Estimation of the contribution of ionization and excitation to the lethal effect of ionizing radiation

Summary A simple theoretical model is proposed for estimating the differential contribution of ionization and excitation to the lethal effect of ionizing radiation. Numerical results were obtained on the basis of published experimental data on the ability of bacterial cellsEscherichia coli to undergo photoreactivation of radiation-induced damage. It was shown that inactivation by excitation may be highly significant for UV-hypersensitive cells capable of photoreactivation; inactivation by excitation increased with the energy of ionizing radiation and the volume of irradiated suspensions. The data are in qualitative agreement with the assumption of a possible contribution of the UV-component of erenkov radiation to the formation of excitations responsible for the lethal effect and the phenomenon of photoreactivation after ionizing radiation. Some predictions from the model are discussed.     

Enhanced survival by photoreactivation and liquid holding following UV damage of TN-368 insect cells

These studies demonstrate that the TN-368 lepidopteran insect cell line, which is extremely resistant to the lethal effects of ionizing radiation, is also quite resistant to 254-nm ultraviolet light. While resistance to ionizing radiation in TN-368 cells has been associated with superior DNA repair processes, previous findings have indicated no correlation between survival ability and amount of unscheduled DNA synthesis in response to ultraviolet light. The present studies were undertaken to define the TN-368 ultraviolet light survival response, the ability of the cells to repair UV-induced damage by photoreactivation, the capacity of the cells to undergo UV repair during liquid holding in the dark, and the relationship between photoreactivation and liquid-holding recovery. Survival was assayed by colony formation. 254-nm irradiations were performed using germicidal lamps and photoreactivation was accomplished using black lights. Photoreactivable sectors of UV damage at 50 and 10% survival are 0.65 and 0.68, respectively. Survival responses, both with and without photoreactivation, have a small initial shoulder followed by an exponential region, and finally the curves continue to decrease but with decreasing slope. F0, Fq, and extrapolation number for the exponential portion of the curves are 77.5 J/m2, 16.8 J/m2, and 1.7 for non-photoreactivated cells and 234 J/m2, 56.1 J/m2, and 1.7 for those exposed to photoreactivating light. In the primarily exponential survival region, the fluences required to produce equivalent levels of survival in photoreactivated cells range from approximately 10.8 to 23.3 times as great as cells receiving UV light alone. The maximum survival enhancement of cells maintained under liquid-holding conditions over cells plated immediately following 100-400 J/m2 irradiations appears to be about 2-fold and occurs at 3-6 h of holding. Photoreactivation alone has a greater enhancement of survival than when photoreactivation follows liquid holding, but when liquid holding follows photoreactivation, the enhancement surpasses that of photoreactivation alone.     

Some effects of radiation hormesis for bacterial and yeast cells

A comparative study of chronic and acute action of ionizing radiation on the processes of aging and dying off of bacterial and yeast cells was carried out. It was ascertained that chronic action of ionizing radiation, 2-10,000 times exceeded the natural background, resulted in slowing down of aging and dying off of both pro- and eukaryotic cells. A single acute irradiation of yeast also resulted in the retardation of dying off of the yeast cells surviving after irradiation. The data is presented demonstrating a great increase in the survival of yeast cells under their repeated irradiation after recovery from potentially lethal radiation.     

Photoreactivation in Saccharomyces cerevisiae cells after irradiation with electrons (25 MeV). The role of photoreactivated damage in the manifestation of oxygen effects in radio- and UV-sensitive mutants of Saccharomyces cerevisiae

Significant photoreactivation was noted in radio- and UV-sensitive rad-mutants of Saccharomyces cerevisiae cells exposed to 25 MeV electrons. In order to make the photoreactivable damage be manifest anoxic conditions of irradiation should be chosen as optimal ones. It was shown that the low oxygen effect was partially associated with the photoreactivable damage involved in the lethal effect of ionizing radiation.     

Dependence of the radiosensitivity of yeast cells on radiation LET. A theoretical analysis

A model is proposed for interpreting the radiosensitivity of yeast cells as a function of linear energy transfer (LET) of ionizing radiation. The model takes into account the role of repair processes in sensitivity of yeast cells to ionizing radiation of different LET. Two types of repair are discussed: (1) a nonspecific repair (characteristic of both haploid and diploid cells), and (2) a diploid-specific repair (characteristic of diploid cells only).     

