Recovery of yeast cells after exposure to ionizing radiation and hyperthermia

Quantitative regularities of recovery of wild-type diploid yeast cells irradiated with gamma-rays (60Co) simultaneously with exposure to high temperatures were studied. It was shown that in conditions of such a combined action the constant of recovery did not depend on the temperature at which the irradiation was carried out. However, with an increase of acting temperature an augmentation in the portion of irreversible component was registered. The analysis of cell inactivation revealed that the augmentation of the irreversible component was accompanied by a continuous increase of cell killing without any postirradiation division after which cells are incapable of recovery. The reproductive death was mainly exerted after ionizing radiation applied alone while in conditions of simultaneous thermoradiation action the interphase killing (cell death without division) predominated. It is concluded on this base that the mechanism of synergistic interaction of ionizing radiation and hyperthermia may be related with cardinal change in mechanisms of cell killing.     

The prognosis of the portion of irreversible radiation damages after simultaneous action of hyperthermia and ionizing radiation on yeast cells

The results of experimental research of diploid yeasts cells survival after simultaneous action of hyperthermia and ionizing radiation (60Co) have been described. It was shown that the cell ability to liquid holding recovery decreased with an increase in the temperature, at which the exposure was carried out. due to the increase in the irreversible component determining the relative part of radiation damage which cells are incapable to recover. To predict theoretically the relative part of irreversible radiation damage after combined action, the mathematical model was suggested taking into account the synergistic interaction of agents. Good correlation between experimental results and model prediction was demonstrated. The importance of the results obtained for the interpretation of the mechanism of synergistic interaction of various factors is discussed.     

Quantitative estimation of the recovery parameters of yeast cells irradiated in the presence of cysteamine

Study of the postirradiation recovery parameters of diploid yeast cells showed that the irreversible component of radiation damage was identical after the cell exposure in the presence and absence of cysteamine. On this basis, it is concluded that the radioprotector equally reduced the number of both irreversible and repairable primary radiation damages. Application of the mathematical model of recovery processes to the results obtained allowed us to draw a conclusion that the probability of cell recovery from the radiation damage per time unit was also identical after cell exposure in the presence and absence of cysteamine.     

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.     

Dark recovery of diploid yeast cells after simultaneous exposure to UV-irradiation and hyperthermia

Quantitative regularities of dark recovery of wild-type diploid yeast cells of Saccharomyces cerevisiae simultaneously treated with UV-light (254 nm) and high temperatures (53-56 degrees C) were studied. Under this combined action, the constant of recovery, which defines the probability of elimination of the UV-radiation induced damage per unit of time, did not depend on the temperature of irradiation. It was shown that both the irreversible component of cell damage and the number of cells that died without division gradually increased as the temperature of exposure increased. It is concluded, on this basis, that the mechanism of synergistic interaction of UV-radiation and hyperthermia is related not to the inhibition of dark recovery itself, but to the increase in the shape of irreversibly damaged cells incapable of recovering from the induced damage.     

Prognosis of yeast cells recovery after simultaneous exposure to UV-radiation and hyperthermia

The results of experimental investigations of survival of diploid yeast cells Saccharomyces cerevisiae (strain XS800) after simultaneous exposure to UV-radiation (254 nm) and hyperthermia (53-57 degrees C) have been described. It was shown that the portion of cells capable of recovery in innutrient medium after the action of these agents decreased with the increasing of temperature under which the irradiation was occurred. Mathematical model taking into account the synergistic interaction was suggested for quantitative prediction of irreversible component after combined actions of these agents. A good correspondence between experimental data and model predictions has been demonstrated. The importance of the results obtained for the interpretation of the synergistic interaction mechanisms are discussed.     

Enhanced expression of heat shock protein and mRNA synthesis by type I interferon in human HL-60 leukemic cells

The effects of IFN and mild hyperthermia on the responses of human promyelocytic HL-60 cells were investigated. Cells subjected to an elevated culture temperature (39.5 degrees-40.5 degrees C instead of 37 degrees C, herein referred to as heat-treated cells) showed an increase in heat shock proteins (HSPs) and corresponding mRNA synthesis, which were additionally potentiated by the presence of IFN. With cells cultured at 37 degrees C, IFN had no effect on HSP expression. The observed inhibition (40-70%) of RNA polymerase II-directed RNA synthesis (based on alpha-amanitin sensitivity) in isolated nuclei of heat-treated cells was also significantly reversed by the simultaneous addition of IFN. These data suggest that the IFN-amplified HSP gene expression may be involved in preventing irreversible damage or in fine tuning the recovery of mammalian cells from heat stress.     

