Exponential or shouldered survival curves result from repair of DNA double-strand breaks depending on postirradiation conditions

The yeast mutant rad54-3 is temperature conditional for the rejoining of DNA double-strand breaks, but cells do proliferate at both the restrictive and permissive temperatures. Thus, after irradiation with 30 MeV electrons, survival curves can be obtained which may or may not involve double-strand break rejoining under certain experimental conditions. Because of this special property of rad54-3 cells, it was possible to demonstrate that rejoining of radiation-induced double-strand breaks under nongrowth conditions yields exponential survival curves the slopes of which decrease as a function of the rejoining time. These survival data suggest that, under nongrowth conditions, the rejoining of double-strand breaks is an unsaturated process and lacks binary misrepair. In contrast, whenever rejoining of double-strand breaks occurs under growth conditions, shouldered survival curves are observed. This is true for immediate plating as well as for delayed plating survival curves. It is proposed that it is the unsaturated rejoining of double-strand breaks under nongrowth conditions, lacking binary misrepair, which is responsible for potentially lethal damage repair.     

Fixation of potentially lethal radiation damage by post-irradiation exposure of Chinese hamster cells to 0.5 M or 1.5 M NaCl solutions.

The effect of 0.05 M and 1.5 M NaCl treatments on CHO cells during and after irradiation has been examined. Treatment with either hypotonic or hypertonic salt solutions during and after irradiation resulted in the fixation of radiation damage which would otherwise not be expressed. The half time for fixation was 4 to 5 min, and the increased expression of the potentially lethal damage by anisotonic solutions was mainly characterized by large decreases in the shoulder of the survival curve, as well as by decreases in DO. Fixation of radiation damage at 37 degrees C occurred to a much greater extent for the hypertonic treatment than for the hypotonic treatment and was greater at 37 degrees C than at 20 degrees C. Although both the hypotonic and hypertonic treatments during and after irradiation reduced or eliminated the repair of sublethal and potentially lethal damage, treatment during irradiation only, radiosensitized the cells when the treatment was hypotonic, and radioprotected the cells when the treatment was hypertonic. These observations are discussed in relation to salt treatments and different temperatures altering competition between repair and fixation of potentially lethal lesions, the number of which depends on the particular salt treatment at the time of irradiation.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of 3-aminobenzamide on DNA strand-break rejoining and cytotoxicity in CHO cells treated with hydrogen peroxide

The influence of the nuclear ADP-ribosyltransferase inhibitor 3-aminobenzamide on the DNA strand-break rejoining kinetics and cytotoxicity in Chinese hamster ovary cells following H2O2 treatment was investigated. For the DNA damage studies, cells were treated on ice with H2O2 (0-20 microM) for 1 h in serum-free medium, after which the H2O2 was removed and the cells were allowed to repair their damage in complete medium at 37 degrees C in the presence or absence of 3-aminobenzamide (5 mM) for periods up to 2 h. The DNA strand breaks remaining as a function of time were then estimated by alkaline elution. A linear relationship between the H2O2 concentration and the initial level of DNA single-strand breaks (zero time allowed for repair) was observed. No double-strand breaks or DNA-protein cross-links were detected at these doses. The rejoining of single-strand breaks after H2O2 (20 microM) alone was characterized by a single exponential process with a t1/2 of approx. 5 min. However, in the presence of 3-aminobenzamide, rejoining was much slower and biphasic, with t1/2 of approx. 10 and 36 min. The inhibitory action of 3-aminobenzamide was concentration-dependent and completely reversible in that, when the 3-aminobenzamide was removed from the treated cultures, the strand-break rejoining kinetics rapidly returned to the t1/2 of 5 min typical of H2O2 alone. Considerably higher concentrations of H2O2 (up to 600 microM) were required for cell killing compared to the DNA damage studies. Cell killing by H2O2 alone was characterized by a shoulderless, exponential survival curve (D0 = 880 microM). The cytotoxicity was potentiated when the cells were treated with 3-aminobenzamide (5 mM) for 1 h after the H2O2 treatment; the survival curve with 3-aminobenzamide also assumed a biphasic character (D0 of 212 microM and 520 microM). These results are consistent with the theory that OH.-induced single-strand breaks do not normally represent lethal lesions to the cell because of their rapid, efficient repair. However, interference with these repair processes (in this case by 3-aminobenzamide) can alter this relationship, possibly allowing lesion fixation.     

