Recovery from sublethal and potentially lethal damage in an X-ray-sensitive CHO cell

It has been suggested that DNA strand breaks are the molecular lesions responsible for radiation-induced lethality and that their repair is the basis for the recovery of irradiated cells from sublethal and potentially lethal damage. EM9 is a Chinese hamster ovary cell line that is hypersensitive to killing by X rays and has been reported to have a defect in the rate of rejoining of DNA single-strand breaks. To establish the importance of DNA strand-break repair in cellular recovery from sublethal and potentially lethal X-ray damage, those two parameters, recovery from sublethal and potentially lethal damage, were studied in EM9 cells as well as in EM9's parental repair-proficient strain, AA8. As previously reported, EM9 is the more radiosensitive cell line, having a D0 of 0.98 Gy compared to a D0 of 1.56 Gy for AA8 cells. DNA alkaline elution studies suggest that EM9 cells repair DNA single-strand breaks at a slower rate than AA8 cells. Neutral elution analysis suggests that EM9 cells also repair DNA double-strand breaks more slowly than AA8 cells. All of these data are consistent with the hypothesis that DNA strand-break ligation is defective in EM9 cells and that this defect accounts for increased radiosensitivity. The kinetics and magnitude of recovery from sublethal and potentially lethal damage, however, were similar for both EM9 and AA8 cells. Six-hour recovery ratios for sublethal damage repair were found to be 2.47 for AA8 cells and 1.31 for EM9 cells. Twenty-four-hour recovery ratios for potentially lethal damage repair were 3.2 for AA8 and 3.3 for EM9 cells. Both measurements were made at approximately equitoxic doses. Thus, the defect in EM9 cells that confers radiosensitivity and affects DNA strand-break rejoining does not affect sublethal damage repair or potentially lethal damage repair.     

Time dependence of Elkind-type recovery in class B oocytes of Drosophila melanogaster.

Recovery from X-ray-induced damage in class B oocytes of Drosophila melanogaster was studied by the dose-fractionation technique. A total dose of 500 R was delivered either as a single exposure or as two fractions of 2000 R and 3000 R separated by increasing time intervals. The use of attached-X females made it possible to study simultaneously the induction of dominant lethals and of chromosome aberrations (detachments of the attached-X chromosome). The same repair kinetics were observed for sublethal damage and for the lesions leading to detachments. The time-response curves are of similar shape: a plateau is reached within 20 to 30 min and half of the repairable damage disappears in 5 to 7 min. It is concluded that the same type of X-ray-induced primary lesion in chromosomes is responsible for the induction of detachments and for dominant lethals. As primary lesions actual chromosome breaks or lesions leading to breaks and chromosome rearrangements are assumed.     

Repairs of X-ray induced sublethal damage in class B oocytes of Drosophila melanogaster.

Repair of X-ray-induced sublethal damage (Elkind-type recovery) in class B oocytes of Drosophila melanogaster was studied. Newly hatched females of two different stocks were treated with either acute or fractionated exposures. For the fractionation experiments a constant time interval of one hour between the dose fractions was used. As genetic endpoints dominant lethality, chromosome aberrations (detachments) and non-disjunction were studied. The repair of X-ray-induced sublethal damage in class B oocytes is expressed as a reappearance on the initial shoulder in the fractionation curve. For dominant lethality it could be shown that less sublethal damage is repaired in oocytes of Berlin wild females than in those of attached-X females (on the average 76 per cent and 101 per cent respectively). Complete repair (about 100 per cent) was observed for detachments in occytes of attached-X females. Within the dose ranges used no radiation effects on non-disjunction could be observed. The results are interpreted to show that in class B oocytes (1) sublethal damage is due to chromosome breaks and/or lesions leading to breaks and (2) X-ray-induced dominant lethality is the consequence of chromosome damage (true dominant lethals).     

Recovery from lethal and mutagenic damage during postirradiation holding and low-dose-rate irradiations of cultured hamster cells

Survival and mutation to thioguanine resistance were measured in V79-4 hamster cells grown to plateau phase without refeeding and irradiated with 60Co gamma rays. The effects of low-dose-rate irradiation and of postirradiation holding on recovery from gamma-ray damage leading to these two responses were also studied. The responses of these plateau (extended G1)-phase cells to acute irradiation were similar to those we previously found for exponentially growing cells, including the linear relationship between induced mutant frequency and (log) surviving fraction. Irradiation at low dose rate (0.34 rad/min) considerably reduced both the lethal and mutagenic effects of given doses of gamma rays, but the linear mutation-survival relationship was approximately the same as for acute irradiation. In contrast, cells given a 5-hr holding period after acute irradiation showed the anticipated recovery from potentially lethal damage but no recovery from damage leading to mutation. These results are discussed in terms of previously proposed cellular repair processes (sublethal damage repair and potentially lethal damage repair) and the possibility that the radiation damage leading to lethality is different from mutagenic damage.     