Qantitative description of mammalian cell recovery after combined exposure to ionizing radiation with chemical agents

The parameters of recovery of mammalian cells after exposure to ionizing radiation combined with chemical agents were calculated based on a mathematical model of post-radiation recovery. The data presented, in contrast to the previously published results, indicate that the inhibition of the recovery may be due either to the damage or disruption of the process of recovery or the increase in the part of irreversible damages from which the cells are incapable to recover; or both of these processes can be realized simultaneously. It is concluded that the combination of ionizing radiation with chemical agents that inhibit the recovery processes through the formation of irreversible damages or affect the probability of recovery can be a perspective way in terms of finding the most effective means to increase radiosensitivity.     

Heat potentiation of radiation damage versus radiation potentiation of heat damage

The enhanced lethality of mammalian cells after combined treatment with hyperthermia and radiation is usually attributed to heat potentiation of radiation damage. However, it has been suggested that the situation may be reversed and that radiation may act as a modifier for heat damage. To test this hypothesis, BP-8 murine sarcoma cells were subjected to sequential radiation and heat treatments and the kinetics and extent of cell death were evaluated with the [125I]-iododeoxyuridine prelabeling assay. Cell death after heating was rapid and essentially complete within 2 days after heat exposure, whereas radiation death was slow and became apparent only after a delay period of 3 days. Combined exposure of cells to radiation and heat caused a pronounced increase in the delayed component of cell death, that is, the radiation component of death. Irradiation of cells before heating did not change the early heat component of cell death even in cells that were exposed to massive radiation doses of up to 300 Gy prior to heating. These results indicate that the increased cell death observed in hyperthermia/radiation-treated cells results from heat potentiation of radiation damage, not radiation potentiation of heat damage.     

Optimizing thermal and radiation effects for bacterial inactivation

The temperatures required for dry-heat spacecraft sterilization have been known to degrade heat-sensitive components. Thermoradiation, the simultaneous application of dry heat and gamma radiation, can provide the same degree of microbial inactivation as dry heat alone while substantially reducing component degradation. This is made possible by the synergistic effects produced when relatively low levels of these agents (e.g., 90 to 350 krads and 60° to 105°C) are applied simultaneously, thus permitting the use of lower temperatures and a reduced duration of heat exposure. The effects of temperature, radiation dose rate, and relative humidity on microbial inactivation during thermoradiation exposure have been established.This experimentation was supported by NASA Contract No. W-12853.     

The influence of chemical inhibitors of DNA repair on the recovery of mammalian cell damages induced by ionizing radiation

Using experimental results published by other authors the irreversible component of radiation damage and recovery constant, characterized the probability of recovery of mammalian cells of various origin from radiation damages per unit time, have been calculated. It was shown that the inhibition of postirradiation recovery, displayed in the decreasing of both the rate and the volume of recovery, has occurred due to the increasing in the portion of radiation damages from which the cell is incapable to recover. At the same time the recovery constant was independent on the conditions of combined action in the most cases, being decreasing in small extent only for hydroxyurea and 3-aminobenzamide. It was concluded that the inhibition of recovery is not the main reason of chemical radiosensibilization, but is a quite expected consequence of the increase in the portion of irreversibly damaged cells.     

Recovery of yeast cells following the combined action of hyperthermia and ionizing radiation

Consecutive action of elevated temperature (50 degrees C) and gamma-irradiation on yeast cells Saccharomyces cerevisiae was studied. It was shown that yeast cells can recover from lethal thermal and radiation lesions after the combined action of the two factors. The efficiency of recovery does not depend upon the sequence of treatments. Heating (50 degrees C) before or after gamma-irradiation increases the radiation response of yeast when plating the cells on a nutrient agar containing 1.5 M KCl. The synergistic effect decreases with yeast cells kept in water at 28 degrees C before plating. The influence of one factor on the effectiveness of recovery from damages induced by the other was estimated.     

Binding activity of glucocorticoid receptors after heat shock

The response of glucocorticoid receptors (GR) to heat was measured by the change in ligand binding activity both in control cells and in cells made tolerant to heat by a prior mild heat exposure. The study was prompted by earlier data showing that one of the heat shock proteins (HSP90) is an essential component of the GR complex and that treatment of mammalian cells with hydrocortisone induces resistance to heat damage. The GR rapidly loses binding activity after commencement of heating. There is a 50% loss of activity after 4 min at 45 degrees C, 8 min at 44 degrees C, or 17 min at 43 degrees C. The reduction in binding is due mainly to a reduction in affinity of binding to the ligand. The ability to bind glucocorticoid recovers quickly after heat treatment. Activity returns to levels 60-80% of normal by 2 h after a heat treatment that initially reduces binding to less than 20% of normal. However, complete restoration of binding activity takes approximately 3 days. The recovery of binding activity does not require protein synthesis. Pretreatment of cells with hydrocortisone, using conditions that induce heat resistance, reduces the activity to 10-20% of control, but residual receptors display a heat sensitivity similar to that of control cells. There was evidence for a limited degree of protection of GR from heat damage in thermotolerant cells.     

