Dependence of the radiosensitivity of yeast cells on the LET of radiations. Experiments on haploid cells

The previously developed model was used to study the dependence of radiosensitivity (D0(-1) of Saccharomyces cerevisiae (the wild type and radiosensitive mutant) on linear energy transfer (LET) of ionizing radiation. D0(-1) (L) of haploid yeasts was shown to be associated, to a certain extent, with the capacity of radiation damages repair. As to the wild-type cells, the above function was represented by a curve showing a maximum, while a descending curve was characteristic of the radiosensitive mutant cells deficient in radiation damages repair. The influence of the repair processes on cell radiosensitivity decreased with increasing LET.     

Induction of prophage lambda during the irradiation of E. coli cells exposed to radiations of various LET

Induction of lambda prophage in lysogenic E. coli cells exposed to ionizing radiation of different LET was studied as a function of dose I(D). Activities of pleiotropic RecA protein were shown to contribute to the shape of the I(D) curve. The experimental data were fitted by the function I(D) = alpha D(1-exp(-D0-1.D]exp(-beta D). Inducibility alpha increased with increasing LET which was related to the increased incidence of DNA lesions being a SOS-system call.     

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).     

Radioprotection by DMSO of mammalian cells exposed to X-rays and to heavy charged-particle beams

Populations of G1-phase Chinese hamster cells in stirred suspensions containing various concentrations of DMSO were irradiated with 250 kV X-rays or various heavy charged-particle beams. Chemical radioprotection of cell inactivation was observed for all LET values studied. When cell survival data were resolved into linear and quadratic components, the extent and concentration dependence of DMSO protection were found to be different for the two mechanisms. The chemical kinetics of radioprotection for single-events were similar for LET values up to those which gave the maximum RBE. DMSO protected to a lesser extent against energetic argon ions at an median LET of approximately 220 keV/micron. These data could indicate the contribution of indirect action by hydroxyl radicals and hydrogen atoms to cell inactivation by single-hit and double-hit mechanisms for various radiation qualities. The decrease in RBE observed at very high LET may result, in part, from reduced yields of water radicals at 10(-9)-10(-8) s resulting from radical recombination mechanisms within the charged particle tracks.     

Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. III. Human diploid fibroblasts.

The induction of inactivation and mutation to thioguanine-resistance in cultured human diploid fibroblasts was studied after exposure to ionising radiations with LET's in the range 20--470 keV micrometer-1. Unique r.b.e. values were obtained for inactivation and mutation induction with nine different qualities of radiation. The plot of r.b.e. verus LET gave humped curves for both endpoints; r.b.e. maxima were in the LET range 90--200 keV micrometer-1 but the maximum r.b.e. value for mutation induction was almost twice that for inactivation. The accuracy of estimates of mutation induction are discussed with regard to possible selective effects against mutants during post-irradiation growth.     

Neoplastic transformation in vitro by low doses of ionizing radiation: role of adaptive response and bystander effects

The shape of the dose-response curve for cancer induction by low doses of ionizing radiation is of critical importance to the assessment of cancer risk at such doses. Epidemiologic analyses are limited by sensitivity to doses typically greater than 50-100 mGy for low LET radiation. Laboratory studies allow for the examination of lower doses using cancer-relevant endpoints. One such endpoint is neoplastic transformation in vitro. It is known that this endpoint is responsive to both adaptive response and bystander effects. The relative balance of these processes is likely to play an important role in determining the shape of the dose-response curve at low doses. A factor that may influence this balance is cell density at time of irradiation. The findings reported in this paper indicate that the transformation suppressive effect of low doses previously seen following irradiation of sub-confluent cultures, and attributed to an adaptive response, is reduced for irradiated confluent cultures. However, even under these conditions designed to optimize the role of bystander effects the data do not fit a linear no-threshold model and are still consistent with the notion of a threshold dose for neoplastic transformation in vitro by low LET radiation.     