Relationship between the recovery from sublethal X-ray damage and the rejoining of chromosome breaks in normal human fibroblasts

Using plateau-phase cultures of AG1522 normal human fibroblasts, we examined relationships between the breakage and rejoining of chromosomes and the induction and repair of sublethal damage (SLD) following fractionated doses of X rays. The rate constant for the rejoining of breaks in prematurely condensed interphase chromosomes, measured previously, accurately predicts both the rate of change in survival due to potentially lethal damage (PLD) repair and the rate of change in survival for dose fractionation due to SLD repair. Further, changes in the frequency of chromosome-type deletions and asymmetrical exchange aberrations measured in the first postirradiation mitosis corresponded closely with changes in cell killing when doses were fractionated, and a dose-fractionation- or dose-rate-independent alpha component of damage was similar for aberration and cell killing end points. These results substantiate the hypothesis that sublethal damage repair results from the rejoining of breaks in interphase chromatin produced by a first dose so they no longer are capable of interacting with those produced by a second dose. The fact that the repair of potentially lethal damage is also readily explained on the basis of chromosome break rejoining (M. N. Cornforth and J. S. Bedford, Radiat. Res. 111, 385-405 (1987)) strongly suggests that PLD and SLD repair are different manifestations of the same basic process operating on the same basic lesions.     

Ligase-deficient yeast cells exhibit defective DNA rejoining and enhanced gamma ray sensitivity.

Yeast cells deficient in DNA ligase were also deficient in their capacity to rejoin single-strand scissions in prelabeled nuclear DNA. After high-dose-rate gamma irradiation (10 and 25 krads), cdc9-9 mutant cells failed to rejoin single-strand scissions at the restrictive temperature of 37 degrees C. In contrast, parental (CDC9) cells (incubated with mutant cells both during and after irradiation) exhibited rapid medium-independent DNA rejoining after 10 min of post-irradiation incubation and slower rates of rejoining after longer incubation. Parental cells were also more resistant than mutant cells to killing by gamma irradiation. Approximately 2.5 +/- 0.07 and 5.7 +/- 0.6 single-strand breaks per 10(8) daltons were detected in DNAs from either CDC9 or cdc9-9 cells converted to spheroplasts immediately after 10 and 25 krads of irradiation, respectively. At the permissive temperature of 23 degrees C, the cdc9-9 cells contained 2 to 3 times the number of DNA single-strand breaks as parental cells after 10 min to 4 h of incubation after 10 krads of irradiation, and two- to eightfold more breaks after 10 min to 2.5 h of incubation after 25 krads of irradiation. Rejoining of single-strand scissions was faster in medium. After only 10 min in buffered growth medium and after 10 krads of irradiation, the number of DNA single-strand breaks was reduced to 0.32 +/- 0.3 (at 23 degrees C) or 0.21 +/- 0.05 (at 37 degrees C) per 10(8) daltons in parental cells, but remained at 2.1 +/- 0.06 (at 23 degrees C) or 2.3 +/- 0.07 (at 37 degrees C) per 10(8) daltons in mutant cells. After 10 or 25 krads of irradiation plus 1 h of incubation in medium at 37 degrees C, only DNA from CDC9 cells was rejoined to the size of DNA from unirradiated cells, whereas at 23 degrees C, DNAs in both strains were completely rejoined.     

