Exponential Survival Curves Research Articles

Resistance of spores of Clostridium botulinum 33A to combinations of ultraviolet and gamma rays.

Spores of Clostridium botulinum 33A exhibit a sigmoidal survival curve if subjected to gamma radiation. The present investigation was concerned with two questions: (i) what is the form of an ultraviolet (UV)-survival curve and (ii) what is the combined effect of UV- and gamma radiation? The UV-survival curve was found to be of sigmoidal type with a "shoulder" width of 675 ergs/mm(2) and a D(10) (exp) of 2,950 ergs/mm(2). To test the combination effect, spores were subjected to UV doses of 225, 450, 675, and 900 ergs/mm(2) followed by a series of increasing doses of gamma rays from 200 to 2,000 krad in 200-krad steps. The gamma ray-survival curves showed that increasing UV pretreatment caused a gradual loss of the "Prodiginine" yielding straight line exponential survival curves after preirradiation with UV doses of 675 ergs/mm(2) and above. Simultaneously the D(10) value for gamma-ray irradiation was reduced, e.g. UV preirradiation with 900 ergs/mm(2) reduced the D(10) by 40%. This observation emphasizes the potential practical advantage of combining UV and gamma rays for sterilization of heat-sensitive commodities.

Resistance of Spores of Clostridium botulinum 33A to Combinations of Ultraviolet and Gamma Rays1

Spores of Clostridium botulinum 33A exhibit a sigmoidal survival curve if subjected to gamma radiation. The present investigation was concerned with two questions: (i) what is the form of an ultraviolet (UV)-survival curve and (ii) what is the combined effect of UV- and gamma radiation? The UV-survival curve was found to be of sigmoidal type with a "shoulder" width of 675 ergs/mm(2) and a D(10) (exp) of 2,950 ergs/mm(2). To test the combination effect, spores were subjected to UV doses of 225, 450, 675, and 900 ergs/mm(2) followed by a series of increasing doses of gamma rays from 200 to 2,000 krad in 200-krad steps. The gamma ray-survival curves showed that increasing UV pretreatment caused a gradual loss of the "Prodiginine" yielding straight line exponential survival curves after preirradiation with UV doses of 675 ergs/mm(2) and above. Simultaneously the D(10) value for gamma-ray irradiation was reduced, e.g. UV preirradiation with 900 ergs/mm(2) reduced the D(10) by 40%. This observation emphasizes the potential practical advantage of combining UV and gamma rays for sterilization of heat-sensitive commodities.

Fractionated Heavy Ion Irradiation of Cultured Human Cells

Cultured human kidney T1 cells were allowed to recover between paired doses of X-rays and accelerated heavy ions. Recovery from sublethal doses of X-rays and heavy ions having LET's up to $1650 {\rm MeV}\text{-}{\rm cm}^{2}/{\rm gm}$ was evident on the basis of colony formation. No recovery could be detected between paired doses of accelerated ions having higher LET's, and very little between paired 150-rad doses of X-rays. Survival curves for colony formation were also determined; recovery occurred whenever survival curves were sigmoid and was absent whenever survival curves were exponential. There was no obvious reduction in the extent of recovery at intermediate LET's, although it may have been slightly delayed. The results indicate that exponential survival curves correspond to irreversible inactivation and sigmoid survival curves correspond to lethality due to the accumulation of reversible sublethal lesions. The probability of the former appears to increase more rapidly with LET than that of th...

Studies on the Lethal Effects of Sulfur Mustard on Dividing Mammalian Cells

The initial effect of sulfur mustard on a dividing population of L cells was inhibition of DNA synthesis. The progression of G2 and G1 cells was not immediately affected, so that the result was an accumulation of cells that synthesized DNA slowly. After about 24 hours the cells contained, on the average, the premitotic amount of DNA, but cell division did not occur. This level of DNA was maintained for at least another 60 hours. Cells that had been released from an FUDR block and treated with mustard at or near the time of release exhibited a purely exponential survival curve. If the release was delayed relative to the mustard treatment, survival was enhanced. The survival curve obtained under these conditions was parallel to the former curve but exhibited a shoulder. When a fractionated-dose technique with mustard was used no evidence for repair of sublethal damage was found; rather, the two doses of mustard, separated in time, were more toxic than a single equivalent dose.

