Yield Of DNA Single-strand Breaks Research Articles

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.

Alterations in repair of alkylating agent-induced DNA damage in polyamine-depleted human cells

Treatment of HeLa cells with the polyamine biosynthesis inhibitors difluoromethylornithine (DFMO) and/or methylglyoxal bis(guanylhydrazone) (MGBG) results in marked depression in levels of the cellular polyamines putrescine, spermidine and spermine. Cells in this polyamine-depleted state exhibited increased sensitivity to monofunctional alkylating agents, manifested as decreased cloning ability and retardation of the DNA excision repair process. DFMO treatment did not alter the initial level of interaction of radiolabeled alkylating agent with cellular DNA, but combined treatment with DFMO and MGBG reduced covalent binding, probably through effects on cell cycling. Polyamine supplementation had no effects on initial yield of DNA single-strand breaks in drug-treated cells. The repair defect appeared similar to that observed previously in polyamine-depleted cells following X-irradiation and UV irradiation, namely retarded sealing of DNA strand breaks. It was not possible to reverse the effects of these inhibitors by short periods of polyamine loading, despite the fact that all three polyamines could be restored to near-normal levels. These findings provide the first demonstration of altered response of polyamine-depleted cells to monofunctional alkylating agents and contribute to our understanding of altered responses of polyamine-depleted cancer cells to a variety of DNA-reactive chemotherapeutic drugs.

Analysis of DNA strand break induction and repair in plants from the vicinity of Chernobyl

For 3 years following the Chernobyl accident, DNA repair efficiency was studied in irradiated and control populations of various plant species. Compared with the control populations, some irradiated populations exhibited increases in the yield of DNA single-strand breaks per unit dose of challenge radiation. The effect was registered in low-dose-rate alpha-irradiated populations, but was absent in plant populations growing in conditions of low-dose-rate beta-irradiation. The efficiency of single-strand DNA repair was identical in control and irradiated populations and approximated 100%.

Chlorpromazine reduces UV-induced squamous cell carcinogenesis in hairless mice and enhances UV-induced DNA damage in cultured cells

Administration of the photoactivable compound chlorpromazine (CPZ) to SKH-1 hairless mice via their drinking water (CPZ, 0.01%) significantly reduced the rates of accumulation and yields of squamous cell carcinomas induced by long-term repeated exposures of these animals to solar UV radiation. This protective effect of CPZ was partially reversed in mice given a single injection of ethyl nitrosourea at birth. In in vitro studies, the presence of CPZ (0.2 mM) in mammalian cell cultures enhanced the yield of DNA single-strand breaks induced in the cells by exposure to monochromatic UVA radiation at 334 nm. Collectively, the results suggest that CPZ may exert antineoplastic effects against UV-induced skin tumours by the induction of DNA damage.

Cellular Glutathione Content andKValues. II

After exposure to radiation in the presence of different oxygen concentrations, the OER values were determined for human fibroblasts cell strains with differing intrinsic GSH content, and cells depleted of GSH to different extent by treatment with BSO. The yield of DNA single-strand breaks was used as the measure of radiosensitivity. The K values were found to be related specifically to the GSH concentration in the cell nuclei. A good agreement was found between the experimental observations and the prediction of a variant of the competition model in which a second type of damage, not influenced by oxygen, was included.

The Application of Rapid-Lysis Techniques in Radiobiology: IV. The Effect of Glycerol and DMSO on Chinese Hamster Cell Survival and DNA Single-Strand Break Production

MILLAR, B. C., SAPORA, O., FIELDEN, E. M., AND LOVEROCK, P. S. The Application of Rapid-Lysis Techniques in Radiobiology. IV. The Effect of Glycerol and DMSO on Chinese Hamster Cell Survival and DNA Single-Strand Break Production. Radiat. Res. 86, 506-514 (1981). A rapid-lysis technique has been used to compare the initial yield of DNA single-strand breaks (ssb) measured 0.2 sec after irradiation and cell survival for Chinese hamster cells in vitro. Both DMSO and glycerol protected cells irradiated in air, and there was a simple relationship between relative radiosensitivity and the initial number of ssb. Under oxic conditions at high concentrations (2 M) of either agent the yield of ssb was reduced by a factor of 3 and the slope of the survival curve by a factor of 2. Under hypoxic conditions much less protection was noted, and even at high concentrations of either agent (2 M) there was only a small degree of protection against cell inactivation and ssb. The results are in agreement with previous work on bacteria using this technique and provide further evidence that if these agents exert their protective effects by interfering with indirect damage then such damage is more important under oxic conditions.