CONTINUING STUDIES ON THE SOUTH AMHERST DROSOPHILA MELANOGASTER NATURAL POPULATION DURING THE 1970'S AND 1980'S

Continuing investigations on the South Amherst Drosophila melanogaster natural population following the significant decline and recovery of lethal (le) and semilethal (sle) frequencies in the late 1960's (Ives, 1970) show that the population has been remarkably stable although it contains MR (male recombination) and/or P DNA elements (Kidwell et al., 1977a; Green, 1980). A 13-year study affirms that the lethals present are nonrandomly distributed along the second chromosome and deficient on the right; they differ significantly in distribution from spontaneous (Ives, 1973) and δ-induced lethals (Minamori and Ito, 1971). Between 1970 and 1977, a total of 4,083 second chromosomes from the Markert subpopulation were analyzed; 28.9% of the chromosomes were lethal and 7.25% were semilethal in homozygous condition. Frequencies are similar for early summer and late fall collections although the rate of allelism among lethals is significantly higher in early summer than in late fall. For the large fall (1970–1979) Porch site population, 2,519 second chromosomes were analyzed; 29.5% were lethal and 8.0% were sublethal as homozygotes; the rate of allelism among lethals was 1.50%. At Hockanum, 1977–1983, lethal and semilethal frequencies were lower; the rate of allelism among lethals was 1.43%. The chromosome map distribution of lethals does not change between summer and late fall at Markert. The overall distributions of lethals at the Markert and Hockanum sites are similar. In tests for male recombination (MR) activity in the population over a 6-year period, a total of 0.47% recombinants were observed; these were uniformly distributed along the second chromosome. Comparisons are made with other long studied D. melanogaster populations.     

The evaluation of the role of recovery from potentially lethal and sublethal damages in the RBE of densely ionizing radiation

The new data confirming the relations between RBE and recovery of cells are presented. The quantitative evaluation of the contribution of potentially lethal and sublethal damage recovery in radiosensitivity of cells of various origin after exposure to low- and high-LET ionizing radiation was carried out. The conclusion about the greater contribution of potentially lethal damage recovery in the magnitude of RBE in comparison with sublethal damage recovery was made.     

Variations in the radiosensitivity of oocyte chromosomes during meiosis in Drosophila melanogaster

The synaptic stages of meiosis in Drosophila melanogaster females are very resistant to the induction of dominant lethal mutations by ionizing radiation. It is assumed that dominant lethals result from interstitial chromatid deletions, and that almost all potential chromatid breaks are repaired in synaptic cells. The type of dose response curve shown by oocytes at later developmental stages is a function of the degree of chromatid coiling and the presence or absence of an investing nuclear envelope.     

Mutagenesis in CAENORHABDITIS ELEGANS. II. a Spectrum of Mutational Events Induced with 1500 R of γ-RADIATION

We previously established a gamma-ray dose-response curve for recessive lethal events (lethals) captured over the eT1 balancer. In this paper we analyze the nature of lethal events produced, with a frequency of 0.04 per eT1 region, at a dose of 1500 r. To do so, we developed a protocol that, in the absence of cytogenetics, allows balanced lethals to be analyzed for associated chromosomal rearrangements. A set of 35 lethal strains was chosen for the analysis. Although the dosage was relatively low, a large number of multiple-break events were observed. The fraction of lethals associated with rearrangements was found to be 0.76. Currently most X- and gamma-ray dosages used for mutagenesis in C. elegans are 6000-8000 r. From our data we conservatively estimated that 43% of rearrangements induced with 8000 r would be accompanied by additional chromosome breaks in the genome. With 1500 r the value was 5%. The 35 lethals studied were derived from 875 screened F1's. Among these lethals there were (1) at least two unc-36 duplications, (2) at least four translocations, (3) at least six deficiencies of chromosome V (these delete about 90% of the unc-60 to unc-42 region) and (4) several unanalyzed rearrangements. Thus, it is possible to recover desired rearrangements at reasonable rates with a dose of only 1500 r. We suggest that the levels of ionizing radiation employed in most published C. elegans studies are excessive and efforts should be made to use reduced levels in the future.     