Radiation-induced somatic mutations and the loss of reproductive integrity in Tradescantia stamen hairs

Inflorescences of Tradescantia (clone 02), a diploid hybrid heterozygous for flower color, were given a 16-hr exposure (25–500 R) of 60Co γ-rays from a 3200 Ci source. Morphological abnormalities induced in stamen hairs were noted for a period of 20 days and records kept of the frequency and position of mutant cells and mutant events and of the killing effect (loss of reproductive integrity) in the hairs. The results show that these effects are highly dependent upon the developmental stage of the hair at the time of exposure. Giant cells and abnormally shaped cells were induced both apically and interstitially at the higher levels of radiation exposures. Branching of the hairs was also observed. Such effects occur very rarely in unirradiated flowers. An increased number of mutant pink cells per stamen appeared about six days postirradiation. They increased with increasing exposure and time, reached a peak about 14 days after treatment, then decreased in number up to 20 days. The frequency of mutant events (single cell or multicellular mutations) per stamen reached the highest value for each exposure at about 10 or 11 days after treatment, leveled off, and declined after about 13 days. This indicates that at the time of irradiation these buds contained the maximum number of meristematic stamen hair cells. Mutation rate varied with exposure and time, but at 11 days after irradiation with 100 R it was estimated to be 5·29 × 10 −4 per R per meristematic cell. The percentage of stunted hairs (those that had lost their reproductive integrity) per stamen showed an increase with increasing exposure. But at exposures up to 300 R some sort of recovery phenomenon was noticed. A typical exponential survival curve was found with a D 37 of 270 R and and extrapolation number of 1·81. Exposures of 200–300 R are optimal for producing mutations and are not sufficiently high to induce lethality to an extent which would mask the mutation sensitivity of stamen hair cells. The results thus indicate that there exists, over a limited time and within a certain exposure range, an approximately linear relationship between radiation exposures and the frequencies of mutant cells and of mutant events.

Contribution of host resistance to radiosensitivity of an isologous murine lymphoma in vivo.

SummaryLSA ascites lymphoma cells were irradiated in vitro and assayed in vivo in isologous recipients. Results with the TD50 method were correlated with a separate assay using an inoculum containing a fixed total number of tumour cells and measuring the 50 per cent sterilization dose (SD50) of x-radiation. Comparison of sham- and pre-irradiated recipients showed differences which were consistent with the presence of isoantigenic tumour antigens, using the SD50 method. Host-resistance against the tumour was reduced by irradiation of the recipients, but significant differences were only evident when cell inocula were large. Isologous tumour resistance was found to become of progressively greater significance with larger numbers of radiation-killed cells. Host-resistance factors appear to increase apparent shoulder size (or extrapolation number), decrease D0, and convert a linear exponential survival curve to a more parabolic one. Host-resistance factors may contribute to cell-survival response in vivo with...

Growth rate and radiosensitivity of human tumours—II: Radiosensitivity of human tumours

Pulmonary metastases from different types of tumour in Man show constant growth rates when measured accurately on successive roentgenograms of the chest. The influence of ionizing radiation on these metastatic tumours could be shown very clearly. Fractionated irradiation resulted in a concentric reduction in the size of the metastases, the outlines of which remained remarkably well defined. This made it possible to calculate the percentage of reduction in volume by measuring the change in diameter. Data on such pulmonary metastases in sixteen of our patients were suitable for quantitative analysis. It was found that volume reduction became evident a few days after the first dose was administered. Repetition of the same daily dose caused percentages of reduction, giving an exponential survival curve. After the last dose of a series was given, the tumour resumed in growth immediately, or at least within a few days, at approximately the same rate as before irradiation. The surviving fractions found in these cases did not conflict with the assumption of an exponential survival curve, as constructed by Elkind for fractionated irraditions of cell cultures. In this way, the mean lethal dose D 37 defined as the dose leaving 37 per cent of the original volume capable of resuming growth, could be calculated for these irradiations, in which daily doses of 100–150 rads were applied using 250 kV roentgen and Cobalt- 60. The results showed wide variations in radiosensitivity amongst tumours of different types (Table 1). Values of D 37 extending from 300 up to 2550 rads were encountered. These differences, which correspond to a factor of 8·5 cannot be explained by the assumption that all cells have the same intrinsic radiosensitivity and that all differences observed are caused by differences in oxygen tension in tissues. Good correlation was found to exist between the radiosensitivity ( D 37) and the growth rate of a tumour, expressed in terms of the doubling time, T 2. For all cases, the equation log D 37 = 1·780 + 0·652 log T 2 fits the data within fair limits. Excluding the value of D 37 for two tumours, i.e. those with the lowest growth rate ( T 2 > 150 days), all value s of D 37 fitted well in a linear correlation according to the equation, D 37 = 220 + 10 T 2. Possible explanations of the phenomena found are discussed, taking into account the concepts of modern experimental radiobiology. The significance of these relationships for clinical radiobiology and the practice of radiotherapy is considered. The possibility of estimating the lethal dose for a tumour if its volume and growth rate are known is of particular interest. Many additional investigations in various fields will be required to reach this goal, however.