Iodine Contrast Medium Sensitizes Cultured Mammalian Cells to X Rays but Not to γ Rays

The influence of an iodine contrast medium on several responses to radiation was examined in mammalian cells in culture (L5178Y). The presence of the medium at the time of irradiation enhanced cell killing, frequency of micronuclei, and yield of DNA single-strand breaks induced by x rays, depending on the concentration used, whereas no such effect was found with ..gamma.. rays. It was concluded that the contrast medium sensitizes mammalian cells in culture primarily by means of the photoelectric effect, thereby increasing the absorbed dose of x rays in the cells.

The Yield of Radiation-Induced DNA Single-Strand Breaks in Escherichia coli and Superinfecting Phage λ at Different Dose Rates. Repair of Strand Breaks in Different Buffers

Cells of E. coli K-12 strain AB 1886 were irradiated in oxygenated phosphate buffered saline at 2°C with electrons from a 4-MeV linear accelerator. The yield of DNA single-strand breaks was determined as a function of the dose rate between 2.5 and 21,000 krad/min. For dose rates over 100 krad/min the yield was found to be constant. Below 10 krad/min the yield of breaks decreases drastically. This is explained by rejoining of breaks during irradiation. Twenty percent of the breaks induced by acute exposure are repaired within 3 min at 2°C. Superinfecting phage λ DNA is repaired at the same rate as chromosomal DNA. In contrast to the results obtained with phosphate-buffered saline, an increase in the number of breaks after irradiation is observed when the bacteria are suspended in tris buffer. It is suggested that buffers of low ionic strength facilitate the leakage through the membrane of a small-molecular-weight component(s) necessary for DNA strand rejoining.

The Yield and Repair of X-Ray-Induced Single-Strand Breaks in the DNA of Escherichia coli K-12 Cells

The yield of single-strand breaks present in chromosomal DNA of Escherichia coli cells after aerobic x irradiation was determined using alkaline sucrose gradient techniques. Experimental conditions were used that avoided a centrifugation speed dependence effect on DNA sedimentation, and allowed either minimal or complete enzymatic repair to occur. The results confirm the qualitative conclusions made in earlier reports from this laboratory, but indicate that the extent of DNA strand breakage was previously underestimated by factors of 3.4 to 7.6, depending on the strain and repair conditions used. Technical and interpretive difficulties in the measurement of DNA single-strand breakage using alkaline sucrose gradient techniques are discussed briefly. In the present experiments, the initial yield of DNA single-strand breaks was found to be 32.4/genome krad/sup -1/ (9.0 eV/break).

The effect of nitroimidazole and nitroxyl radiosensitizers on the post-irradiation synthesis of DNA.

The modification of DNA damage by three radiosensitizing drugs, present during gamma-irradiation of hypoxic Chinese hamster cells, was investigated. Both 2-methyl-5-nitroimidazole-1-ethanol (metronidazole) and 1-(2-nitro-1-imidazole)-3-methoxy-2-propranol (Ro-07-0582) were found to cause large increases in the yield of DNA single-strand breaks (SSB); triacetoneamine-N-oxyl (TAN) was found to have only a small effect on SSB production. The three drugs tested did not inhibit the rejoining of SSB. A pulse label and chase procedure was used to examine post-irradiation DNA synthesis. TAN present during irradiation under hypoxia was found to cause interruptions in subsequent DNA synthesis. Metronidazole and Ro-07-0582 had no effect on post-irradiation DNA synthesis. In addition, the effects of pre- and post-irradiation exposure to TAN were investigated, since these treatments have shown increased cell-killing in survival studies. TAN pre- and post-treatments were found to have no significant effect on subsequent DNA synthesis.

Absence of ultrafast processes of repair of single-strand breaks in mammalian DNA.