Recovery after exposure to near-ultraviolet light of cells containing 5-bromodeoxyuridine.

The survival of synchronized V79 Chinese hamster cells irradiated with near-ultraviolet light after a 1-h labeling with 5-bromodeoxyuridine (BrdUrd) is highly dependent upon the cell's position in the cell cycle at the time of irradiation (Hagan, M., and M. M. Elkind. Biophys. J. 1979. 27:75-86). In this report, we show that cells irradiated in the same S phase after BrdUrd incorporation demonstrate an ability to repair sublethal damage, in contrast to the lack of an increase in survival with dose fractionation in template-labeled cells (Ben-Hur, E., and M. M. Elkind. Mutat. Res. 1972. 14:236-245). In addition, we show that pulse-labeled cells in S phase can repair potentially lethal damage expressed by caffeine. The kinetics of these recovery processes and the absence of a caffeine effect on the repair of sublethal damage indicate that these two processes are to a large degree unrelated. We conclude that in template-labeled cells inadequate time to effect prereplicational repair precludes effective contributions to cell survival from other kinds of DNa repair processes.     

Occurrence of spontaneous and nitrosoethylurea-induced mutations and potential changes in repair-deficient Drosophila strains

X-linked recessive lethal frequencies in mature spermatozoa were studied in repair-deficient Drosophila strains. Frequencies of spontaneous and ethylnitrosourea-induced lethals were enhanced in mus(I)104DI+ and unchanged in mei-9IL+. In addition, the majority of lethals was fixed in stages preceding mature spermatozoa. It was shown that premutational lesions (spontaneous and ethylnitrosourea-induced in both mutants) arise in germ cells, these lesions being realized into mutations in the next generations.     

Recovery in the survival capacity of ultraviolet-irradiated 3T3 mouse cells at G0 cannot be solely dependent on the excision of pyrimidine dimers

Mouse cells (3T3 line) excised at most 20% of the pyrimidine dimers introduced into their DNA by a dose of short-wavelength ultraviolet radiation that allows a significant fraction of the cells to survive. When irradiation was delivered at the pre-replicative stage, a significant repair of lethal events was observed, as the cells progressed toward S phase. The recovery in survival cannot be accounted for solely by excision of pyrimidine dimers. Therefore, either another lesion produced by ultraviolet radiation is critical in terms of lethality, or the dimer, which may trigger the lethal event, becomes no longer an obstacle for the replication system after a certain period of time.     

Genetic damage induced by X-rays or neutrons in spermatozoa of Drosophila melanogaster; differential processing in the oocytes of females carrying the DNA-repair-deficient mutants mei-9a and mus-101d1

Radiation-induced premutational lesions on the chromosomes of irradiated mature spertozoa of Drosphila are processed when the sperm nucleus the egg cytoplasm at fertilization. This processing depends on enzymatic repair systems, which are built up in ocytes under the control of the maternal genotype. The present study is concerned with 2 repair-deficient mutants, mei-9^a and mus-101^D1. Irradiated Basc males were crossed to homozygous mei-9^a or mus-101^D1 females, or to repair-proficient control females. The frequencies of recovered sex-link recessive lethal mutations and of II–III translocations were used to assess the effects of impaired maternal repair. Neutrons, as a densely ionizing radiation, and X-rays as a sparsely ionizing one, were used to induce the premutational lesions.The question being asked was whether different radiation qualities cause specific types of lesion that are processed differentially under conditions of impaired maternal repair. The results indicate that this may be so. In comparison with the control, with repair-proficient females, all major effects caused by impaired maternal repair led to frequency reductions in the recovery of lethals and translocations. These reductions in yield were pronounced in all neutron experiments, whereby mus-101^D1 had a stronger effect than mei-9^a. Two possible explanations are considered. The first is based on the idea that specific lesions are processed in a specific way, resulting in a specific mutational end-product, which may not be recovered when repair is impaired. The second is based on the notion that energy deposition in cells exposed to neutrons is not uniform, which leads to clustered damage. Impaired repair may select againts multiply damaged cells much more powerfully than normal repair. Consequently, the surviving fraction of cells is likely to have received less than the average dose. With X-rays, no or only spurious effects of the repair-defective mutants were detected, except in the following case: recovery of translocations (but not of lethals) was strongly reduced when irradiated males were crossed to mus-101^D1 females. It is assumed that mus-101^D1 is defective in repair of DNA double-strand damage, and that the formation of translocations may depend particularly on this repair function.     

