A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291–298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a “comet” with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate “features” of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The “tail moment”, the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0–15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.

The thyroid gland of children is especially vulnerable to the carcinogenic action of ionizing radiation. To provide insights into various modifying influences on risk, seven major studies with organ doses to individual subjects were evaluated. Five cohort studies (atomic bomb survivors, children treated for tinea capitis, two studies of children irradiated for enlarged tonsils, and infants irradiated for an enlarged thymus gland) and two case-control studies (patients with cervical cancer and childhood cancer) were studied. The combined studies include almost 120,000 people (approximately 58,000 exposed to a wide range of doses and 61,000 nonexposed subjects), nearly 700 thyroid cancers and 3,000,000 person years of follow-up. For persons exposed to radiation before age 15 years, linearity best described the dose response, even down to 0.10 Gy. At the highest doses (>10 Gy), associated with cancer therapy, there appeared to be a decrease or leveling of risk. For childhood exposures, the pooled excess relative risk per Gy (ERR/Gy) was 7.7 (95% CI = 2.1, 28.7) and the excess absolute risk per 10⁴ PY Gy (EAR/10⁴ PY Gy) was 4.4 (95% CI = 1.9, 10.1). The attributable risk percent (AR%) at 1 Gy was 88%. However, these summary estimates were affected strongly by age at exposure even within this limited age range. The ERR was greater (P = 0.07) for females than males, but the findings from the individual studies were not consistent. The EAR was higher among women, reflecting their higher rate of naturally occurring thyroid cancer. The distribution of ERR over time followed neither a simple multiplicative nor an additive pattern in relation to background occurrence. Only two cases were seen within 5 years of exposure. The ERR began to decline about 30 years after exposure but was still elevated at 40 years. Risk also decreased significantly with increasing age at exposure, with little risk apparent after age 20 years. Based on limited data, there was a suggestion that spreading dose over time (from a few days to >1 year) may lower risk, possibly due to the opportunity for cellular repair mechanisms to operate. The thyroid gland in children has one of the highest risk coefficients of any organ and is the only tissue with convincing evidence for risk at about 0.10 Gy.

This continues the series of periodic general reports on cancer mortality in the cohort of A-bomb survivors followed by the Radiation Effects Research Foundation. The follow-up is extended by the 5 years 1986–1990, and analysis includes an additional 10,500 survivors with recently estimated radiation doses. Together these extensions add about 550,000 person-years of follow-up. The cohort analyzed consists of 86,572 subjects, of which about 60% have dose estimates of at least 0.005 Sv. During 1950–1990 there have been 3086 and 4741 cancer deaths for the less than and greater than 0.005 Sv groups, respectively. It is estimated that among these there have been approximately 420 excess cancer deaths during 1950–1990, of which about 85 were due to leukemia. For cancers other than leukemia (solid cancers), about 25% of the excess deaths in 1950–1990 occurred during the last 5 years; for those exposed as children this figure is nearly 50%. For leukemia only about 3% of the excess deaths in 1950–1990 occurred in the last 5 years. Whereas most of the excess for leukemia occurred in the first 15 years after exposure, for solid cancers the pattern of excess risk is apparently more like a life-long elevation of the natural age-specific cancer risk. Taking advantage of the lengthening follow-up, increased attention is given to clarifying temporal patterns of the excess cancer risk. Emphasis is placed on describing these patterns in terms of absolute excess risk, as well as relative risk. For example: (a) although it is becoming clearer that the excess relative risk for those exposed as children has declined over the follow-up, the excess absolute risk has increased rapidly with time; and (b) although the excess relative risk at a given age depends substantially on sex and age at exposure, the age-specific excess absolute risk depends little on these factors. The primary estimates of excess risk are now given as specific to sex and age at exposure, and these include projections of dose-specific lifetime risks for this cohort. The excess lifetime risk per sievert for solid cancers for those exposed at age 30 is estimated at 0.10 and 0.14 for males and females, respectively. Those exposed at age 50 have about one-third these risks. Projection of lifetime risks for those exposed at age 10 is more uncertain. Under a reasonable set of assumptions, estimates for this group range from about 1.0–1.8 times the estimates for those exposed at age 30. The excess life-time risk for leukemia at 1 Sv for those exposed at either 10 or 30 years is estimated as about 0.015 and 0.008 for males and females, respectively. Those exposed at age 50 have about two-thirds that risk. Excess risks for solid cancer appear quite linear up to about 3 Sv, but for leukemia apparent nonlinearity in dose results in risks at 0.1 Sv estimated at about 1/20 of those for 1.0 Sv. Site-specific risk estimates are given, but it is urged that great care be taken in interpreting these, because most of their variation can be explained simply by imprecision in the estimates.