Rejoining of gamma-ray-induced DNA damage in Cryptosporidium parvum measured by the comet assay

Cryptosporidium parvum is a well-known waterborne intracellular protozoan that causes severe diarrheal illness in immunocompromised individuals. This organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits one of the highest known resistances to gamma irradiation. We investigated rejoining of gamma-ray-induced DNA damage in C. parvum by neutral comet assay. Oocysts were gamma irradiated at various doses (1, 5, 10, and 25 kGy) and were incubated for various periods (6-96 h) after exposure to 10 kGy. The comet tail moment showed that the number of DNA double-strand breaks increased concomitantly with the gamma irradiation dose. When investigating rejoining after irradiation at 10 kGy, double-strand breaks peaked at 6 h postirradiation, and rejoining was highest at 72 h postirradiation. The observed rejoining pattern suggests that repair process occurs slowly even when complex DNA double-strand breaks in C. parvum were induced by high dose irradiation, 10 kGy.     

Thermal enhancement of DNA damage by an alkylating agent in human cells

Human skin cells were incubated at various temperatures during and after treatment with methyl methanesulfonate and the number of single-strand breaks introduced into the cellular DNA then estimated by alkaline sucrose sedimentation. Elevation of temperature above 37° greatly enhanced damage to the DNA caused by methyl methanesulfonate. Inactivation of an essential step in the repair of DNA was indicated by the observation that rejoining of breaks in the DNA was halted above a critical temperature (about 41.5°). Enhancement of damage to DNA increased with temperature, especially above 42°. Similar results were obtained for Chinese hamster cells. A correlation of these results with cell viability is discussed.     

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.     

Repair of near-ultraviolet (365 nm)-induced strand breaks in Escherichia coli DNA. The role of the polA and recA gene products.

The action of near-ultraviolet (UV-365 nm) radiation in cellular inactivation (biological measurements) and induction and repair of DNA strand breaks (physical measurements) were studied in a repair-proficient strain and in polA-, recA-, uvrA-, and polA uvrA-deficient strains of Escherichia coli K-12. The induction of breaks in the polA and polA uvrA strains was linear with dose (4.0 and 3.7 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively). However, in the recA-, uvrA-, and repair-proficient strains, there was an initial lag in break induction at low doses and then a linear induction of breaks at higher doses with rates of 4.6, 2.8, and 3.2 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively. We interpret these strain differences as indicating simultaneous induction and repair of breaks in polymerase 1 (polA)-proficient strains under the 0 degrees C, M9 buffer irradiation conditions that, for maximum efficiency, require both the polA and recA gene products. Strand-break rejoining also occurred at 30 degrees C in complete growth medium. We propose that at least three (and possibly four) distinct types of pathways can act to reduce the levels of 365-nm radiation-induced strand breaks. A quantitative comparison of the number of breaks remaining with the number of lethal events remaining after repair in complete medium at 30 degrees C showed that between one and three breaks remain per lethal event in the wild-type and recA strains, whereas in the polA strain one order of magnitude more breaks were induced.     

Induction and rejoining of DNA double-strand breaks in human cervix carcinoma cell lines of differing radiosensitivity

Five recently established cell lines of human carcinoma of the cervix of varying radiosensitivity have been used to determine whether the induction or rejoining of DNA double-strand breaks (dsb) shows any correlation with radiosensitivity or radiation recovery capacity. Double-strand DNA breaks have been measured using neutral filter elution at pH 9.6. The number of breaks induced immediately after irradiation with doses of 10 to 40 Gy 60Co gamma rays appeared to show some correlation with radiosensitivity particularly after 10 Gy; the two more radiosensitive lines incurred more breaks than the more radioresistant lines. In addition, the shape of the induction curve with dose was linear for the two sensitive lines but curvilinear for the resistant lines. Despite the dose scales being different, this mirrored their respective cell survival curve shapes. After 30 or 50 Gy irradiation, rejoining of breaks appeared to be rapid and almost complete within 60 min at 37 degrees C for the three resistant lines. However, for the sensitive lines, one line (HX160c) in particular exhibited a reduced rate of dsb rejoining. In addition, a residual level of dsb was present in this line even after allowing rejoining for 3 h. While induction and rejoining of DNA dsb therefore appears to be a factor in determining radiosensitivity, at doses relevant to cellular survival (up to 10 Gy), the greater induction of DNA dsb in radiosensitive lines may play a significant role in determining the cellular response to ionizing radiation.     

