-strand DNA-binding protein (SSB). It was demonstrated that large quantities of SSB enhance the capacity of the unmodified replisome to use the UV-damaged template for DNA synthesis. DNA thus synthesized is of low molecular weight, as shown by sedimentation in alkaline sucrose gradients. It is therefore suggested that SSB actively participates in the replisome translocation past dimers and/or the initiation of new DNA chains downstream of these lesions.

DNA Repair↗

PubMed1990

Comparative effect of the thiols dithiothreitol, cysteamine and WR-151326 on survival and on the induction of DNA damage in cultured Chinese hamster ovary cells exposed to gamma-radiation.

We compared the ability of three thiols--dithiothreitol (DTT), cysteamine and WR-151326--to protect aerated Chinese hamster ovary cells from the lethal and DNA-damaging effects of gamma-radiation. These results were compared with earlier measurements for WR-1065 and WR-255591. The time-course and the concentration dependence of protection against cell killing was determined after 10 Gy of gamma-rays. The aminothiols cysteamine and WR-151326 protected at much lower extracellular concentrations than the simple thiol DTT; however, there was no clear difference between the behaviour of cysteamine, WR-151326, WR-1065 and WR-255591 in this respect. Protection by DTT and cysteamine was complete within 1 min, whereas for WR-151326, WR-1065 and WR-255591 about 30 min was required before protection began to reach a plateau. Based on these data, complete radiation survival curves were generated for each thiol and protection factors calculated. Effects on the induction of DNA single-strand breaks (ssb) and double-strand breaks (dsb) by gamma-rays were measured using alkaline (pH 12.1) and neutral (pH 7.0 and 9.6) elution, respectively. All three thiols protected against ssb induction, although to a significantly lower extent than against cell killing measured under identical conditions. Each thiol also protected against dsb induction. After high radiation doses the protection factors for dsb induction were also less than the protection factors for cell survival; however, when dsb were assayed using the low-dose replicate plating neutral elution method, the relative effect of each thiol on cell survival and on dsb induction appeared to be equivalent. The hierarchy of protection against both ssb and dsb induction (based on the extracellular thiol concentration required to produce a given degree of protection) was similar to that for cell survival, i.e., WR-151326 congruent to cysteamine less than DTT.

Animals↗

PubMed1990

Determination of the constants of the Alper formula for single-strand breaks from kinetic measurements on DNA in aqueous solution and comparison with data from cells.

On the basis of rate constants measured in aqueous solution for (i) DNA single-strand break (ssb) formation induced by OH radicals, (ii) prevention of ssb formation by reaction of DNA radicals with glutathione, and (iii) addition of O2 to DNA radicals, oxygen enhancement ratios (OER) and K values of the Alper equation have been calculated. The values obtained were compared with OER and K values determined for ssb formation in lambda DNA irradiated in Escherichia coli as a function of the oxygen concentration. Without adjustment of any parameter the two sets of data are similar when the corrected Alper formula is used. The results support the oxygen fixation-thiol repair model of Howard-Flanders and Alper, and indicate that under selected conditions DNA in aqueous solution may serve as a model system for DNA in cells.

Bacteriophage lambda↗

PubMed1990

DNA radiolysis by fast neutrons.

The effects of fast neutron irradiation on DNA were studied using DNA of the pBR322 plasmid (4362 base pairs), and the results compared to those obtained with 60Co gamma rays. Irradiation of the plasmid DNA in solution with a neutrons beam (p34+Be) of the CERI (CNRS Orléans) cyclotron (with a flat energy spectrum from 34 MeV to low energies) results in half the yield of single-strand breaks (ssb), and 1.5 times higher yield of double-strand breaks (dsb) for neutrons as compared to gamma-rays. Possible specificity of the neutron-induced breaks was examined: the scavenging of OH. radicals by 0.1 mol dm-3 ethanol inhibits all neutron-induced ssb, but only 85 per cent of the dsb. For gamma-irradiation, both ssb and dsb are completely inhibited in these conditions. These results suggest at least three different origins for neutron-induced dsb. The occurrence of around 30 per cent of dsb can be explained by a radical transfer mechanism (proposed by Siddiqi and Bothe (1987) for gamma-irradiation). Around 55 per cent of dsb may be due to the non-random distribution of radicals in high-density tracks of the secondary particles of neutrons, which results in a simultaneous attack of the two strands by OH. radicals. These first two processes are both OH.-mediated and thus are sensitive to ethanol. The direct effect of fast neutrons and their secondaries (recoil protons, alpha-particles and recoil nuclei) can account for the remaining 15 per cent of dsb, not inhibited by 0.1 mol dm-3 ethanol.