Repair mechanisms and variations in UV sensitivity within the cell cycle

1. 1. Studies were made of the UV sensitivity of a series of stages in the cell cycle of synchronous populations of diploid meiotic spores of Chlamydomonas reinhardi. 2. 2. Early stages had sigmoid survival curves, but at successively later stages of development, D Q values were continuously reduced, and the latest stages had exponential survival curves. D O values, though changing only to a minor extent, were at a maximum in mid-cycle. 3. 3. From studies of the effects of various post-irradiation treatments and in particular of the effect of inhibitors of dark-repair processes, it was concluded that much of the variation in stage sensitivity was attributable to different repair activities. Those stages farthest from division had the longest period in which to repair UV lesions, whereas successively later stages had less time and thus exhibited an enhanced sensitivity.

REPAIR OF X-RAY IN MICROCOCCUS RADIODURANS.

Micrococcus radiodurans , a red-pigmented bacterium, is very resistant both to ionizing and to ultra-violet radiations. The dose-response curves in both cases are sigmoidal with high extra-polation numbers. Two non-pigmented mutants of M . radiodurans have been isolated. One of these shows the same type of survival curve as the wild type while the other has an exponential survival curve. Energy transfer reactions including the possible role of pigment have been excluded as a mechanism of resistance. Similarly, resistance is not due to unusual amounts or unique properties of the bacterial DNA . Survivors of both sensitive and resistant bacteria show a lag period following irradiation. In the case of sensitive bacteria no change occurs in the radiosensitivity of the cells during this period. On the other hand, M . radiodurans recovers during the lag period, at the end of which it has become as resistant as the unirradiated cells. All available evidence for the loss of viability in bacteria points to DNA as the target. Recovery of M . radiodurans from radiation damage, therefore, is concluded to be due to repair of damaged DNA by an active cellular, probably enzymic process which occurs during the lag period. The large shoulder of the survival curve and the high extrapolation number are a reflexion of the efficiency of the repair system. The significance of the sigmoidal and exponential survival curves of resistant bacteria is discussed and a theory put forward which postulates two types of damage to the genetic structures, each having a specific repair system. Some experimental evidence in support of this theory is produced.

THE RESPONSE OF MAMMALIAN TUMOR CELLS IN VIVO TO RADIATIONS OF DIFFERING IONIZATION DENSITIES (LET).

The response of mammalian tumor cells to ionizing radiation was studied in vivo, using the transplantability of leukemic ascites cells as a measure of cell-reproductive capacity. The relative effects of radiations having a wide range of ionization densities (LET) were assessed under oxygenated and anoxic irradiation conditions. Low-LET radiations give dose-response curves with a shoulder, indicating ability of cells to recover from sublethal irradiation effects when doses are fractionated, while highLET radiation produces an exponential survival curve and no recovery between fractions. Low-LET radiations are much influenced by the presence or absence of oxygen (oxygen-effect ratio 2.1: 2.3), while high-LET radiation effects are little modified by the presence or absence of oxygen (oxygen effect ratio 1.2). Finally, considerations for the use of high-LET (densely ionizing) radiations in radiotherapy of human cancer are discussed.

SMALL-CELL SEGREGANTS FROM A POSSIBLY HOMOZYGOUS DIPLOID STRAIN OF ESCHERICHIA COLI.

Ogg, James E. (Colorado State University, Fort Collins) and Ronald D. Humphrey. Small-cell segregants from a possibly homozygous diploid strain of Escherichia coli. J. Bacteriol. 85:801-807. 1963.-Evidence was presented to show that a homozygous possibly diploid strain of Escherichia coli, when grown in a chemically defined medium under the conditions described in this report, will yield small-cell segregants that have haploid characteristics. The segregants have one-half as much deoxyribonucleic acid per cell as the diploid, give exponential survival curves when exposed to X rays, and have fermentative characteristics similar to the original haploid parent strain of the homozygous diploid strain. No detectable changes in surface antigens were associated with the segregation phenomenon. The haploidlike segregants can be converted to the diploid radiation-resistant state by growing them in the presence of camphor vapors.

