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.     

Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay.

In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.     

Single-strand and double-strand deoxyribonucleic acid breaks produced by several bleomycin analogues

Production of single-strand breaks (ssb) and double-strand breaks (dsb) of PM2 phage DNA by several structurally related bleomycin (BLM) analogues was studied by gel electrophoresis. BLM A2 and BLM B2 produced a comparable extent of dsb. In various experiments, BLM A2 and BLM B2, at 22-41 ng/mL, degraded 50% of the form I DNA into 33-38% form II and 12-17% form III DNA. BLM B1' produced ssb and dsb at a ratio similar to that of BLM A2, but both at a rate less than half that of BLM A2. Phleomycin (PLM) D1 induced an equivalent amount of ssb but only one-eighth of dsb induced by BLM B2. The relatively lower extent dsb production for PLM D1 was observed either in borate buffer (pH 9.5) or in Tris-HCl buffer (pH 7.5) and in the presence or absence of exogenous Fe(II). Deamido-BLM A2 produced ssb to an extent approximately half that of BLM A2 and dsb to less than one-eighth that of BLM A2. The following conclusions were drawn. (1) BLM analogues produced ssb and dsb to different extents and ratios. (2) The ratio of dsb to ssb varied depending on the analogue, indicating a lack of a direct correlation between ssb and dsb. (3) The extent of ssb and dsb was affected by modifications on both the C- and N-terminal half-molecules of BLM: modification of either the N-terminal amide or the bithiazole greatly reduced dsb, whereas changes in structure or charge in the C-terminal amine affected ssb and dsb to a similar extent.     

Cell inactivation and DNA single- and double-strand breaks in cultured mammalian cells irradiated by a thermal neutron beam

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.     

2-Chlorodeoxyadenosine inhibits the repair of DNA double-strand breaks and does not inhibit the repair of DNA single-strand breaks in X-irradiated Chinese hamster V79 cells.

The exposure of log-phase Chinese hamster V79 cells to 2-chlorodeoxyadenosine (CdA) for 3 h after X irradiation enhanced the lethal effects of X-rays in a concentration-dependent manner. The enhancement of the killing efficiency of X-rays by CdA was mainly observed in the reduction of quasi-threshold doses (Dq) of the dose-response curves. When the ability of CdA to inhibit the repair of X-ray-induced double- and single-strand breaks (dsb and ssb) of DNA was investigated by neutral- and alkaline-filter elution techniques, respectively, it was observed that 90% of dsb were rejoined in the absence of CdA within 30 min after X irradiation and 15-40% of dsb rejoining was suppressed by co-incubation of the cells with 5-10 microM of CdA for 3 h after X irradiation, whereas almost 100% of ssb were rejoined within 15 min regardless of the presence or absence of CdA. From these results it was concluded that CdA interfered exclusively with the repair of DNA dsb in X-irradiated Chinese hamster V79 cells and thereby increased the lethality of X-rays.     

Enhanced strand break induction of DNA by resonant metal-innershell photoabsorption

We determined the number of single and double strand breaks (ssb and dsb) in a DNA-chloroterpyridine platinum complex induced by resonant photoabsorption in the L(III) innershell of a platinum atom. The number of ssb and dsb were measured in supercoiled plasmids (AG30) versus the chloroterpyridine platinum concentration, i.e., the ratio of intercalated molecules to the number of phosphate sites in DNA. A significant increase in the number of ssb and dsb was observed when the DNA contained intercalated molecules. This technique is an efficient way to induce ssb and dsb triggered by the atomic Auger effect.     

Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells

Interstitial Telomeric Repeat Sequence (ITRS) blocks are recognized as hot spots for spontaneous and ionizing radiation-induced chromosome breakage and recombination. Background and ionizing radiation-induced DNA breaks in large blocks of ITRS from Chinese hamster cell lines were analyzed using the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. Our results indicate an extremely alkali-sensitivity of ITRS. Furthermore, it appears that ITRS blocks exhibit a particular chromatin structure, being enriched in short unpaired DNA segments. These segments could be liable to severe topological stress in highly compacted areas of the genome resulting in their spontaneous fragility and thus explaining their alkali-sensitivity. The induction and repair kinetics of DNA single-strand breaks (ssb) and DNA double-strand breaks (dsb) induced by ionizing radiation were assessed by DBD-FISH on neutral comets using Chinese hamster cells deficient in either DNA-PKcs or Rad51C. Our results indicate that the initial rejoining rate of dsb within ITRS is slower than that in the whole genome, in wild-type cells, demonstrating an intragenomic heterogeneity in dsb repair. Interestingly, in the absence of DNA-PKcs activity, the rejoining rate of dsb within ITRS is not modified, unlike in the whole genome. This was also found in the case of Rad51C mutant cells. Our results suggest the possibility that different DNA sequences or chromatin organizations may be targeted by specific dsb repair pathways. Furthermore, it appears that additional unknown dsb repair pathways may be operational in mammalian cells.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.     