Lethal and potentially lethal lesions induced by radiation--a unified repair model

A model of radiation action is described which unifies several of the major existing concepts which have been applied to cell killing. Called the lethal and potentially lethal (LPL) model, it combines the ideas of lesion interaction, irreparable lesions caused by single tracks, linear lesion fixation, lesion repair via first-order kinetics, and binary misrepair. Two different kinds of lesions are hypothesized: irreparable (lethal) and repairable (potentially lethal) lesions. They are tentatively being identified with DNA double-strand breaks of different severity. Two processes compete for depletion of the potentially lethal lesions: correct repair following first-order kinetics, and misrepair following second-order kinetics. Fixation of these lesions can also occur. The model applies presently only to plateau (stationary)-phase cells. Radiobiological phenomena described include effects of low dose rate, high LET, and repair kinetics as measured with repair inhibitors such as hypertonic solution and beta-arabinofuranosyladenine (beta-araA). One consequence of the model is that repair of sublethal damage and the slow component of the repair of potentially lethal damage are two manifestations of the same repair process. Hypertonic treatment fixes a completely new class of lesions which normally repair correctly. Another consequence of the model is that the initial slope of the survival curve depends on the amount of time available for repair after irradiation. The "dose-rate factor" occurring in several linear-quadratic formulations is shown to emerge when appropriate low-dose and long-repair-time approximations are made.     

A BASIC program on heterokaryosis and survival in Phycomyces

We have written a BASIC program about survival and heterokaryosisin Phycomyces for APPLE II series. Surviving and heterokaryoticunit fraction of spores as a consequence of several lethal effectssuch as mitotic, dominant and recessive lethals can be computedby the system, and also the uninucleate fraction of spores whichwere originated by mitotic and recessive lethals. The probability of a mitotic lethal per nucleus can be alsocomputed starting from the only surviving unit fraction of sporesThe program can also compute the relationship between the mutationrates due to mitotic and recessive lethals acting together concurrently,introducing surviving and heterokaryotic unit fraction of spores The program can be easily modified to run on different microcomputerswith similar versions of BASIC Received on September 17, 1985; accepted on January 13, 1986     

Growth Hormone Mitigates against Lethal Irradiation and Enhances Hematologic and Immune Recovery in Mice and Nonhuman Primates

Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 µg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.     

Exposure of cells to nonlethal concentrations of hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal destabilization

The cytotoxicity of hydrogen peroxide is, at least partly, mediated by the induction of intralysosomal iron-catalyzed oxidative reactions with damage to lysosomal membranes and leakage of destructive contents. We hypothesize that minor such leakage may be nonlethal, and the ensuing cellular degeneration repairable. Consequently, we investigated, using a model system of cultured J-774 cells, the effects of hydrogen peroxide in moderate concentrations on cellular viability, lysosomal membrane integrity, morphology, and ATP and reduced glutathione concentrations. These parameters were initially estimated directly after a 30 min exposure to a bolus dose of hydrogen peroxide in phosphate buffered saline at 37°C, and then again following subsequent recovery periods of different lengths under ordinary culture conditions. All cells survived an exposure to 250 μM hydrogen peroxide for 30 min, whereas 350 and 500 μM exposure was lethal to a small fraction of cells. The oxidative stress caused early, time- and dose-dependent, partial relocalization of the lysosomotropic weak base acridine orange from the lysosomal compartment to the cytosol. This phenomenon is known to parallel leakage of damaging lysosomal contents such as hydrolytic enzymes. There were also signs of cellular damage in the form of surface blebbing and increased autophagocytosis, more marked with the higher doses of hydrogen peroxide. Also found was a rapid depletion of ATP and GSH. These alterations were all reversible, as long as cells were exposed to nonlethal amounts of hydrogen peroxide. Based on these and previous findings, we suggest that lysosomes are less stable organelles than has hitherto been assumed. Restricted lysosomal leakage might be a common event, for example, during sublethal oxidative stress, causing reversible, degenerative alterations, which are repaired by autophagocytosis.     