Sensitivity and Protection of Mouse Bone-marrow Cells X-irradiatedin Vitro

SummaryThe 30-day survival of mice exposed to a lethal dose (798 r) of x-rays was a function of the number of isologous bone-marrow cells injected. From this calibration curve it was possible to determine the surviving fraction in a suspension of marrow cells irradiated in vitro. The in vitro radiation-response of such suspensions could be represented by a typical exponential survival curve the linear part of which indicated a D37 of 84 r and an extra-polation number of 2·5.Cysteine, S,2-aminoethylisothiuronium chloride, and anoxia protected marrow cells irradiated in vitro and gave dose reduction factors ranging from 1·7–2·1. Noradrenaline did not protect the cells.

Effects of Different Ionizing Radiations on Human Cells in Tissue Culture: I. Irradiation Techniques and Dosimetry

Effects of alpha, beta and x radiation on the inhibition of clone formation of human cells have been measured. With alpha radiation an exponential survival curve was found, whereas with x and beta radiation a more complicated curve was obtained. The sensitive area calculated from the experiments with alpha radiation is equal to 42 mu/sup 2/, which is shown to be approximately identical with the cross-sectional area of the nucleus. The RBE of alpha radiation is found to range from 2.5 periments on the addition of alpha and x radiation are explained on the assumption that the passage of one alpha particle through the nucleus of a cell is sufficient to prevent this cell from developing into a clone. (auth)

Lethal effect of visible light on a mutant strain of haploid yeast. I. General dependencies

1. 1. The ability to develop a photolethal sensitivity to visible light has been demonstrated for a respiration-deficient mutant of a haploid strain of Saccharomyces cerevisiae. This ability is present in rapidly dividing cells as opposed to resting cells of the same strain or respiring diploid S. cerevisiae. 2. 2. The rate of light-sensitivity development in a non-nutrient buffer is dependent on the conditions of storage. The rate increases markedly with temperature and pH, and decreases with the molarity of the buffer. Incubation with dextrose prior to storage markedly reduces the subsequent development of light sensitivity. 3. 3. Photodynamic action is indicated by a strong dependence of photolethality on oxygen tension. 4. 4. Although the correlation is not 1:1, the lethality produced by light strongly affects CO 2 production compared to the affects on cell metabolism of other types of radiation. 5. 5. The sigmoid shape of the light-survival curve compared to the exponential x-ray survival curve, as well as other considerations, indicate that the sites of the photo effect is probably non-genetic.

RADIOKINETIC EVIDENCES FOR THE BIPARTITE OR DIPLOID NUCLEUS IN CONIDIA OF STREPTOMYCES GRISEOFLAVUS

In contrast to several species of Streptomyces which revealed exponential survival curves by X-ray inactivation tests, Str. Griseoflavus and auxotrophic mutants of the strain gave apparently two-hit curves. On the ground of the interrelation between radiokinetic and biological findings, a proposal that the conidium of Str. Griseoflavus comprises a bipartite or diploid nucleus in each cell was presented.

A New Approach to the Mathematical Theory of the Action of High-Energy Radiation on Single Cells: II. Consequences of the Theory

The understanding of the mechanism of the interaction between radiation and living matter has been greatly promoted by radiation experiments on unicellular organisms. Among these the yeast cell plays an important role, partly because of its availability in various ploidies, from haploid to tetraploid (1). The following discussion is centered around the yeast cell, but its domain of applicability is believed to extend to other microorganisms. Yeast cells of different ploidy respond very differently to radiation. Haploid yeast shows an exponential survival curve, whereas diploid, triploid, and tetraploid yeast exhibit sigmoid survival curves (2-11). This has been interpreted as strong evidence that the lethal effect of high-energy radiation is of genetic origin. The details of the interaction between radiation and genetic material still constitute one of the major problems. The different possible ways in which this interaction can take place may be divided in two main groups, sometimes labeled direct action and indirect action, respectively (2, 4, 12). The latter is sometimes called diffusion model or migration model (3, 4). By direct action is meant that the radiation ionizes or excites molecules in the genetic material through the electric fields of the charged particles of which the radiation consists, or which are produced by the radiation. The direct action has been treated extensively by Lea (13) and by Timofeeff-Ressovsky and Zimmer (14). The indirect action, on the other hand, explains the genetic effects of radiation by postulating that the ultimate genetic damage is done by certain chemical agents, which in their turn are the indirect products of the radiation. We shall call these agents intermediaries. Speculations as to the nature of these intermediaries have