Cobalt Radioisotopes↗

PubMed1991

DNA radiolysis by fast neutrons. II. Oxygen, thiols and ionic strength effects.

Plasmid DNA was irradiated with fast neutrons, and the protection by cysteamine against strand breakage (ssb and dsb) was evaluated in the presence and absence of oxygen. In the absence of cysteamine no radiosensitizing effect of oxygen was observed. In anoxia the protection factors, PF(ssb) and PF(dsb) of 1 mM cysteamine (in 50 mM potassium phosphate solution) were lower than the PFs observed with gamma-irradiation. The results agree with the radical repair model, in which the thiol competes with the oxygen produced inside the anoxic neutron-irradiated system, according to the 'oxygen-in-the-track' hypothesis. At low ionic strength in air-saturated solutions, positively charged cysteamine protects more efficiently than negatively charged thiolactate. The dependence of the PFs on the charge of the thiol can be explained by the condensation of counter-ions and depletion of co-ions around DNA, predicted by Manning's theory. Based on the same theory, we propose here an explanation of the ionic strength or by thiolactate at low ionic strength is largely due to scavenging of OH. radicals in the bulk solution. At low ionic strength the PF(ssb) of cysteamine is higher for neutrons than for gamma-irradiation. This might suggest different primary lesions for the two types of irradiation.

Cysteamine↗

PubMed1992

A comparison of the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

One of the important temporal stages of radiation action in cellular systems is the chemical phase, where oxygen fixation reactions compete with chemical repair reactions involving reducing agents such as GSH. Using the gas explosion technique it is possible to follow the kinetics of these fast (greater than 1 ms) reactions in intact cells. We have compared the chemical repair kinetics of the oxygen-dependent free radical precursors leading to DNA single-strand and double-strand breaks, measured using filter elution techniques, with those leading to cell killing in V79 cells. The chemical repair rates for DNA dsb (670s-1 at pH 7.2 and 380s-1 at pH 9.6) and cell killing (530s-1) were similar. This is in agreement with the important role of DNA dsb in radiation induced cell lethality. The rate for DNA ssb precursors was significantly slower (210s-1). The difference in rate between DNA ssb and dsb precursors may be explained on the basis of a dsb free radical precursor consisting of a paired radical, one radical on each strand. The instantaneous probability of one or other of these radicals being chemically repaired and not proceeding to form a dsb will be twice that of ssb radical precursor. This agrees well with the concept of locally multiply damaged sites (LMDS) produced from clusters of ionizations in DNA (Ward 1985).

Animals↗

PubMed1996

DNA double- and single-strand breaks induced by accelerated He2+ and N6+ ions in human cells: relative biological effectiveness is dependent on the relative contribution of the direct and indirect effects.

Double (dsb) and single-(ssb) strand breaks induced by accelerated He2+ ions (LET from 10 to 220 keV/micron with > 70% in the range of 35-55 keV/micron, mean LET about 40 keV/micron) and N6+ ions (LET 125 keV/micron) have been determined in three DNA substrates derived from human cells in culture. He ions were 50% more efficient than N ions to induce dsb in intact cells. In the presence of a high concentration of an OH-radical scavenger, dimethylsulphoxide (DMSO), the dsb were reduced by a factor of 4.5 after He ions but only a factor of 1.4 after N ions, i.e. N ions were about a factor of three more efficient in a substrate where direct hits in the DNA dominated. With gamma rays DMSO gives a reduction by a factor of 3.6. In nucleoids, where the histones were removed by salt treatment, dsb induction increased 22, 18 and 12 times for gamma rays, He ions and N ions respectively. Under these conditions dsb is about 1.5-fold higher after He compared with N ions. Total number of breaks, ssb and dsb, were determined for intact cells. Compared with gamma-rays the breaks were reduced by a factor of 4 after He ions and a factor of 14 after N ions, i.e. ssb and dsb seem to be in the same range after nitrogen ions. Although our measurements do not allow an absolute determination of relative biological effectiveness (RBE) for He and N ions induced dsb, we can show that the RBE for N ions increases by a factor of 2.5 if OH radicals are removed, and decreases by a factor of 2 if most of the protection against OH radicals is removed, altogether a variation of a factor of 5. RBE for He ions, on the other hand, is almost independent of irradiation conditions.

