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.     

Photoreactivation of damage induced by ionizing radiation in yeast cells

Summary Experimental data on photoreactivation of damage induced by ionizing radiation in yeast cells are presented. The value of photoreactivation was found to be the highest for the following conditions predicted by us as optimum ones: large volume of irradiated suspension, hypoxia and high energy sparsely ionizing radiation. A comparison of data for yeast and bacterial cells shows that Cerenkov emission from ionizing radiation may produce photoreactivated pyrimidine dimers in both prokaryotic and eukaryotic cell systems.     

Some factors affecting the sensitivity of cultured human cells to high-LET radiation

Summary Comparative effects of decay of DNA-bound¹²⁵I, of-radiation and of tritiated water on survival of the proliferative ability of cultured cells were examined. The results confirm a previous report that cells frozen to -196° C in the presence of 2M glycerol have lost a considerable proportion of their intracellular water. The data also suggest that the fraction of the lethal damage caused by deposition of radiation energy in intracellular water close to the DNA is greater for-radiation than for the decay of DNA-bound¹²⁵I.Inherited differences in the sensitivity of untransformed fibroblasts from individual humans to ionizing radiations and other DNA-damaging agents are being explored. The ratios of the sensitivities of various cell lines to particular agents can vary several-fold. Thus the RBE of various radiations is affected not only by the irradiation conditions and the water content of the cells but also by inherited abnormalities in the DNA repair systems in human cells.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

Role of DNA damage in the inactivation of Saccharomyces cerevisiae yeasts by 313-nm ultraviolet light

The relative contribution of respiration photoinhibition and DNA damage in the lethal effect induced by 313 nm ultraviolet light (UV) has been investigated in some strains of the yeast Saccharomyces cerevisiae. It has been shown that cells inactivation is essentially due to photo-induced damage to DNA. By photoreactivation experiments it has been found that dimers of the pyrimidine bases are the main lethal photoproducts induced in the DNA by 313 nm ultraviolet light.     

Dissection of functional domains in Escherichia coli DNA photolyase by linker-insertion mutagenesis.

Summary The phr gene, which encodes protein of 472 amino acid residues, is required for light-dependent photoreactivation and enhances light-independent excision repair of ultraviolet light (UV)-induced DNA damage. In this study, dodecamer HindIII linker insertions were introduced into the cloned phr gene and the functional effects of the resulting mutations on photoreactivation and light-independent dark repair in vivo were studied. Among 22 mutants obtained, 7 showed no photoreactivation as well as no enhancement of light-independent repair. Four of these were located in amino acid residues between Gln333 and Leu371 near the 3 end of the gene, two were located in a small region at Glu275 to Glu280 near the middle of the gene and the remaining one was between Pro49 and Arg50. Three mutants that had insertions located in the 42 by segment from 399 to 441 by of the phr coding sequence (corresponding to amino acid residues Ile134 to Lys149) lost the light-independent repair effect but retained photoreactivation. These results suggest that (i) Escherichia coli DNA photolyase contains several critical sites that are distributed over much of the enzyme molecule, and (ii) a functional domain required for the effect on light-independent repair is at least in part distinct from that necessary for light-dependent photoreactivation.     

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.     

Different rates of ultraviolet-induced DNA damage in the epidermis and dermis of a platyfish model for carcinogenesis

The purpose of this study was to compare fluence-response relationships for the production of cyclobutane pyrimidine dimers in epidermal or dermal DNA of platyfishXiphophorus hybrids irradiated with UVB, and to determine photoreactivation from black light on dimers producedin situ. This was accomplished by quantitative gel electrophoresis of unlabeled DNA following extraction of the DNA and treatment with an enzyme specific for the detection of pyrimidine dimers. The dermis was the target tissue for UV-induced DNA damage inXiphophorus hybrid fish skin. Shapes of dimer-fluence response data following filtered sunlamp irradiation ( > 290 nm) or monochromatic wavelength 302 nm in the epidermis or dermis were different. In the epidermis there was an initial steep upward slope followed by a plateau, whereas in the dermis a linear relationship was observed. The final values of dimers at the high doses were, however, nearly equal in the epidermis and dermis exposed to either radiation. These differences in fluence-response relationships are probably attributable to the intertwining of the epidermis and to the shielding effect of the epidermal layer, with scales leading to a heterogenous population of cells which are exposed to different UV doses. Photoreversal of dimers was readily observed by black light irradiation in both epidermis and dermis irradiated with either > 290 nm or 302 nm.This research was supported by the Office of Health and Environmental Research of the U.S. Department of EnergyThe author is recipient of the National Academy of Sciences' Kobelt Fund Grant     