A New Approach to the Mathematical Theory of the Action of High-Energy Radiation on Single Cells: I. Formulation of the Theory

The understanding of the mechanism of the interaction between radiation and living matter has been greatly promoted by radiation experiments on unicellular organisms. Among these the yeast cell plays an important role, partly because of its availability in various ploidies, from haploid to tetraploid (1). The following discussion is centered around the yeast cell, but its domain of applicability is believed to extend to other microorganisms. Yeast cells of different ploidy respond very differently to radiation. Haploid yeast shows an exponential survival curve, whereas diploid, triploid, and tetraploid yeast exhibit sigmoid survival curves (2-11). This has been interpreted as strong evidence that the lethal effect of high-energy radiation is of genetic origin. The details of the interaction between radiation and genetic material still constitute one of the major problems. The different possible ways in which this interaction can take place may be divided in two main groups, sometimes labeled direct action and indirect action, respectively (2, 4, 12). The latter is sometimes called diffusion model or migration model (3, 4). By direct action is meant that the radiation ionizes or excites molecules in the genetic material through the electric fields of the charged particles of which the radiation consists, or which are produced by the radiation. The direct action has been treated extensively by Lea (13) and by Timofeeff-Ressovsky and Zimmer (14). The indirect action, on the other hand, explains the genetic effects of radiation by postulating that the ultimate genetic damage is done by certain chemical agents, which in their turn are the indirect products of the radiation. We shall call these agents intermediaries. Speculations as to the nature of these intermediaries have

The inactivation of the infectious centers of tobacco mosaic virus by ultraviolet light

The early events of tobacco mosaic virus infection were studied by following the radiosensitivity of infection as a function of time after inoculation. At 20°, the survival of infection as a function of ultraviolet light dose is exponential for approximately 5 hours after inoculation, after which the appearance of new intracellular infectious particles is indicated by a change to a multitarget survival curve. During the first 5 hours after inoculation, the infection is characterized by three discernible phases of sensitivity to ultraviolet light (UV): (1) a period during which the infection has essentially the same sensitivity as the virus in vitro; (2) a period during which resistance to UV increases; and (3) a period during which a secondary level of resistance is maintained. The timing of these phases is markedly dependent on temperature. The exponential survival curve during the first phase is independent of inoculum concentration, indicating that an exclusion mechanism is operative which permits only one infectious virus particle to initiate an infection.

Death-rate of populations of Bact. lactis aerogenes II. Environmental and other factors influencing the form of the survival curve

Populations which have been grown with an ample supply of glucose give approximately exponential survival curves at high acidities, adaptive adjustments, revealed in tests at lower concentrations, being apparently suppressed. Shortage of glucose during previous growth favours survival in the test, and changes the distribution of survival times, the decline of the population being initially delayed. Serial subculture of the bacterial strain in a synthetic medium also leads to initial delays. Ageing the culture before preparation of the suspension for test increases the overall resistance, but has little influence on the initial form of the survival curve. The curves for drug-induced filamentous cultures are closely similar to those for normal cultures. Neither low permeability nor co-operative protective effects between different cells seem to be general causes of delayed lethal action. Damage to the cells during preparation of the test population does not explain why the delay is sometimes absent.

Death rate of populations of Bact. lactis aerogenes. II. Environmental and other factors influencing the form of the survival curve.

Populations which have been grown with an ample supply of glucose give approximately exponential survival curves at high acidities, adaptive adjustments, revealed in tests at lower concentrations, being apparently suppressed. Shortage of glucose during previous growth favours survival in the test, and changes the distribution of survival times, the decline of the population being initially delayed. Serial subculture of the bacterial strain in a synthetic medium also leads to initial delays. Ageing the culture before preparation of the suspension for test increases the overall resistance, but has little influence on the initial form of the survival curve. The curves for drug-induced filamentous cultures are closely similar to those for normal cultures. Neither low permeability nor co-operative protective effects between different cells seem to be general causes of delayed lethal action. Damage to the cells during preparation of the test population does not explain why the delay is sometimes absent.