Protective role of superoxide dismutases against ionizing radiation in yeast

The protective role of superoxide dismutases (SODs) against ionizing radiation, which generates reactive oxygen species (ROS) harmful to cellular function, was investigated in the wild-type and in mutant yeast strains lacking cytosolic CuZnSOD (sod1Delta), mitochondrial MnSOD (sod2Delta), or both SODs (sod1Deltasod2Delta). Upon exposure to ionizing radiation, there was a distinct difference between these strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein, intracellular H2O2 level, as well as lipid peroxidation. When the oxidation of 2',7'-dichlorofluorescin was used to examine the hydroperoxide production in yeast cells, the SOD mutants showed a higher degree of increase in fluorescence upon exposure to ionizing radiation as compared to wild-type cells. These results indicated that mutants deleted for SOD genes were more sensitive to ionizing radiation than isogenic wild-type cells. Induction and inactivation of other antioxidant enzymes, such as catalase, glucose 6-phosphate dehydrogenase, and glutathione reductase, were observed after their exposure to ionizing radiation both in wild-type and in mutant cells. However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells. These results suggest that both CuZnSOD and MnSOD may play a central role in protecting cells against ionizing radiation through the removal of ROS, as well as in the protection of antioxidant enzymes.     

Effect of elevated temperatures on the radiation sensitivity of yeast cells of different species

Summary The influence of hyperthermia on the survival of irradiated yeast cells of different species has been studied. The experiments reported in the paper have shown: (1) simultaneous action of ionizing radiation and high temperatures appeared to increase the radiation response by a factor of approximately 2.7 for diploid and only by a factor of 1.5 for haploid cells of wild-type; (2) the combined action of high temperature and ionizing radiation had no synergistic effect for rad51 mutant diploid yeast cells; (3) heating before or after irradiation did not alter the radiation response of yeast cells; (4) enhancement of yeast cell sensitivity by simultaneous action of hyperthermia and²³⁹Pu--particles was negligible; (5) the magnitude and the rate of liquid holding recovery is lowered with increasing of irradiation temperature. On this basis, it was concluded that possible mechanism for thermal sensitization of yeast cells may involve the reduced capacity of cells to recover damages resulted from the combined action of both modalities.     

Release of Inorganic Phosphate from Irradiated Yeast: Radiation Biodosimetry and Evaluation of Radioprotective Compounds

When cells of bakers' yeast, Saccharomyces cerevisiae, were irradiated with ionizing radiation, inorganic phosphate, ninhydrin-reactive material, and substances absorbing at 260 mmu were released into the suspending medium. The amount of inorganic phosphate released depended on the radiation dose and on the temperature and pH during irradiation. The concentration of yeast cells did not affect the phosphate yield per milligram of yeast. It is suggested that the release of phosphate may serve as an index of the total radiation environment (i.e., as a biodosimeter) where radiation inactivation of microrganisms is of primary importance, e.g., in radiation preservation of foods. The somewhat limited range of the yeast biodosimeter (ca. 0.5 to 1.75 Mrad) may be extended by use of other more resistant microorganisms, such as bacterial spores. Compounds which have been reported as protecting microorganisms and mammals against the lethal effect of ionizing radiation also inhibited the radiation-induced release of inorganic phosphate from yeast. This phosphate release system is proposed as the basis for an economical, rapid supplement to screening procedures in the evaluation of radioprotective compounds.     

The Cosmic Silence experiment: on the putative adaptive role of environmental ionizing radiation

Previously we reported that yeast and Chinese hamster V79 cells cultured under reduced levels of background environmental ionizing radiation show enhanced susceptibility to damage caused by acute doses of genotoxic agents. Reduction of environmental radiation dose rate was achieved by setting up an underground laboratory at Laboratori Nazionali del Gran Sasso, central Italy. We now report on the extension of our studies to a human cell line. Human lymphoblastoid TK6 cells were maintained under identical in vitro culture conditions for six continuous months, at different environmental ionizing radiation levels. Compared to “reference” environmental radiation conditions, we found that cells cultured in the underground laboratories were more sensitive to acute exposures to radiation, as measured both at the level of DNA damage and oxidative metabolism. Our results are compatible with the hypothesis that ultra-low dose rate ionizing radiation, i.e. environmental radiation, may act as a conditioning agent in the radiation-induced adaptive response.     