The effect of substrate on the radiation resistance of yeasts isolated from sausage meat

J.-A, McCARTHY AND A.P. DAMOGLOU. 1996. The radiation resistance of a selection of yeasts isolated from sausages was assessed in phosphate-buffered saline and in sausage meat. The yeasts Candida zeylanoides, Debaryomyces hansenii and Trichosporon cutaneum exhibited sigmoidal survival curves in both substrates whilst the more sensitive Sporobolomyces roseus exhibited an exponential survival curve in buffer but a sigmoidal curve in meat. Irradiating C. zeylanoides, D. hansenii and T. cutaneum in sausage meat changed the shape of their survival curves to significantly alter the calculated parameters D _s (the dose in kGy that must be achieved before reduction in numbers occurs) and D ₁₀^sig(the dose in kGy required after the shoulder to achieve a 1 log cycle reduction in numbers). The D _s values were reduced while higher D ₁₀^sigvalues were obtained demonstrating that the sausage meat contributed a protective effect to these yeasts at higher irradiation doses. For the yeast S. roseus , similar numbers of survivors were recovered from both substrates at initial low irradiation doses (0–0.5 kGy) with the protective effect being demonstrated again at higher doses (> 2 kGy). These findings should be considered when defining a commercial process to reduce the numbers of yeasts in these products.     

Biological effectiveness of ionizing radiations of various quality in Escherichia coli bacteria (a theoretical analysis). Cell radiosensitivity and DNA repair

On the basis of the ideas reported earlier a study was made of the dependence of radiosensitivity (D0-1) of isogenic mutations of E. coli upon LET. This dependence was shown to be conditioned not only by the physical parameters of radiation but also by the ability of cells to repair definite types of DNA lesions. The influence of the balance in the activity of repair enzymes in E. coli on the shape of D0-1 (LET) curve is discussed.     

Relation of the radiosensitivity of yeast cells to the LET of the radiation. Experiments with diploid cells

A study was made of the dependence of radiosensitivity D0-1 of diploid yeast cells on linear energy transfer L of radiation, and the obtained results were analyzed in terms of the previously developed model concepts. A diploid-specific repair of radiation damages was shown to be equally effective with different L. This mode of repair had no direct effect on D0-1 (L) function. The observed contribution of the diploid-specific repair to cell radioresistance decreased with increasing L. This, however, was due to physical and geometric factors rather than to the decreased efficiency of diploid-specific repair.     

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.     

Survival of diploid yeasts after gamma irradiation: genetic control of the maturation effect

A study was made of the influence of rad mutation, leading to radiosensitivity increase, on the effect of additional growth in diploid Saccharomyces cerevisiae yeast exposed to gamma-radiation. The most radiosensitive mutants of this series, rad52/rad52 and rad54/rad54, did not virtually vary from the wild type cells in the value of the additional growth effect. Some other mutants, for instance, rad53/rad53 and rad55/rad55, exhibited a significantly lesser effect of additional growth. It was shown that the effect of additional growth did not depend upon the rate of rapid and slow postirradiation recovery of the wild type cells. The results of the studies prompt the conclusion that the processes responsible for the additional growth effect and those responsible for recovery of cells from radiation damages are mutually independent.     

Effect of salt solutions on the radiosensitivity of mammalian cells as a function of the state of adhesion and the water structure.

The radiation isodose survival curve of attached Chinese hamster (V79) cells, subjected to a wide concentration range of salt or sucrose solutions, is characterized by two maxima separated by a minimum. Cells are radioprotected at the maxima (high and low hypertonic salt concentrations) while they are radiosensitized at the minimum (intermediate hypertonic salt concentrations). Both cations and anions can alter the cellular radiosensitivity above and beyond the (osmotic) effect observed for cells treated with sucrose solutions. However, the basic curve shape, except in the case of sulphate salts, remains the same. When these experiments are repeated with single cells in suspension, the isodose survival curve is quite different in that high salt concentrations (greater than 0.9 M) do not protect cells in suspension unlike the case with attached cells. The curve shape is also altered in that the second maximum is absent with many salt solutions. If multicellular spheroids are used for these experiments, the data resemble those for single cell suspensions rather than for attached cells. The radiation survival data for cells in suspension in salt solutions correlate with water proton spin-lattice relaxation time (T1) and, in hypo- and iso-tonic solutions, with cell volume.     

Power saturation of ESR signal in ammonium tartrate exposed to 60Co gamma-ray photons, electrons and protons

In this paper we present an investigation of the electron spin resonance (ESR) line shape of ammonium tartrate (AT) dosimeters exposed to radiation with different linear energy transfer (LET). We exposed our dosimeters to gamma-ray photons ((60)Co), 7 MeV and 14 MeV initial energy electrons, and 19.3 MeV initial energy protons. The differences in the power saturation behavior of ESR spectra of AT irradiated with photons, electrons and protons could be correlated to the effective LET of the radiation beams. We analyzed the behavior of peak-to-peak amplitude as a function of microwave power, and we developed a fitting procedure that permits us to obtain the dependence of the homogeneity parameter of the line shape on the LET of the radiation using the Castner saturation theory. This simple procedure allows us to distinguish the LET of the radiation beam.     

Inactivation and mutation of cultured mammalian cells by aluminium characteristic ultrasoft X-rays. II. Dose-responses of Chinese hamster and human diploid cells to aluminium X-rays and radiations of different LET.

The induction of inactivation and mutation to thioguanine-resistance of two types of cultured mammalian cells, V79 Chinese hamster and HF19 human diploid, was studied after irradiation with aluminium K characteristic ultrasoft X-rays, helium ion track intersections of different LET, 42 MeV d-Be neutrons, and hard X- or gamma-rays. The form of the dose-response curves was different for the two cell-types, and there was an overall difference in radiosensitivity, the human cells being the more sensitive to all radiations. However, for both inactivation and mutation-induction, the relative responses of both cell-types to these radiations was similar. Aluminium X-rays were considerably more effective than hard X- or gamma-rays and were at least as effective as helium ions of 20-28 keV micron-1, although aluminium X-rays produce tracks of very limited range (less than about 0.07 micron). Single track effects by aluminium X-rays cannot, therefore, extend beyond about 0.07 micron, and the subcellular sites involved in inactivation and mutation cannot be greater than this dimension or else the effectiveness of aluminium X-rays would be similar to that of low-LET radiations. This observation is in contradiction to models of radiation action which require relatively large sensitive sites; for example the 'theory of dual radiation action' requires a site diameter of about 0.4 micron to explain the shape of the dose-response curves for V79 hamster cells.     

Effects of heavy ions and energetic protons on normal human fibroblasts

At the low particle fluences of radiation to which astronauts are exposed in space, "non-targeted" effects such as the bystander response may have increased significance. The radiation-induced bystander effect is the occurrence of biological responses in unirradiated cells near to or sharing medium with cells traversed by radiation. The objectives of this study were to establish the responses of AG01522 diploid human fibroblasts after exposure to several heavy ions and energetic protons, as compared to X-rays, and to obtain initial information on the bystander effect in terms of cell clonogenic survival after Fe ion irradiation. Using a clonogenic survival assay, relative biological effectiveness (RBE) values at 10% survival were 2.5, 2.3, 1.0 and 1.2 for 1 GeV/amu Fe, 1 GeV/amu Ti, 290 MeV/amu C and 1 GeV/amu protons, respectively, compared to 250 kVp X-rays. For induction of micronuclei (MN), compared to the low LET protons, Fe and Ti are very effective inducers of damage, although C ions are similar to protons. Using a transwell insert system in which irradiated and unirradiated bystander cells share medium but are not touching each other, it was found that clonogenic survival in unirradiated bystander cells was decreased when irradiated cells were exposed to Fe ions or X-rays. The magnitude of the decrease in bystander survival was similar with both radiation types, reaching a plateau of about 80% survival at doses of about 0.5 Gy or larger.     

Responses of synchronous L5178Y S/S cells to heavy ions and their significance for radiobiological theory

Synchronous suspensions of the radiosensitive S/S variant of the L5178Y murine leukaemic lymphoblast at different positions in the cell cycle were exposed aerobically to segments of heavy-ion beams (20Ne, 28Si, 40Ar, 56Fe and 93Nb) in the Bragg plateau regions of energy deposition. The incident energies of the ion beams were in the range of 460 +/- 95 MeV u-1, and the calculated values of linear energy transfer (LET infinity) for the primary nuclei in the irradiated samples were 33 +/- 3, 60 +/- 3, 95 +/- 5, 213 +/- 21 and 478 +/- 36 keV microns-1, respectively; 280 kVp X-rays were used as the baseline radiation. Generally, the maxima or inflections in relations between relative biological effectiveness (RBE) and LET infinity were dependent upon the cycle position at which the cells were irradiated. Certain of those relations were influenced by post-irradiation hypothermia. Irradiation in the cell cycle at mid-G1 to mid-G1 + 3 h, henceforth called G1 to G1 + 3 h, resulted in survival curves that were close approximations to simple exponential functions. As the LET infinity was increased, the RBE did not exceed 1.0, and by 478 keV microns-1 it had fallen to 0.39. Although similar behaviour has been reported for inactivation of proteins and certain viruses by ionizing radiations, so far the response of the S/S variant is unique for mammalian cells. The slope of the survival curve for X-photons (D0: 0.27 Gy) is reduced in G1 to G1 + 3 h by post-irradiation incubation at hypothermic temperatures and reaches a minimum (Do: 0.51 Gy) at 25 degrees C. As the LET infinity was increased, however, the extent of hypothermic recovery was reduced progressively and essentially was eliminated at 478 keV microns-1. At the cycle position where the peak of radioresistance to X-photons occurs for S/S cells, G1 + 8 h, increases in LET infinity elicited only small increases in RBE (at 10% survival), until a maximum was reached around 200 keV microns-1. At 478 keV microns-1, what little remained of the variation in response through the cell cycle could be attributed to secondary radiations (delta rays) and smaller nuclei produced by fragmentation of the primary ions.     

The clonogenic response of bovine aortic endothelial cells in culture to radiation

The effect of ionizing radiation on the survival of bovine aortic endothelial (BAE) cells was determined by the in vitro colony formation method. The BAE cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, antibiotics, and growth factors obtained from the culture of mouse S-180 cells. The cultured BAE cells were positive to the staining of antibodies against human factor VIII and formed clones in plastic culture flasks with a plating efficiency of about 11%. The survival curve of the BAE cells following an exposure to a single dose of X rays was characterized by D0 = 101 rad, Dq = 65 rad, and an extrapolation number (n) of 1.9. These parameters were not modified by the absence of growth factors at the time of irradiation. The response of BAE cells to radiation was dose-rate dependent. The split-dose studies demonstrated that the BAE cells were able to repair sublethal radiation damage within 1 h after irradiation.     

Evidence for reduced capacity for damage accumulation and repair in plateau-phase C3H 10T1/2 cells following multiple-dose irradiation with gamma rays

The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates.     

The influence of dose rate on the lethal and mutagenic effects of X-rays in proliferating L5178Y cells differing in radiation sensitivity

The lethal and mutagenic effects of ionizing radiation delivered at high (53 Gy/h) and low (0.02 Gy/h) dose rates were measured in two closely related strains of mouse lymphoma L5178Y cells differing in radiation sensitivity (LY-R and LY-S). Strain LY-R was more resistant to the lethal effects of radiation than strain LY-S when exposed at either the high or low dose rate. The survival of strain LY-R was markedly enhanced by the reduction in dose rate. The dose-rate dependence of the survival of strain LY-S was less clear, because of the biphasic nature of its survival curve following low dose-rate radiation. However, if the initial slope of the low dose-rate survival curve is compared to the slope of the high dose-rate survival curve for strain LY-S, only a slight increase in survival at the low dose rate is apparent. Although more sensitive to the lethal effects of radiation, strain LY-S was less mutable at the hypoxanthine/guanine phosphoribosyl transferase locus by both low dose-rate and high dose-rate radiation than strain LY-R. Little dose-rate dependence was exhibited by either strain with regard to the mutagenic effects of radiation. Thus, for strain LY-R, which showed marked dose-rate dependence for survival but not for mutation, the ratio of mutational to lethal lesions was much greater following exposure to low dose-rate than to high dose-rate radiation.     

Mutagenic action of heavy ions on Escherichia coli cells

Induction of direct mutations in the lactose operon of E. coli cells by gamma-radiation and accelerated heavy ions with different LET was studied. The experiments were performed with the wild-type PolA and LexA strains. A quadratic dependence of the mutation rate on the dose of different radiations for the wild-type strain and the PolA mutant was observed. However, different types of radiation showed different relative genetic effectivenesses (RGE). The dependence of RGE on LET for the wild-type and PolA strain has a maximum. A LexA strain showed much reduced mutation rates and a linear dose response. The RGE decreased with increasing LET of ionizing radiation.