Town, Smith and Kaplan (1972) reported that the yield of DNA single-strand breaks (SSB) in E. coli is largely independent of the presence of molecular oxygen during irradiation. They suggested that the oxygen enhancement ratio (o.e.r.) normally observed is due to the presence of an ultrafast repair mechanism acting (in bacterial cells) mainly on anoxically-produced breaks. To determine whether similar mechanisms exist in mammalian cells, we carried out comparable experiments on two cell-lines, one from Chinese hamster, the other from mouse. Both heat inactivation and chemical inhibition were treatment inactivated all the enzymatic processes assayed, it did not alter the o.e.r. for SSB production, which remained about 3-0. The presence of sodium cyanide, hydroxyurea, iodoacetic acid, EDTA and quinacrine all failed to alter significantly the o.e.r. Isolated nuclei also demonstrated the full o.e.r. For these cell-lines at least, ultrafast reprir does not seem to exist. Isolated Adenovirus 2, which presumably lacks enzymic activity, demonstrated an o.e.r. of 3-6 for SSB production. From these results and others it seems unlikely that the so-called ultrafast enzymic repair is a general phenomenon accounting for the o.e.r. in a wide range of biological systems. Rather, the o.e.r. for SSB seems to result from differences in the direct physico-chemical effects of radiation under aerobic and anoxic conditions in most organisms.

On the nature of the oxygen effect on x-ray-induced DNA single-strand breaks in mammalian cells.

SummaryThe initial yield of DNA single-strand breaks in X-irradiated Chinese hamster ovary cells was about 4-fold higher in aerated samples than in those under extreme hypoxia (<10 p.p.m. O2), with DNA breakage yields of 3·6 × 10−9 and 0·85 × 10−9 dalton−1 krad−1 for air and N2 irradiations, respectively. In cells in which metabolism had been inhibited by heating before irradiation, a maximal DNA break yield of about 8 × 10−9 dalton−1 krad−1 was found for samples irradiated in air or under moderate hypoxia (200 p.p.m. O2), while extreme hypoxia (<10 p.p.m. O2) gave a yield of 4·3 × 10−9 breaks dalton−1 krad−1. When cysteamine was added to heated cells irradiated under extreme hypoxia, the DNA break yield was close to that obtained for viable cells under extreme hypoxia. Similarly, when the reducing agent, Na2S2O4, was added before irradiation to remove O2 chemically, the breakage yield for heated cells was close to that obtained for viable cells under extreme hypoxia. A 10-fold difference in the radiation effect was observed between maximal sensitivity (8 × 10−9 dalton−1 krad−1) seen in heated cells under air or 200 p.p.m. O2 and the near to minimal sensitivity (0·85 × 10−9 dalton−1 krad−1) seen in viable cells under <10 p.p.m. O2. A reasonable interpretation of these findings is that at radiation-induced free radical sites, fast chemical reactions occur which result in the fixation or the restoration of the damage. Thus, an oxygen-enhancement effect exists in mammalian cells for the X-ray-induction of DNA single-strand breaks; this effect appears to depend mainly upon radiation chemical events.

Third annual David A. Karnofsky Memorial Lecture. The compleat clinical oncologist: a new approach to training.

Third annual David A. Karnofsky Memorial Lecture. The compleat clinical oncologist: a new approach to training.

Influence of Ultrafast Repair Processes (Independent of DNA Polymerase I) on the Yield of DNA Single-Strand Breaks in Escherichia coli K-12 X-Irradiated in the Presence or Absence of Oxygen

The rate of production of DNA single-strand breaks has been studied in several polA1 derivatives of Escherichia coli K-12 x-irradiated in the presence or absence of oxygen. For log phase cells irradiated in phosphate-buffered saline, pH 7.3, the rate of production of breaks per single-strand genome per krad was 2.13 in the presence of air, and 0.66 in its absence [an oxygen-nitrogen breaks ratio (ONBR) of 3.2]. To determine if this oxygen effect was due to a difference in the absolute yield of breaks or to a differential ability for repair, the yield of single-strand breaks was studied in cells which had been inactivated either by heat treatment (52°C) or cold shock (0°C). In both cases there was a large increase in the yield of radiation-induced anoxic breaks (2.8-fold) and a small increase (1.25-fold) in aerobic breaks. Attempts were also made to inhibit repair using chemicals. Sodium cyanide (1 mM) used at 0°C had no effect on the anoxic yield of breaks. However, in the presence of quinacrine (0.2 mM) ...

DNA polymerase required for rapid repair of x-ray--induced DNA strand breaks in vivo.

A much higher yield of DNA single-strand breaks was obtained in the DNA polymerase-deficient mutant Escherichia coli K-12 pol A1 after a given dose of x-rays than had been found before in Escherichia coli. The increased yield of single-strand breaks was due to the absence of a rapid repair system, which had not been described in Escherichia coli K-12. This absence probably accounts for the x-ray sensitivity of the pol A1 mutant. The rapid repair system can be reversibly inhibited in pol+ cells.