Comparative study of RBE of densely ionizing radiation for various types of cell death in yeast

A comparative study of the relative biological effectiveness (RBE) of alpha-particles 249Pu for reproductive and interphase forms of killing of haploid and diploid yeast cells of wild-type and their radiosensitive mutants has been carried out. The correlation between the RBE of alpha-particles and cell repair capacity was confirmed for reproductive death: it was the highest for diploid cells, smaller for haploid cells and the smallest for their radiosensitive mutants. To achieve the interphase cell killing much higher irradiation doses were used after which cells were incapable of liquid-holding recovery during the storing of exposed cells in non-nutrient media at 30 degrees C. The RBE values for this form of killing were significantly lower in comparison with reproductive death. These data are an additional argument supporting the point of view that the RBE of densely ionizing radiation is determined not merely by physical processes of energy absorption as it is traditionally believed but also by ability of cells to recover from DNA damages inflicted by ionizing radiation.     

New photo-induced effects of reactivation and protection of yeast cells under lethal UVB radiation

Brief exposure of yeasts to low-intensity monochromatic light (400–730 nm) has revealed the effects of photoreactivation and photoprotection of the cells inactivated by medium wave UVB radiation (290–320 nm). The red spectral region with a maximum at 680 nm has been found to be the most active in the initiation of photoreactivation and photoprotection. It has been noted that, according to the regularities investigated, these processes differ fundamentally from the known processes of enzymatic photoreactivation and photoprotection, which have a spectral response limited by, respectively, blue (<450 nm) and near (<380 nm) UV light. The data obtained make possible to consider the observed effects of photoreactivation and photo-protection as the manifestation of functioning of some light-dependent defense system capable of increasing the resistance of cells to UVB radiation.     

The Saccharomyces Cerevisiae Ku Autoantigen Homologue Affects Radiosensitivity Only in the Absence of Homologous Recombination

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G(1) and G(2) or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.     

Yeast survival following gamma irradiation: delayed colony formation and determination of the proportion of undamaged cells

The experimental method is proposed to determine both a relative number of cells without damages and the effect of the delayed appearance of colonies of haploid and diploid yeast after exposure to ionizing radiation.     

Radiosensitization of yeast cells by inhibition of histone h4 acetylation

Deletion of genes for proteins involved in histone H4 acetylation produces sensitivity to DNA-damaging agents in both Saccharomyces cerevisiae and mammalian cells. In the present studies, we show that treating wild-type yeast cells with histone acetyl transferase (HAT) inhibitors, which are chemicals that cause a global decrease in histone H4 acetylation, sensitizes the cells to ionizing radiation. Using HAT inhibitors, we have placed histone H4 acetylation into the RAD51-mediated homologous recombination repair pathway. We further show that yeast cells with functionally defective HAT proteins have normal phospho-H2A (gamma-H2A) induction after irradiation but a reduced rate of loss of gamma-H2A. This argues that HAT-defective cells are able to detect DNA double-strand breaks normally but have a defect in the repair of these lesions. We also show that cells treated with HAT inhibitors have intact G1 and G2 checkpoints after exposure to ionizing radiation, suggesting that G1 and G2 checkpoint activation is independent of histone H4 acetylation.     

Liquid holding recovery kinetics in wild-type and radiosensitive mutants of the yeast Saccharomyces exposed to low- and high-LET radiations

Three wild-type diploid yeast strains Saccharomyces ellipsoideus and Saccharomyces cerevisiae and five radiosensitive mutants of S. cerevisiae in the diploid state were irradiated with gamma-rays from 60Co and alpha-particles from 239Pu in the stationary phase of growth. Survival curves and the kinetics of the liquid holding recovery were measured. It was shown that the irreversible component was enhanced for the densely ionizing radiation in comparison to the low-LET radiation while the probability of the recovery was identical for both the low- and high-LET radiations for all the strains investigated. It means that the recovery process itself is not damaged after densely ionizing radiation and the enhanced RBE of the high-LET radiation may be caused by the increased yield of the irreversible damage. A parent diploid strain and all its radiosensitive mutants showed the same probability for recovery from radiation damage. Thus, the mechanism of the enhanced radiosensitivity of the mutant cells might not be related to the damage of the repair systems themselves but with the production of some kind of radiation damage from which cells are incapable to recover.     