Radiosensitive mutants of yeast-Saccharomyces with disruptions in the cell division cycle. Cell inactivation by restrictive temperature, ultra-violet and ionizing radiation in relation to the passage of the generated cycle

Three thermo- and radiosensitive mutants of yeast-Saccharomyces were used to study cell inactivation under the effect of elevated temperature, UV-light, and ionizing radiation. The forms of cell inactivation were shown to be identical with all the factors under study and to resemble lethal "terminal phenotypes" of Hartwell cds mutants. It is suggested that the cell division cycle is blocked due to the disorders in the system of gene product synthesis and not to defects in the enzymes themselves.     

Effect of gamma irradiation on membranes of normal and pathological erythrocytes (beta-thalassemia)

Summary The influence of ionizing radiation on the membrane of human normal erythrocytes has extensively been studied and a variety of effects including changes in the cation fluxes [3, 9] or in non-electrolytes permeability [5, 6, 11], in membrane fluidity, in peroxidation of unsaturated lipids as well as chemical composition or structural modifications [4, 7, 8] has been observed.However, only few studies deal with the effects of ionizing radiation on pathological red blood cells.In this work, we have investigated by means of electron spin resonance (ESR) spectroscopy the effects of⁶⁰Co-radiation on the normal and homozygous-thalassemic human erythrocyte membranes [2, 13].Presented in part at the VII Congress of the Società Italiana di Biofisica Pura ed Applicata Lipari (Italy) 1985     

Photoreactivation in Methanobacterium thermoautotrophicum

The sensitivity of three methanogenic bacteria towards ultraviolet irradiation was similar to the UV-sensitivity of Escherichia coli. The lethal effects of UV-irradiation in Methanobacterium thermoautotrophicum Marburg and in Methanobacterium thermoautotrophicum H but not in Methanococcus vannielii were reversed by exposure to visible light. In cell suspensions of Methanobacterium thermoautotrophicum that had been irradiated to 0.1% survival, 90% of the UV-caused damage was photorepairable. The in vivo action spectrum for photoreactivation suggests that in this organism a deazaflavin, probably F₄₂₀, functions as the chromophore of the photoreactivating enzyme.     

Radiolysis,racemization and the origin of molecular asymmetry in the biosphere

Summary An investigation has been undertaken to determine whether ionizing radiation might engender racemization of optically active amino acids, along with their usual radiolysis. As prototypes, crystalline D- and L-leucine, as well as aqueous solutions of their sodium salts were exposed to the radiation from a 3000 Ci⁶⁰Co-ray source.-ray doses which caused about 68% radiolysis of solid leucine left a residue which was about 5% racemized, while racemization proved even greater at lower doses for the dissolved sodium salts. In aqueous solution both percent degradation and percent racemization of the sodium salts were proportional to-ray dosage within the range employed (1–27 · 10⁶ rads). Implications of these observations for the origin of molecular asymmetry by the-decay parity violation mechanism are discussed.     