Various radiation responses in mammalian cells depend on the position of the cell within its generation cycle (that is, its age) at the time of irradiation. Studies have most often been made by irradiating synchronized populations of cells in vitro. Results in different cell lines are not easy to compare, but an attempt has been made here to point out similarities and differences with regard to cell killing and division delay. In general, survival data obtained so far show that, in cells with a short G₁, cells are most sensitive in mitosis and in G₂, less sensitive in G₁, and least sensitive during the latter part of the S period. In cells with a long G₁, in addition to the above, there is usually a resistant phase early in G₁ followed by a sensitive stage near its end. (The latter may be as sensitive as mitosis.) Exceptions to the above, especially in some L cell sublines, have been noted, and a possible explanation is given.

In Chinese hamster cells, maximum survival after irradiation occurs during S, but it does not coincide with the time of the maximum rate of DNA synthesis or with the time of the maximum number of cells in DNA synthesis, and changes in survival also occur in cells inhibited from synthesizing DNA. Rather, survival depends on the position the cell has reached in the cycle at that time, which involves not only DNA synthesis but other processes as well. Survival is not completely correlated with DNA synthesis, since halting DNA synthesis just before or just after irradiation only slightly affects survival at its maximum.

Division delay exhibits a pattern of response which is similar in most cell lines. Delay is considerable for cells irradiated in mitosis, is small for cells in G₁, increases to a maximum for cells during S, and declines for cells in G₂. L cells or human kidney cells may have a longer delay for cells irradiated in G₂ than for those irradiated in S. The results can be explained in terms of a two-component model of division delay. One component results from the prolongation of the S period due to the reduced rate of DNA synthesis, and the other, a block in G₂, is independent of DNA synthesis. The proportion of the two components may vary in different cell lines.

Multiple-fraction experiments have been carried out to determine the response to repeated small doses of 240 kV X rays down to 45 rad per fraction, using the mouse skin reaction system. A method of irradiating without anesthetic was developed so that up to 64 fractions could be given within 8 days; over this time, proliferation was negligible.

It was found that the total dose required to produce a given reaction continued to rise with the number of fractions above 30 fractions, in contradiction to the recent conclusions of Dutreix and colleagues. The plot of reciprocal total dose against size of each fraction was shown to be linear. This finding led to an analysis in terms of a function F_e, which is proportional to the slope of the chord of the appropriate cell survival curve from the origin to the dose per fraction used.

The cell survival curve derived here was well fitted by an equation of the form

The initial slope was 1/690 rad and the slope at 2340 rad was 1/126 rad. Thus, 1 rad at a dose approaching 0 rad has 18% of the effect of 1 rad at a single dose of 2340 rad for mouse skin reactions. A cell survival theory based on Neary's theory of chromosome aberrations is presented and the current results are consistent with the postulate that cell death results from the formation of chromosome aberrations.

X-irradiation of Chinese hamster cells at temperatures above 37°C results in enhanced killing response. The magnitude of this thermal effect increases with increasing temperature and varies inversely with dose rate during the exposure of the cells to the combined effects of elevated temperature and ionizing radiation. Postirradiation incubation at an elevated temperature is also effective in enhancing the response but not preirradiation hyperthermia. Split-dose experiments demonstrate that hyperthermia also inhibits the repair of sublethal damage for temperatures up to ∼41°C. Above 41°C, lethal damage expression is enhanced as well. Fluctuations in the age-response structure of cells x-irradiated at 42°C are reduced, a result consistent with a reduced capacity for sublethal damage when cells are hyperthermic during irradiation.