Effect of the dose rate on the synergism of combined ionizing radiation and hyperthermia

In experiments with yeast cells it was shown that the synergistic effect of a combination of ionizing radiation and hyperthermia is a function of dose rate. It was demonstrated that the temperature at which radiation is delivered should be elevated to obtain the maximum synergistic effect with the increasing dose rate.     

Repair-induced Changes in Yeast Radiosensitivity

Potentially lethal X-ray or ultraviolet damage in the diploid yeast, Saccharomyces cerevisiae, can be reversed if the irradiated cells are incubated in distilled water or buffer for a number of hours prior to plating. This phenomenon is called liquid-holding recovery. We found that the liquid-holding procedure served not only to restore the viability of the irradiated cells, but also to alter their sensitivity to further doses of radiation. Specifically, the ultraviolet sensitivity of cells which had undergone liquid-holding recovery was markedly decreased, whereas their X-ray sensitivity appeared to be slightly increased. These sensitivity changes were qualitatively the same irrespective of whether the initial radiation exposure was to X rays or ultraviolet light. (In contrast, the radiation sensitivity of cells which had undergone maximal photoreactivation was essentially the same as that of untreated controls.) It is suggested that these changes in radiosensitivity are the result of structural alterations induced in the cell's deoxyribonucleic acid by the execution of at least the initial steps of a deoxyribonucleic acid repair process during the liquid-holding period.     

Genetic instability of colonies' morphologic characteristics in the yeast Saccharomyces cerevisiae. The influence of mutations of radiosensitivity]

The exposure to ionizing radiation of radiosensitive mutants of diploid yeast Saccharomyces cerevisiae deficient in double-strand break repair results in formation of morphologically unstable colonies. Some characteristics of this process were studied. The results obtained are consistent with the hypothesis on relationship between DNA double-strand breaks or their repair with the formation of unstable clones of diploid yeast cells.     

Hyperthermic inactivation of diploid yeast and the interaction of damage caused by hyperthermia and ionizing radiation.

Inactivation of diploid yeast by hyperthermia has been studied. DO and Dq decrease with temperature for euoxic and anoxic conditions. The Arrhenius plot shows a break at 52 degrees C; the inactivation energies above and below this temperature are 153 and 94kcal/mol respectively. Hyperthermia (20 min at 51 degrees C) also potentiates the lethal action of gamma rays in diploid yeast cells under both euoxic and anoxic conditions. The interaction between hyperthermic and radiation damage appears to be largely at the sublethal level. The euoxic cells, the hyperthermic potentiation decreases with increasing time between the application of hyperthermia and radiation, being completely lost after 24 hours. However, in the anoxic cells there was no decrease in the hyperthermic potentiation with increasing time interval. These results suggest that yeast cells are capable of repairing hyperthermic sublethal damage, but require oxygen to do so. Thus there is a similarity in the process of repair of sublethal damage caused by ionizing radiation on the one hand and hyperthermia on the other.     

Evaluation of homology between cloned Escherichia coli and yeast DNA photolyase genes and higher eukaryotic genomes

Repair of ultraviolet-induced pyrimidine dimers by photoreactivation is catalyzed by a single enzyme, DNA photolyase. However, the process of photoreactivation is difficult to detect reproducibly in cultured mammalian cells. We have used clones containing yeast and Escherichia coli DNA photolyase genes to determine whether their sequences are conserved and whether there is homology between either cloned sequence and chick or human genomic DNA and mRNA sequences. The cloned sequences failed to hybridize to each other even under nonstringent conditions, indicating little conservation of sequence between the yeast and E. coli genes. Furthermore, only weak hybridization under nonstringent conditions was found between the cloned photoreactivating genes and human or chick genomic DNA or mRNA. This indicates that there is negligible homology between the cloned probes and mammalian DNA, but we are unable to conclude whether this indicates sequence divergence for prokaryotic and eukaryotic photoreactivation genes or the absence of such genes from the mammalian genome.