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.     

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

The repair of ionising-radiation-induced DNA double-strand break type damage was measured by Kohn neutral elution in an X-ray-sensitive mutant of V79-4, irs1. This was done in order to investigate further the likelihood that irs1 carries a defect which leads to error-prone repair of DNA damage, and not simply a reduced ability to rejoin DNA double-strand breaks. The mutant displayed an equal increase in sensitivity to the lethal effects of neutrons, as compared to X-rays. Both irs1 and V79-4 showed an increased sensitivity to the killing effects of neutrons of around 2 at 10% survival. irs1 also showed an exponential survival after either X-rays or neutrons. The induction of DNA double-strand breaks was measured in both cell lines over a dose range of 10-40 Gy using Kohn neutral filter elution. Induction of breaks by X-rays in irs1 seemed to increase slightly with dose, relative to induction in V79-4, so that at 40 Gy 1.5 times more DNA double-strand breaks were measured in irs1 cells than in V79-4. Neutron irradiation resulted in a more similar level of induction in either strain after 10-40 Gy. This difference in induction of damage may be due to a different cell-cycle composition in either cell line. The rejoining of X-ray induced double-strand breaks showed a very similar pattern (on a percentage rejoined basis) in both cell lines, although from the induction data at 40 Gy, the dose at which rejoining was measured, fewer breaks were rejoined in V79-4 but also fewer breaks remained unsealed. Neutron-induced breaks, however, were rejoined more efficiently in irs1 again on a percentage basis, but also in absolute terms since similar induction was seen after 40 Gy. This data, together with the differences seen in the rejoining of X-ray compared to neutron induced breaks, may indirectly support the proposal that irs1 is a misrepair mutant.     

Transfection of a human gene for the repair of X-ray- and EMS-induced DNA damage

EM9 cells are a line of Chinese hamster ovary cells that are sensitive to killing by ethylmethanesulfonate (EMS) and X ray, since they are unable to repair the DNA damage inflicted by these agents. Through DNA-mediated gene transfer, human DNA and a selectable marker gene, pSV2neo, were transfected into EM9 cells. Resistant clones of transfected cells were selected for by growth in EMS and G418 (an antibiotic lethal to mammalian cells not containing the transfected neo gene). One primary clone (APEX1) and one secondary clone (TEMS2) were shown to contain both marker and human DNA sequences by Southern blot. In cell survival studies, APEX1 was shown to be as resistant to EMS and X ray as the parental cell type AA8 (CHO cells). TEMS2 cells were found to be partially resistant to EMS and X ray, displaying an intermediate phenotype more sensitive than AA8 cells but more resistant than EM9 cells. Alkaline elution was used to assess the DNA strand-break rejoining ability of these cells at 23 degrees C. APEX1 cells showed DNA repair capacity equal to that of AA8 cells; 75% of the strand breaks were repaired with a rejoining T 1/2 of 3 min. TEMS2 showed similar levels of repair but a T 1/2 for repair of 9 min. EM9 cells repaired only 25% of the breaks and showed a T 1/2 for repair of 16 min. The DNA repair data are consistent with the survival data in that the more resistant cell lines showed a greater capacity for DNA repair. The data support the conclusion that APEX1 and TEMS2 cells contain a human DNA repair gene.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparison of chromosome repair kinetics in G(0) and G(1) reveals that enhanced repair fidelity under noncycling conditions accounts for increased potentially lethal damage repair

Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G(0) phase or allowed to enter G(1) phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G(0). By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G(1). The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G(1)) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G(1)) than for noncycling cells (G(0)). Since most repair in G(0)/G(1) occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.     