Protective Role of Glutathione Synthesis on Radiation-Induced DNA Damage in Rabbit Brain

1. Radiotherapy has attracted increasing interest in recent years. It is known that ionizing radiation induces oxygen radical injury, whereas oxidative stress by the radiation can cause cellular responses to defense cellular injury. In this study, the metabolism of antioxidants in response to ionizing radiation to the brain was studied in the brain using experimental rabbits.2. Ionizing radiation to the hemicerebrum caused an increase in the levels of glutathione (GSH) and the activity of a GSH synthesizing enzyme, -glutamylcysteine synthetase (-GCS), and Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Ionizing radiation also induced DNA-damage estimated by the formation of 8-hydroxydeoxyguanosine. These changes were dependent on the radiation dose.3. Previous intrathecal-administration of buthionine sulfoximine (100 M), a specific inhibitor of -GCS, increased DNA damage by radiation in the radiated hemicerebrum. That of S-methyl GSH, on the other hand, resulted in a significant reduction of DNA damage by radiation.4. These results suggest that synthesis of GSH and Cu,Zn-SOD is responsive to ionizing radiation and this induction of antioxidants may play a role in reducing tissue damage in radiotherapy.     

Investigation of the Functional Role of Ctp Proteins in the Cyanobacterium Synechocystis sp. PCC 6803

To understand the functional role of CtpB and CtpC proteins, which are similar to the C-terminal processing CtpA peptidase, the effect of the insertional inactivation of the ctpB and ctpCgenes on the phenotypic characteristics of Synechocystis sp. PCC 6803 was studied. The inactivation of the ctpC gene was found to be lethal to the cyanobacterium, which indicates a vital role of the CtpC protein. The mutant with the inactivated ctpB gene had the same photosynthetic characteristics as the wild-type strain. The double mutant ctpActpB with the two deleted genes was identical, in the phenotypic characteristics, to the mutant with a knock-out mutation in the ctpAgene, which was unable to grow photoautotrophically. The data obtained suggest that, in spite of the high similarity of the Ctp proteins, they serve different functions in Synechocystis sp. PCC 6803 cells and cannot compensate for each other.     

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.     

Hybrid dysgenesis in Drosophila: The mechanism of T-007-induced male recombination

Summary The term hybrid dysgenesis describes a syndrome of genetic effects which sometimes results when Drosophila melanogaster from wild populations are outcrossed; this syndrome often includes male recombination as well as enhanced rates of genic and chromosomal mutation, sterility, and transmission ratio distortion. In this study, we have examined the mechanism of T-007-induced male recombination by genetically characterizing third chromosomes generated by an exchange in a well-marked euchromatic region. Most recombinant chromosomes were sequentially normal, and no recessive lethal events at the point of exchange were recovered. The results demonstrate that although some recombinants may be generated by nonhomologous chromosome (or chromatid) breakage and reunion, the predominant effect of T-007 is through an enhanced rate of normal mitotic exchange. The rate of mitotic exchange is also increased by ionizing radiation and chemical mutagens; we suggest that the common factor in all three cases is the induction of single strand breaks.     

Induction of SOS repair by ionizing radiation

Summary ß-galactosidase and alkaline phosphatase activities of Escherichia coli strain PQ37 carrying the fusion gene of sulA and lacZ treated with different types of ionizing radiation were examined. The induction factor (ratio ofß-galactosidase to alkaline phosphatase activity), reflecting the SOS-induction potency, increases significantly with radiation dose. Maximum effectiveness to induce SOS-response has been found for deuterium and helium ions in comparison to -rays, carbon or krypton ions. Increased energy of helium ions leads to greater SOS-induction potency of radiation.     

The values of alpha 2-macroglobulin in the serum of persons at work with X-rays

Summary A group of 86 persons (65 women, 21 men) working at the risk of ionizing radiation (IR) was examined for the levels of alpha 2-macroglobulin ( ₂-M), and compared to a group of an age matched healthy population. Whereas the difference in men did not reach a level of significance, significant difference was assessed in women: in a subgroup older than 40 y exposed to the IR a rise of ₂-M levels was noted in correlation with the age. Multiple correlation coefficients showed a synergistic influence of both age and exposure to IR. The results are discussed from the point of view of the hypothesis that in exposed women a shifting realizes of usual old age changes to younger age group. A possibility is suggested to use the examination of ₂-M levels for preventive control of workers at the risk of IR.     

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.