Increment kinetics of recessive lethal mutations and dominant lethals in offspring of Drosophila melanogaster on storage of methyl-methanesulfonate-treated sperm in females

The frequencies of sex-linked recessive lethal mutations in F1 males after feeding adult male Drosophila melanogaster with 0.25 and 0.5 mM methyl methanesulfonate (MMS) orally for 24 h increased approximately linearly with storage of the treated spermatozoa in females, whereas the number of hits of dominant lethals in the sperm after feeding 0.3 and 0.5 mM MMS increased approximately with the square of the storage time. Chromosome losses and mosaics in F1 males also increased with the dose of MMS to males, but their yields were too low to be analyzed quantitatively, only indicating a slight increase of chromosome loses and a slight decrease of mosaics with the time of storage of sperm. Maternal non-disjunctions (or chromosome losses), detected in F1 males, decreased with the dose of MMS to spermatozoa and their yield decreased with the time of storage of sperm of both MMS-treated and the control groups. A unitary model is proposed to explain the effect of storage on the dominant lethals and recessive lethal mutations.     

Radiosensitization of GL261 glioma cells by tetraiodothyroacetic acid (tetrac)

We studied effects of tetrac (tetraiodothyroacetic acid) on survival of GL261, a murine brain tumor cell line, following single doses of 250 kVp x-rays and on repair of damage (sublethal and potentially lethal damage repair; SLDR, PLDR) in both exponential and plateau phase cells. Cells were exposed to 2 μM tetrac (1 h at 37oC) prior to x-irradiation. At varying times after irradiation, cells were re-plated in medium without tetrac. Two weeks later, colonies were counted and results analyzed using either the linear-quadratic (LQ) or single-hit, multitarget (SHMT) formalisms. Tetrac sensitized both exponential and plateau phase cells to x-irradiation, as shown by a decrease in the quasi-threshold dose (Dq), leading to an average tetrac enhancement factor (ratio of SF2 values) of 2.5. Tetrac reduced SLDR in exponential cells by a factor of 1.8. In plateau phase cells there was little expression of SLDR, but tetrac produced additional cell killing at 1-4 h after the first dose. For PLDR expression in exponential cells, tetrac inhibited PLDR by a factor of 1.9, and in plateau phase cells, tetrac decreased PLDR expression by a factor of 3.4. These data show that the decreased Dq value seen after single doses of x-rays with tetrac treatment is also accompanied by a significant decrease in recovery from sublethal and potentially lethal damage.     

Radiosensitization in multifraction schedules. I. Evidence for an extremely low oxygen enhancement ratio

Stable monolayers of contact-inhibited C3H 10T1/2 cells were used in multifraction radiation experiments to measure the oxygen enhancement ratio (OER) at low doses/fraction under conditions where cell cycle effects (repopulation, redistribution) were minimal. Consistent with there being a dose-dependent reduction in the OER at low doses, an extremely low OER of 1.34 was measured after 20 fractions of 1.7 Gy every 12 h. The sparing effects of fractionating radiation doses were not apparent for cells irradiated under hypoxic conditions (i.e., multifraction survivals were lower than acute single-dose values) until doses exceeding 15 Gy were reached. This result suggested a deficiency in the recovery from sublethal and/or potentially lethal damage might exist after hypoxic irradiations, thereby reducing the OER. The capacity to repair potentially lethal damage was found to be nearly the same after hypoxic as compared to aerobic irradiations. However, there was an apparent absence of sublethal damage repair by 10T1/2 cells between two hypoxic irradiations which could be a major contributing factor to the extremely low OER value measured in this multifraction schedule.     

Reparable and irreparable damage in yeast cells induced by sparsely ionizing radiation.

It is shown that in diploid yeast there are significant differences in the extent of irreparable damage after irradiation with X-rays, 60Co-gamma-rays and 30 MeV electrons. At extremely low dose rates, 60Co-gamma-rays were found to produce almost no irreparable damage at least up to 1200 Gy. X-rays, however, at the same low dose rate caused irreparable damage in the same dose range yielding a surviving fraction of 0.25 at 1200 Gy. For irradiations at high dose rate followed by liquid holding recovery the relative biological effectiveness of X-rays amounted to at least 4 for absorbed doses of up to 1000 Gy. With 30 MeV electrons at high dose rates an accumulation of sublethal and potentially lethal damage resulting in irreparable damage occurred above 1000 Gy. It is suggested that irreparable damage in yeast is due to a cooperative effect of neighbouring track ends.