Hyperthermia in a differentiating murine erythroleukemia cell line: cell killing by heat and radiation

The survival response of Friend erythroleukemia cells (a differentiating cell system) to heat and radiation has been examined. The Friend erythroleukemia cells (FELC) were more heat and radiation sensitive than V79 cells, and the heat and radiation survival curves possessed shoulders, showing the ability of the cells to accumulate sublethal damage. Thermal tolerance was expressed after prolonged heating at 41.0-42.0 degrees C. Thermal radiosensitization by heating at 42.0 or 45.0 degrees C was greatest for simultaneous heat and radiation treatments, and recovery occurred when the cells were incubated at 37 degrees C between the heat and radiation or radiation and heat treatments. Arrhenius analysis of the FELC heat survival data showed that the curve for thermal inactivation possessed a break at about 43.0 degrees C and that the thermal inactivation energies above and below the break point were comparable to those for V79 cells and other cell lines reported in the literature.     

Temperature regulation in the unrestrained rabbit during exposure to 600 MHz radiofrequency radiation

Six male New Zealand white rabbits were individually exposed to 600 MHz radiofrequency (RF) radiation for 90 min in a waveguide exposure system at an ambient temperature (Ta) of 20 or 30 degrees C. Immediately after exposure, the rabbit was removed from the exposure chamber and its colonic and ear skin temperatures were quickly measured. The whole-body specific absorption rate (SAR) required to increase colonic and ear skin temperature was determined. At a Ta of 20 degrees C the threshold SAR for elevating colonic and ear skin temperature was 0.64 and 0.26 W/kg, respectively. At a Ta of 30 degrees C the threshold SARs were slightly less than at 20 degrees C, with values of 0.26 W/kg for elevating colonic temperature and 0.19 W/kg for elevating ear skin temperature. The relationship between heat load and elevation in deep body temperature shown in this study at 600 MHz is similar to past studies which employed much higher frequencies of RF radiation (2450-2884 MHz). On the other hand, comparison of these data with studies on exercise-induced heat production and thermoregulation in the rabbit suggest that the relationship between heat gain and elevation in body temperature in exercise and from exposure to RF radiation may differ considerably. When combined with other studies, it was shown that the logarithm of the SAR required for a 1.0 degree C elevation in deep body temperature of the rabbit, rat, hamster, and mouse was inversely related to the logarithm of body mass. The results of this study are consistent with the conclusion that body mass strongly influences thermoregulatory sensitivity of the aforementioned laboratory mammals during exposure to RF radiation.     

The dynamics of the induction and regression of puffs 87A, 87C and 93D in the polytene chromosomes of Drosophila melanogaster under the action of anoxia and high temperature]

A study was made of the heat shock puff activity in salivary glands of Drosophila melanogaster larvae after 5 and 20 min treatments with anoxia (dipping into physiological solution), heat shock (37 degrees C), and simultaneously with both the agents. The simultaneous treatment with heat shock and anoxia, as well as treatment with anoxia only blocked the induction of heat shock puffs. They appeared 10-15 min after the treatment during recovery under aerobic conditions. There was a super-additive effect of the simultaneous treatment on the heat shock puffing duration. A specific regulation of the 93D locus was observed. The 93D puff was induced by a 5 min simultaneous treatment with anoxia and heat shock and, as a rule, was not induced by the analogous 20 min treatment. The role of anoxia in blocking heat shock puff induction under simultaneous effects of heat shock and anoxia is discussed.     

Reduction in metabolic heat production during exposure to radio-frequency radiation in the rat

The purpose of this study was to assess the ability of the rat to reduce metabolic rate when exposed to deep-penetrating radio-frequency (RF) radiation. Male Sprague-Dawley rats were maintained at an ambient temperature (Ta) of 10 degrees C and exposed to 600-MHz radiation while metabolic rate (MR) was measured by indirect calorimetry. RF radiation exposures were made in a waveguide-type system that permitted the continuous control of specific absorption rate (SAR). SAR's of 2-5 W/kg led to significant reductions in MR when averaged from 30 to 60 min after the initiation of RF radiation exposure. The total decrease in MR during RF radiation exposure accounted for approximately 37% of the total RF heat load. Exposure of another group of rats to the same SAR's at a Ta of 10 degrees C resulted in a significant elevation in colonic temperature. Thus, despite the decrease in MR, heat gain still exceeded heat loss during RF radiation exposure, with a resultant elevation in deep body temperature. In conclusion, in a cold environment the rat exposed to RF radiation decreases its MR. However, the response time and efficiency of the response is not adequate to prevent an increase in body temperature.     