Effects of inhibitors on repair of DNA in normal human and xeroderma pigmentosum cells after exposure to x-rays and ultraviolet irradiation

Experiments were carried out to obtain direct evidence for the hypothesis that in human cells the repair of UV-damaged DNA is initiated by an incision step, and that this step is defective in cells from patients having Xeroderma pigmentosum (XP). The alkaline sucrose gradient centrifugation technique was used to detect breaks in the DNA.A decreased sedimentation velocity of the DNA was found after exposure of normal and XP cells to high doses of UV (5000 erg/mm²). Breaks were induced in the DNA by the UV irradiation without the action of an enzyme. After exposure of both types of cell to UV doses of 100–500 erg/mm², breaks that might occur by enzymic incision were not observed, possibly because of immediate rejoining.After single-strand breaks had been induced by X-rays, rejoining did not occur at temperatures lower than 22°. Rejoining was inhibited by KCN, 2,4-dinitrophenol, EDTA, iodoacetate and crystal violet. Actinomycin D, acriflavine and phleomycin, also tested as potential inhibitors of the repair process, induced breaks or conformational changes in the DNA of unirradiated normal and XP cells.Application to UV-exposed cells of conditions that inhibit the rejoining of breaks did not cause accumulation of breaks in the DNA. The results suggest a coordinated and sequential performance of the steps in the repair of each UV lesion by repair enzymes which may act as a complex.     

Depletion of glutathione after gamma irradiation modifies survival

The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells. Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM). Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting. Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95%. Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures. Analysis of survival curves indicated that the Dq was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation. The DEM/BSO exposure did not affect D0. To study postirradiation conditions, cells were exposed to 10 microM DEM prior to and during irradiation, which was performed at ice temperatures. Levels of GSH were depleted by 75% by this protocol. Immediately after irradiation, the cells were rapidly warmed by the addition of 37 degrees C growth medium containing either 10 or 90 microM DEM. Addition of 10 microM DEM after irradiation did not affect the degree of depletion, which remained constant at 75%. In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 microM DEM. Addition of 90 microM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 microM DEM. In a second depletion protocol, cells were exposed to 100 microM DEM at room temperature for 5 min, irradiated, incubated at 37 degrees C for 1 h, washed, and then incubated in 50 microM BSO for 24 h. This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO. Survival was not affected if the cells were exposed to the DEM or BSO alone. This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained. The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure. These experimental results in addition to previous ones (Freeman and Meredith, Int. J. Radiat. Oncol. Biol. Phys. 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.     

Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay

The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis. However, it is not known whether different lysis conditions identify different types of DNA damage or simply measure the same damage with different efficiencies. Results support the latter interpretation for radiation, but not for the alkylating agent MNNG. For X-ray-induced damage, cells showed the same amount of damage, regardless of lysis pH (12.3 compared to >13). However, increasing the duration of lysis at 5 degrees C from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent damage was observed by conducting lysis at room temperature (22 degrees C) for 6 h, but at the expense of a higher background level of DNA damage. The oxygen enhancement ratio and the rate of rejoining of single-strand breaks after irradiation were similar regardless of pH and lysis time, consistent with more efficient detection of strand breaks rather than detection of damage to the DNA bases. Conversely, after MNNG treatment, DNA damage was dependent on both lysis time and pH. With the higher-pH lysis, there was a reduction in the ratio of oxidative base damage to strand breaks as revealed using treatment with endonuclease III and formamidopyrimidine glycosylase. Therefore, our current results support the hypothesis that the increased sensitivity of longer lysis at higher pH for detecting radiation-induced DNA damage is due primarily to an increase in efficiency for detecting strand breaks, probably by allowing more time for DNA unwinding and diffusion before electrophoresis.     

Relationship of DNA repair to chromosome aberrations, sister-chromatid exchanges and survival during liquid-holding recovery in X-irradiated mammalian cells

The repair of X-ray induced DNA single strand breaks and DNA—protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined sungle strand breaks was approx. proportional to the X-ray dose. DNA—protein cross-links were removed at a slower rate (T1/2 approx. 10–12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed.