Theory of survival of bacteria exposed to ionizing radiation. I. X and gamma rays

A new model for the survival of bacteria exposed to ionizing radiation is constructed in the framework of a target theory based on microdosimetric concepts, where single- and double-strand breaks of DNA and their repair in vivo can be described consistently in terms of the microdosimetric quantity j (number of effective primary events per track per target). In this model, the ability of cells to repair DNA damage is taken into consideration in terms of the repair capacities for single- and double-strand breaks of DNA, xi 1 and xi 2 (0 less than or equal to xi 1, xi 2 less than or equal to 1). To apply this model to Escherichia coli K-12 strains with different repair abilities, values of the repair capacity for single-strand breaks, xi 1, were derived from experimental survival curves. The theoretical survival curves for 60Co gamma rays were found to be effectively insensitive to the value of xi 2. Experimental survival curves for the wild-type, uvr, and rec strains of E. coli K-12 were well reproduced in this model. From these results, it is concluded that the theoretical formulation for the survival fraction of bacteria can afford a quantitative method for analysis of the repair process for radiation-induced single-strand breaks in DNA in vivo.     

Mechanistic analysis of the contributions of DNA and protein damage to radiation-induced cell death

Protein oxidation can contribute to radiation-induced cell death by two mechanisms: (1) by reducing the fidelity of DNA repair, and (2) by decreasing cell viability directly. Previously, we explored the first mechanism by developing a mathematical model and applying it to data on Deinococcus radiodurans . Here we extend the model to both mechanisms, and analyze a recently published data set of protein carbonylation and cell survival in D. radiodurans and Escherichia coli exposed to gamma and ultraviolet radiation. Our results suggest that similar cell survival curves can be produced by very different mechanisms. For example, wild-type E. coli and DNA double-strand break (DSB) repair-deficient recA- D. radiodurans succumb to radiation doses of similar magnitude, but for different reasons: wild-type E. coli proteins are easily oxidized, causing cell death even at low levels of DNA damage, whereas proteins in recA- D. radiodurans are well protected from oxidation, but DSBs are not repaired correctly even when most proteins are intact. Radioresistant E. coli mutants survive higher radiation doses than the wild-type because of superior protection of cellular proteins from radiogenic oxidation. In contrast, wild-type D. radiodurans is much more radioresistant than the recA- mutant because of superior DSB repair, whereas protein protection in both strains is similar. With further development, the modeling approach presented here can also quantify the causes of radiation-induced cell death in other organisms. Enhanced understanding of these causes can stimulate research on novel radioprotection strategies.     

Targets for radiation-induced cell death: when DNA damage doesn't kill

Chinese hamster ovary (CHO) K1 and radiosensitive CHO irs-20 cells were synchronized in S phase and labeled for 10 min with 5-[(125)I]-iodo-2'-deoxyuridine ((125)IdU). The cells were washed, incubated in fresh medium for 1 h for incorporation of the intracellular radionucleotides into DNA, and then frozen (-80 degrees C) for accumulation of (125)I decays. At intervals after freezing, when the cells had accumulated the desired number of decays, aliquots of the frozen cells were thawed and plated to determine survival. The survival curves for K1 and irs-20 cells were similar from 100% to 30% survival. At higher (125)I doses (more decays/cell), the survival of K1 cells continued to decline exponentially, but the survival of X-ray-sensitive irs-20 cells remained at approximately 30% even after the cells had accumulated 1265 decays/cell. The results contradict the notion that increased DNA damage inevitably causes increased cell death. To account for these findings, we propose a model that postulates the existence of a second radiation target. According to this model, radiation damage to DNA may be necessary to induce cell death, but DNA damage alone is not sufficient to kill cells. We infer from the survival response of irs-20 cells that damage to a second (non-DNA) structure is involved in cell death, and that this structure directly affects the repair of DNA and cell survival.     

Radiation survival of vascular smooth muscle cells as a function of age

Late damage to normal tissues is an important consideration in determining the dose of radiation which can be delivered to a given target volume in clinical radiation therapy. The response of large blood vessels to radiation injury is undoubtedly complex and is influenced by (1) the cellular composition of the vessel wall, (2) the slow turnover of vascular cells, and (3) vascular repair mechanisms. As a first order model for radiation effects in large vessels, we have studied the radiobiologic properties of cultured vascular smooth muscle cells. We have measured survival curves and repair of sublethal radiation damage in exponentially growing cultures of rat aortic smooth muscle cells as a function of animal age and site of origin (thoracic versus abdominal aorta). Radiation survival parameters (utilizing two different mathematical models for the survival curve) and repair of sublethal damage did not appear to vary significantly as a function of animal age (3-23 months) or site or origin.     

Effects of continuous low dose-rate irradiation: computer simulations

The effects of continuous low dose-rate irradiation are studied with a computer model that incorporates cell kinetics and the accumulation and repair of radiation damage. This theoretical approach independently explores the effects on survival curves of a phase block, inherited damage and proliferation by dying cells. The computer model is a Monte Carlo simulation which follows the evolution in time of the family trees of a growing cell population under continuous irradiation. The model uses as input the measured phase-specific survival curves for acute exposures and the cell kinetic parameters to generate survival curves for continuous low dose-rate irradiations. Cell survival curves for Chinese hamster lung cells (V79) for dose rates ranging from 15 to 500 cGy/h have been generated using various model assumptions. The model shows that for these cells a G2 block will maximize cell killing for an optimum dose rate near 75 cGy/h. The effect on survival curves of inherited damage, as well as that of the proliferation by dying cells, is shown to increase monotonically with decreasing dose rates, and to be quite large at low dose rates.     

The level of induced DNA double-strand breaks does not correlate with cell killing in X-irradiated mitotic and G1-phase CHO cells

The neutral (pH 9.6) filter elution technique was used to evaluate DNA damage induced in CHO cells irradiated at mitosis or in G1-phase under various incubation and postirradiation treatment conditions. Mitotic and G1/S border cells were more sensitive to radiation than G1 cells with respect to cell killing, but showed similar (G1/S) or lower (M) DNA elution dose--response curves. Similar cell survival and DNA/elution dose--response curves were obtained with plateau-phase cultures containing mainly G1-cells, as well as with G1 cells obtained after division of mitotic cells in either fresh or conditioned medium. However, survival of plateau-phase cells could be modified substantially by delayed-plating or postirradiation treatment with araA. These results, together with previously published observations, indicate that induction of DNA dsb cannot be invoked as an explanation for the variations in radiosensitivity observed through the cycle, or as an explanation for the formation of the survival curve shoulder. It is proposed that repair and fixation of radiation-induced DNA damage, expressed at the cell survival level as repair and fixation of alpha-PLD, are responsible for these effects.     

The relevance of the nuclear division cycle to radiosensitivity in yeast

Summary To investigate whether the nuclear division cycle could be related to cycle-specific changes in repair of ionizing radiation damage, we have determined the survival curves after -irradiation of samples taken frequently from synchronously dividing cultures of Saccharomyces cerevisiae cells. Survival was low in G1 and increased during S, reaching a maximum around the end of the S phase, which was maintained in G2. The shape of the survival curves for samples taken from later stages revealed a rapid cycle-specific drop in the radioresistance of individual cells. A simple model was formulated on the assumption that survival is greatly enhanced by the action of an enzymatic repair mechanism which requires duplicated but unsegregated DNA as a substrate. By taking into account the measurable age heterogeneity of samples taken from the synchronous cultures, this model was shown to fit the survival data closely. For an individual cell, the increasing survival during the S phase is thus attributable to a rising fraction of duplicated genome, whereas the rapid decrease in radioresistance at a later stage in the cell cycle may be interpreted as due to the final physical separation of sister chromatids. The start of the latter event was timed to the stage in mitosis when the nucleus begins to move towards the neck of the bud. The data are consistent with the hypothesis that the high radioresistance of cells in late S and G2 is due to the repair of double-stranded DNA breaks by a process involving recombination between sister chromatids.     

Effects of Continuous Low Dose-Rate Irradiation: Computer Simulations

Abstract. The effects of continuous low dose-rate irradiation are studied with a computer model that incorporates cell kinetics and the accumulation and repair of radiation damage. This theoretical approach independently explores the effects on survival curves of a phase block, inherited damage and proliferation by dying cells. the computer model is a Monte Carlo simulation which follows the evolution in time of the family trees of a growing cell population under continuous irradiation. the model uses as input the measured phase-specific survival curves for acute exposures and the cell kinetic parameters to generate survival curves for continous low dose-rate irradiations. Cell survival curves for Chinese hamster lung cells (V79) for dose rates ranging from 15 to 500 cGy/h have been generated using various model assumptions. the model shows that for these cells a G₂ block will maximize cell killing for an optimum dose rate near 75 cGy/h. the effect on survival curves of inherited damage, as well as that of the proliferation by dying cells, is shown to increase monotonically with decreasing dose rates, and to be quite large at low dose rates.     

A radiation target method for size determination of supercoiled plasmid DNA

Supercoiled DNA plasmids were exposed in the frozen state to high-energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.     

Differential response to gamma radiation of human stomach cancer cells in vitro

In vitro effects of radiation were studied in two permanent cell lines (AGS and SII) from two patients with adenocarcinoma of the stomach and three permanent sublines from each cell line. Radiation survival parameters for AGS and SII parent cell lines and sublines were determined after in vitro irradiation of their cells with 0.5 to 10 Gy of 60Co gamma rays. The AGS and SII cell lines had different growth properties, DNA contents and radiation survival curves. Surviving fractions of SII parent cells (76 chromosomes) after 2.0 and 10 Gy were 1.22 and 17.8 times greater, respectively, than values for AGS parent cells (47 chromosomes). Sensitivities (D0) were 1.08 and 1.45 Gy for AGS and SII parent lines, respectively. The D0 values for AGS parent cells and sublines were similar (1.01 to 1.08 Gy), but SII parent cells and sublines had D0 values of 1.45, 1.36, 1.37 and 1.12 Gy (for SII-A). Also, the SII parent cells had survival fractions after 2.0 and 10 Gy that were 1.3 and 11.3 times greater, respectively, than values for the SII-A cells. These data show differences in radiation responses among stomach cancer cell lines and sublines that may relate to DNA content, but there was no consistent correlation between radiation response and a particular cell characteristic.     

A comparison of five effects of ultraviolet light on the Arbacia egg

The demonstration of five different effects of ultraviolet radiation on the sea urchin's egg indicates that more than one basic photochemical process goes on there. Photorecovery is observed in only one of these. The need for caution in interpreting such effects is obvious. Evidence for a different mechanism for the timing of cleavage in eggs activated by ultraviolet radiation as compared to normally fertilized eggs is presented. The bearing of these studies on survival curves for microorganisms is discussed.     

Probing lethal damage expression in cytochalasin B-induced polykaryons by radiation quality

The polykaryon-forming unit (PFU) cell survival assay is based on the postirradiation flow cytometric analysis of the DNA content accumulated in high-ploidy cells (polykaryons) induced by the cytokinesis inhibitor cytochalasin B and can provide a meaningful measure of cell radiosensitivity. In this assay, cell survival is defined as the ability to form a polykaryon of a given ploidy after irradiation. The slope of the polykaryon dose response has been shown to be highly correlated with the initial slope of the clonogenic survival curves after gamma irradiation, which implies a common subset of lethal lesions. We reported previously on an apoptotic mode of cell death in the polykaryon system and on the heritability of small variations in polykaryon radioresponse. We now show that exposure of PFUs to a given dose of alpha particles results in a greater reduction in the proportion of cells able to reach at least 16C when compared to the same dose of low-LET radiation. This reduction is less than that observed in the low-dose (alpha term) region of the clonogenic curve. On the basis of published LET-dependent spectra of radiation-induced DNA damage, we suggest that this behavior reflects a differential expression of lethal damage that can be probed by varying the LET of the radiation and that base damages contributing additional complexity to clustered DNA lesions may be more deleterious in PFUs than in clonogens.     

HO·和γ射线对细胞致死效应的比较

利用双链断裂模型比较研究了羟自由基和γ射线对B16、L0 2、SMMC - 772 1和V79四种细胞的致死效应。结果表明 :1.HO·处理和γ射线照射对四种细胞有都明显的致死效应 ,而且剂量越大致死作用越强。 2 .HO·处理和γ射线照射存活曲线都存在肩区 ,说明两种处理过程都有亚致死损伤的修复。 3.HO·诱导DNA双链断裂一般都需要两次击中而γ射线辐照既有一次击中的成分 ,也有两次击中的成分。 4.四种细胞对HO·的敏感性与对γ射线的辐射敏感性顺序相反 ,说明γ射线对细胞的作用不能简单地解释为自由基的行为。     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

Radiosensitivity of normal human epidermal cells in culture

Using an in vitro culture system we have derived radiation survival curves for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells.     

The radiation response of a human colon adenocarcinoma grown in monolayer, as spheroids, and in nude mice

A human colon adenocarcinoma cell line, WiDr, has been grown in monolayer, as multicellular spheroids, and as xenografted tumors in immune-deprived mice. The growth and radiation responses of the cells under these different growth conditions were compared. The mean doubling time of monolayer cultures was 0.8 day and the initial volume doubling times of spheroids and xenografts averaged 1.2 and 6 days, respectively. The mean total viable cell plating efficiencies were 82, 63, and 7% for cells from monolayers, spheroids, and xenografted tumors, respectively. The radiation responses of single cell suspensions prepared from WiDr tumors (8-10 mm in diameter), exponentially growing monolayer cultures (5 days growth), and spheroids (1200 microns in diameter) irradiated in air at 4 degrees C were similar. Values for D0 were 1.5 Gy and for n between 3 and 5. Nitrogen curves were characterized by a D0 of 5 Gy and n between 3 and 6. Oxygen enhancement ratios were approximately 3.3. Both spheroids and tumors had radioresistant components to the 37 degrees C/air-breathing survival curves with estimated hypoxic fractions of 8 and 12%, respectively. The final portion of the survival curves for irradiations in nitrogen and under normal growth conditions were parallel for both tumors and spheroids. Thus WiDr spheroids appear to model accurately the radiation sensitivity of WiDr tumors.     

Induction of sperm head abnormalities by incorporated radionuclides: dependence on subcellular distribution, type of radiation, dose rate, and presence of radioprotectors

In contrast to the biological effects caused by exposure to external beams of radiation, the effects of tissue-incorporated radionuclides are highly dependent on the type of radiation emitted and on their distribution at the macroscopic, microscopic, and subcellular levels, which are in turn determined by the chemical nature of the radionuclides administered. Induction of abnormalities of sperm heads in mice is investigated in this work after the injection of a variety of radiochemicals including alpha emitters. When the initial slopes of the dose-response curves are used to compare the relative biological effectiveness (RBE) of different radiocompounds, the alpha particles emitted in the decay of 210Po are more effective than Auger electrons emitted by 125I incorporated in the DNA of the spermatogonial cells, and both emissions are more effective than X rays. It is also shown that the Auger emitters (125I, 111In) distributed in the cell nucleus are more efficient in producing abnormalities than the same radionuclides localized in the cytoplasm. These findings are consistent with our earlier observations, where spermatogonial cell survival is assayed as a function of the testicular absorbed dose. Further, chronic irradiation of testis with gamma rays from intratesticularly administered 7Be is about three times more effective in causing abnormalities than a single acute exposure to 120-kVp X rays. The resulting RBE values correlate well with our data on sperm head survival with the same radiocompounds. Finally, the radioprotector cysteamine, when administered in small, nontoxic amounts, significantly reduces the incidence of sperm abnormalities from alpha-particle radiation as well as emissions from 125I incorporated into DNA, the dose reduction factors being 10 and 14, respectively.     

Mathematical model of interphase cell death. Biophysical justification and generalization

The authors propose a biophysical justification of a radiation-induced injury and interphase death of cells. The injury to certain units of the microtrabecular network and cytoskeleton is considered to be a primary biological effect of radiation on cells. The role of these structural changes in the development of the specific radiation response is discussed. It is found possible to describe formally, by the defined parameters of the proposed model, the survival curves for not only interphase but also reproductive cell death.     

Linkage effects in a model for cell survival after radiation

A thermodynamic treatment for the effects of radiation on cell survival is proposed. The treatment is an extension of the linear-quadratic model (K.H. Chadwick and H.P. Leenhouts, Phys. Med. Biol. 13 (1973) 78) following the principles of linkage thermodynamics (E. Di Cera, S.J. Gill and J. Wyman, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 5077). Linkage effects between chemical binding to DNA and radiation action are considered, along with the synergism between different types of radiations. A simple mathematical condition is found for the additivity of radiation doses that result in an isoeffect. The resolvability of the model parameter is investigated by simulations and statistical analysis of the distributions obtained.     

Theoretical analysis of mixed irradiation (3).

As the model we proposed last year was contradictory to experimental data, we revised again the models for mixed irradiation by Zaider and Rossi and by Suzuki, substituting a 'reciprocal-time' pattern of repair function for a first-order one in reduction and interaction factors of the models, although we used a second order repair function last year. The reduction factor, which reduces the contribution of the square of a dose to cell killing in the models, and the interaction factor, which also reduces the contribution of the interaction of two or more doses of different types of radiation, were formulated by using the 'reciprocal-time' pattern of repair function. These newly modified models for mixed irradiation could express or predict cell survival more accurately than the older ones, especially when irradiation is prolonged at low dose rates. We present survival curves of cells calculated from the newly and the older models of assumptive simultaneous mixed irradiation with two or three types of radiation.