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-break repair, radioresistance of DNA synthesis, and camptothecin sensitivity in the radiation-sensitive irs mutants: comparisons to ataxia-telangiectasia cells

Induction and rejoining of DNA single-strand breaks (ssb) and double-strand breaks (dsb) after gamma-irradiation were measured, respectively, by alkaline and neutral sucrose gradient sedimentation methods. The radiosensitive mutants irs1, irs2, and irs3 showed no significant difference from wild-type V79 hamster cells in ability to rejoin either ssb or dsb, while the previously-described xrs-1 mutant showed the expected defect in rejoining dsb. The resistance of DNA synthesis to gamma-irradiation was measured in the 3 irs mutants and, for comparative purposes, in transformed human cell lines from normal and ataxia-telangiectasia (A-T) individuals. The irs2 mutant was found to be very similar in response to the A-T lines, showing a marked decrease in inhibition of DNA synthesis, compared to V79 cells, in both time-course and dose-response experiments. However, irs1 also had some decrease in inhibition at the higher doses used, while irs3 was similar to the wild-type V79 cells. Both irs1 and irs2 were found to be considerably more sensitive to the DNA topoisomerase I-inhibitor camptothecin, while irs3 was only slightly more sensitive than the parent V79 line. These data place the irs mutants in a similar category of radiosensitive phenotype to A-T cells, but we view this as only the beginning of a useful classification of this type of mutant. The irs2 mutant has the strongest links to A-T cells, through its sensitivity profile to DNA-damaging agents and radioresistant DNA synthesis, but irs1 in particular has other similarities to A-T.     

Matching of single-strand breaks to form double-strand breaks in DNA

Single-strand breaks (ssb) in opposite strands of DNA can be sufficiently near that a double-strand break (dsb) results. A theory is presented by which the maximum number h of base pairs which cannot prevent double-strand breakage can be determined from the rates of production of ssb and dsb. The assumptions required to derive the necessary equations as well as the range of validity of the equations are discussed in detail. In the experiments ssb and dsb were produced by x-irradiation in buffers which do not eliminate indirect effects and were measured by analytical ultracentrifugation. Values of h have been determined in low and high ionic strength and in low ionic strength over a range of temperatures. The values, 2.64 and 15.8, were obtained for high and low ionic strength, respectively.     

Differential effects of glutathione depletion and metallothionein induction on the induction of DNA single-strand breaks and cytotoxicity by tert-butyl hydroperoxide in cultured mammalian cells

Induction of DNA single-strand breaks (ssb), their resealing and cytotoxicity by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. The effect of the depletion of cellular glutathione (GSH), iron chelation and induction of metallothionein (MT) on these parameters was studied. t-BuOOH in a concentration range of 0.02-0.5 mM induced DNA ssb in a dose-dependent fashion. Strand breakage increased as a function of time up to 1 h. Divalent iron chelator o-phenanthroline suppressed markedly the induction of DNA ssb while the trivalent iron chelator desferrioxamine had no effect. GSH-depletion increased cytotoxicity of t-BuOOH. In contrast, the depletion of GSH did not affect the efficiency of formation of DNA ssb by t-BuOOH and the rate of resealing of the DNA damage. The induction of MT did not influence the efficiency of formation of DNA ssb by t-BuOOH. In summary, while GSH depletion and MT induction affected the formation of DNA ssb and cytotoxicity differently divalent iron was required for both. Therefore, appears likely that DNA breakage and cytotoxicity by t-BuOOH are caused by independent mechanisms.     

Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells

The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1-1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM t-BuOOH were repaired within 60 min of treatment in both GSH-depleted (GSH-) and non-depleted (GSH+) cells. In contrast, chromosomal aberrations increased a little during the 60 min after treatment in both GSH- and GSH+ cells. The aberrations were then repaired in GSH+ cells but those in GSH- cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.     

Differences in accumulation of blunt- and cohesive-ended double-strand breaks generated by restriction endonucleases in electroporated CHO cells.

Restriction endonucleases (RE) have been used in cytogenetic studies to mimic the DNA double-strand break (dsb)-inducing action of radiation. In the experiments presented here, we have treated electroporated CHO cells with RE and have measured the resulting dsb using the filter elution technique under non-denaturing conditions (pH 9.6). PvuII, which generates blunt-ended dsb, gave rise to a significant number of measurable dsb. The frequency of the dsb induced by PvuII is shown to increase over a 3-12-h post-treatment incubation period, which implies that the RE is active in the cell for a considerable length of time. We postulate that the accumulation of dsb reflects a competition between enzymatic incision and repair of the DNA. The presence of araA, a known inhibitor of DNA synthesis, did not affect the frequency of PvuII-induced breaks indicating a lack of an inhibitory effect of araA on the repair of RE-induced dsb. Two RE which cause cohesive-ended dsb, namely BamHI and EcoRI, were found to be ineffective in giving rise to measurable dsb. Our interpretation of this is that for cohesive-ended dsb (caused by BamHI and EcoRI) the rate at which these breaks are rejoined matches or exceeds the rate of enzymatic incision and hence no dsb were observed. In the case of PvuII, the possibly slower rate of repair of blunt-ended termini would on this hypothesis result in the observed net accumulation of dsb.     