Is DNA damage the signal for induction of thermal resistance? induction by radiation in yeast

Yeast, as well as higher eukaryotes, are induced to increase thermal resistance (thermotolerance) by prior exposure to a heat stress. Prior exposure to an acute dose of either 60Co gamma or 254-nm ultraviolet radiation, at sublethal or fractionally lethal doses, is shown to cause a marked increase in the resistance of Saccharomyces cerevisiae to killing by heat. Following a radiation exposure, thermal resistance increased with time during incubation in nutrient medium, and the degree of resistance reached was proportional to the dose received. Partial induction by radiation followed by maximum induction by heat did not produce an additive response when compared to a maximum induction by heat alone, suggesting that the same process was induced by both heat and radiation. Irradiation with 254-nm uv light followed by an immediate, partial photoreversal of the pyrimidine dimers with long-wavelength uv light resulted in a reduced level of resistance compared to cells not exposed to the photoreversal light, indicating that the cells specifically recognized pyrimidine dimers as a signal to increase their thermal resistance. Exposure to 254-nm uv or ionizing radiation induced thermal resistance in mutants defective in either excision repair (rad3, uv-sensitive) or recombinational repair (rad52, gamma-sensitive), suggesting that recognition and repair of DNA damage by these systems are not a part of the signal which initiates an increase in resistance to heat. The amount of induction, per unit dose, was greater in the DNA repair-deficient mutants than in the wild-type cells, suggesting that an increase in the length of time during which damage remains in the DNA results in an increase in the effectiveness of the induction. These data indicate that types of DNA damage as diverse as those produced by ionizing radiation and by ultraviolet light are recognized as a signal by the yeast cell to increase its thermal resistance. It is therefore suggested that heat-induced alterations in DNA or in DNA-dependent chromosomal organization may be the signal for heat induction of thermotolerance in this and other eukaryotes.     

The eukaryote chaperonin CCT is a cold shock protein in Saccharomyces cerevisiae

The eukaryotic Hsp60 cytoplasmic chaperonin CCT (chaperonin containing the T-complex polypeptide-1) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin. The present research focused mainly on the expression of the CCT subunits, CCTalpha and CCTbeta, in yeast (Saccharomyces cerevisiae). Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock. In this study, messenger ribonucleic acid (mRNA) and protein levels of CCT subunits following exposure to low temperatures, were examined. The Northern blot analysis indicated a 3- to 4-fold increase in mRNA levels of CCTalpha and CCTbeta genes after cold shock at 4 degrees C. Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTalpha protein, which is expressed at 10 degrees C, but not at 4 degrees C. Transfer of 4 degrees C cold-shocked cells to 10 degrees C induced a 5-fold increase in the CCTalpha protein level. By means of fluorescent immunostaining and confocal microscopy, we found CCTalpha to be localized in the cortex and the cell cytoplasm of S. cerevisiae. Localization of CCTalpha was not affected at low temperatures. Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy. The induction pattern of the CCTalpha protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and the transition to growth at higher temperatures, as found for other Hsps during the recovery phase from heat shock.     

Influence of hyperthermia on gamma-ray-induced mutation in V79 cells

Asynchronously growing V79 cells were assayed for mutation induction following exposure to hyperthermia either immediately before or after being irradiated with 60Co gamma rays. Hyperthermia exposures consisted of either 43.5 degrees C for 30 min or 45 degrees C for 10 min. Each of these heat treatments resulted in a survival level of 42%. For all sequences of combined treatment with hyperthermia and radiation, cell killing by gamma rays was enhanced. Mutation induction by gamma rays was enhanced when heat preceded gamma irradiation, but no increase was observed when heat was given after gamma exposures. Treatment at 45 degrees C for 10 min gave a higher yield in mutants at all gamma doses studied compared to treatment at 43.5 degrees C for 30 min. When heat-treated cells were incubated for different periods before being exposed to gamma rays, thermal enhancement of radiation killing was lost after 24 h. In contrast, only 5-6 h incubation was needed for loss of mutation induction enhancement.