Cells, Cultured↗

PubMed1997

Radiosensitivity and double-strand break rejoining in tumorigenic and non-tumorigenic human epithelial cell lines.

Radiosensitivity and repair of DNA damage induced by ionizing radiation and restriction enzymes were investigated in three human epithelial cell lines: two tumorigenic squamous carcinoma cell lines (SCC-4 and SCC-25), and a non-tumorigenic epidermal keratinocyte cell line (RHEK-1). Sensitivity to ionizing radiation was determined using a clonogenic cell survival assay, which showed SCC-4 to be more radiosensitive than SCC-25 and RHEK-1, which in turn displayed about equal sensitivity. Using DNA precipitation under alkaline conditions for the analysis of induction and repair of DNA single-strand breaks (ssb), an increased level of ssb induction was found for SCC-4 while the efficiency of ssb repair was about equal in all three cell lines. Using pulsed-field gel electrophoresis (PFGE) for the measurement of induction and repair of DNA double-strand breaks (dsb), no consistent differences were detected between the three cell lines. A plasmid reconstitution assay was used to determine the capacity to rejoin restriction enzyme-induced dsb in whole-cell extracts prepared from the three cell lines. In these experiments, dsb rejoining was shown to be significantly reduced in the most radiosensitive SCC-4 cell line while it was about equal in RHEK-1 and SCC-25. The results indicate that plasmid reconstitution in cell-free extracts is a sufficiently sensitive assay to detect differences in repair capacity among tumour cell lines of different radiosensitivity which remain undetectable by DNA precipitation and PFGE.

Amino Acid Sequence↗

PubMed2002

Mitochondrial factors modulate the activity of endonuclease G, the major nuclease of Mammalian mitochondria.

Endonuclease G (Endo G), a sugar-nonspecific nuclease preferring single-stranded DNA (ssDNA), is responsible for major nuclease activity in mitochondria. If the enzyme provides an important nicking function for mitochondrial DNA (mtDNA) in vivo, then mitochondrial factors likely exist which modulate the enzyme's activity and prevent cleavage at single-stranded moieties of mtDNA. In the present paper, we report that specific membrane phospholipids, polyamines and single-stranded DNA-binding protein (SSB) appear to exert such effects in vitro. Phosphatidylcholine and phosphatidylethanolamine, the major constituents of the mitochondrial inner membrane, stimulated purified Endo G activity 5- to 10-fold. Spermine at 5-100 microM also stimulated activity about 4-fold. However, at more than 500 microM, the spermine largely inhibited the degradation of ssDNA and duplex DNA. Escherichia coli SSB, which has physicochemical properties analogous to those of mitochondrial SSB (mtSSB), markedly inhibited the degradation of phiX174 ssDNA by Endo G, indicating the possible involvement of mtSSB in vivo in protection of single-stranded regions of mtDNA from nucleolytic attacks.

Animals↗

PubMed2006

DNA oxidative damage and strand breaks in young healthy individuals: a gender difference and the role of life style factors.

The aim of this study was to analyze background levels of DNA damage in young (19-31 years) non-smoking individuals and to correlate damage to gender and life style. DNA single strand breaks (SSB) and alkali labile sites (ALS) were measured in 99 subjects living in Stockholm, Sweden. Further, oxidative DNA damage was analyzed using the DNA repair glycosylase FPG as well as HPLC-ECD for specific analysis of 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). We found that males had higher (P < 0.001) levels of SSB + ALS than females, but no difference was seen for oxidative lesions. There was no correlation between FPG sites and 8-oxodG. For females, there was a positive correlation between FPG levels and body mass index and a negative correlation between SSB + ALS and fruit intake. We conclude that the background level of oxidative DNA damage, analyzed with improved methods, is low and that gender, fruit intake and BMI can affect DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