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.     

The quality of DNA double-strand breaks: A Monte Carlo simulation of the end-structure of strand breaks produced by protons and alpha particles

The quality of DNA damage induced by protons and -particles of various linear energy transfer (LET) was studied. The aim was to single out specific lesions in the DNA molecule that might lead to biological endpoints such as inactivation. A DNA model coupled with a track structure code (MOCA-15) were used to simulate the lesions induced on the two helixes. Four categories of DNA breaks were considered: single-strand breaks (ssb), bluntended double-strand breaks (dsb, with no or few overlapping bases), sticky-ended double-strand breaks (with cohesive free ends of many bases), and deletions (complex lesions which involve at least two dsb within a small number of base pairs). Calculations were carried out assuming various sets of parameters characterizing the production of these different DNA breaks. No large variations in the yields of ssb and blunt- or sticky-ended dsb were found in the LET range between 10 and 200 keV/µm. On the other hand, the yield of deletions increases up to about 100 keV/µm and seems to reach a plateau at higher LET values. In the LET interval from 30 to 60 keV/µm, protons proved to be more efficient than -particles in inducing deletions. The induction of these complex lesions is thus dependent not simply on LET but also on the characteristics of the track structure. Comparison with RBE values for cell killing shows that this special class of dsb might play an important role in radiation-induced cell inactivation.     

Quantitative modelling of DNA damage using Monte Carlo track structure method

This paper presents data on modelling of DNA damage induced by electrons, protons and alpha-particles to provide an insight into factors which determine the biological effectiveness of radiations of high and low linear energy transfer (LET). These data include the yield of single- and double-strand breaks (ssb, dsb) and base damage in a cellular environment. We obtain a ratio of 4–15 for ssb:dsb for solid and cellular DNA and a preliminary ratio of about 2 for base damage to strand breakage. Data are also given on specific characteristics of damage at the DNA level in the form of clustered damage of varying complexity, that challenge the repair processes and if not processed adequately could lead to the observed biological effects. It is shown that nearly 30% of dsb are of complex form for low-LET radiation, solely by virtue of additional breaks, rising to about 70% for high-LET radiation. Inclusion of base damage increases the complex proportion to about 60% and 90% for low- and high-LET radiation, respectively. The data show a twofold increase in frequencies of complex dsb from low-LET radiation when base damage is taken into account. It is shown that most ssb induced by high-LET radiation have associated base damages, and also a substantial proportion is induced by low-energy electrons. Received: 20 September 1998 / Accepted in revised form: 15 December 1998     

Effect of cellular glutathione content on the induction of DNA double strand breaks by 25 MeV electrons

The effect of endogenous glutathione (GSH) on the induction of DNA double strand breaks (dsb) by 25 MeV electrons was investigated using stationary haploid yeast cells defective in gamma-glutamyl-cysteine-synthetase (gsh 1) containing less than 5 per cent of the normal GSH content. In gsh 1 cells the induction of dsb is increased by a factor of 1.5 under oxic and 1.8 under anoxic irradiation conditions: whereas the oxygen enhancement ratio was only slightly decreased (1.9) compared to wild-type cells (2.4).     

Lack of effect of glutathione depletion on cytotoxicity, mutagenicity and DNA damage produced by doxorubicin in cultured cells

Since endogenous glutathione (GSH), the main non-protein intracellular thiol compound, is known to provide protection against reactive radical species, its depletion by diethylmaleate (DEM) was used to assess the role of free radical formation mediated by doxorubicin in DNA damage, cytotoxicity and mutagenicity of the anthracycline. Subtoxic concentrations of DEM that produced up to 75% depletion of GSH did not increase doxorubicin cytotoxicity in a variety of cell lines, including Chinese hamster ovary (CHO) and lung (V-79) cells, LoVo human carcinoma cells and P388 murine leukemia cells. Similarly, the number of doxorubicin-induced DNA single strand breaks in CHO cells and the mutation frequency in V-79 cells were not affected by GSH depletion. The results obtained suggest that mechanisms other than free radical formation are responsible for DNA damage, cytotoxicity and mutagenicity of anthracyclines.     

In vitro study of cytotoxicity by U.V. radiation and differential sensitivity in combination with alkylating agents on established cell systems

The effect of U.V. radiation or alkylating agents, such as actinomycin-D, cycloheximide and mitomycin-C (MMC), was studied on CHO, BHK and HeLa cells. U.V. radiation caused DNA ssb and dsb and were prevented by cycloheximide and actinomycin-D. MMC is known to be cytotoxic in CHO/BHK cells by forming free radical generation. MMC in combination with U.V. radiation enhanced DNA ssb & dsb in these cell types. However, HeLa cells were insensitive to U.V. radiation. This insensitivity to U.V. radiation could be ascribed to the presence of glutathione transferase which is absent in CHO/BHK cell line.