This continues the series of general reports on mortality in the cohort of atomic bomb survivors followed up by the Radiation Effects Research Foundation. This cohort includes 86,572 people with individual dose estimates, 60% of whom have doses of at least 5 mSv. We consider mortality for solid cancer and for noncancer diseases with 7 additional years of follow-up. There have been 9,335 deaths from solid cancer and 31,881 deaths from noncancer diseases during the 47-year follow-up. Of these, 19% of the solid cancer and 15% of the noncancer deaths occurred during the latest 7 years. We estimate that about 440 (5%) of the solid cancer deaths and 250 (0.8%) of the noncancer deaths were associated with the radiation exposure. The excess solid cancer risks appear to be linear in dose even for doses in the 0 to 150-mSv range. While excess rates for radiation-related cancers increase throughout the study period, a new finding is that relative risks decline with increasing attained age, as well as being highest for those exposed as children as noted previously. A useful representative value is that for those exposed at age 30 the solid cancer risk is elevated by 47% per sievert at age 70. There is no significant city difference in either the relative or absolute excess solid cancer risk. Site-specific analyses highlight the difficulties, and need for caution, in distinguishing between site-specific relative risks. These analyses also provide insight into the difficulties in interpretation and generalization of LSS estimates of age-at-exposure effects. The evidence for radiation effects on noncancer mortality remains strong, with risks elevated by about 14% per sievert during the last 30 years of follow-up. Statistically significant increases are seen for heart disease, stroke, digestive diseases, and respiratory diseases. The noncancer data are consistent with some non-linearity in the dose response owing to the substantial uncertainties in the data. There is no direct evidence of radiation effects for doses less than about 0.5 Sv. While there are no statistically significant variations in noncancer relative risks with age, age at exposure, or sex, the estimated effects are comparable to those seen for cancer. Lifetime risk summaries are used to examine uncertainties of the LSS noncancer disease findings.

Radiosensitization efficiencies for seven different 2-nitroimidazoles including Ro-07-0582 and its urinary metabolite, Ro-05-9963, and two 5-nitroimidazoles including metronidazole, have been determined in hypoxic Chinese Hamster cells, line V79-379A, X-irradiated in vitro. All the compounds were active hypoxic cell sensitizers with the enhancement ratios increasing with drug concentration. The 2-nitroimidazoles were all more efficient than the 5-nitroimidazoles. Overall, the efficiencies, defined as the concentration required to give a particular enhancement ratio, varied by a factor of about 200. Electron-affinities of the sensitizers were determined by pulse radiolysis as the one-electron reduction potentials and these correlate well with the sensitization efficiencies of the compounds. The correlation extends beyond the nitroimidazole series as is shown by data for p-nitroacetophenone, nifuroxime (a nitrofuran) and oxygen itself. The nitroimidazoles varied by a factor of 70 in their octanol/water partition coefficients, but the effect of this parameter on sensitizing efficiency is small compared with the influence of electron affinity.

When mammalian cells are treated with alkali of pH at about 12, the cells are lysed and the released DNA starts to uncoil. This process of DNA strand separation is accelerated if the cells have been exposed to ionizing radiation, and the effect is clearly detectable in the dose range 10–100 rads. The rate of strand separation is also influenced by temperature and ionic strength of the alkaline solution. The kinetics of DNA strand separation in alkali is studied for three conditions in terms of ionic strength and temperature, chosen in such a way that the effect of irradiation may conveniently be studied in the dose range 10 rads to 20 krads. The accelerating effect of ionizing radiation on DNA strand separation is probably due to DNA strand breakage and the technique described is thus a sensitive method of studying such damage to DNA. A model for the strand-separation process, based on the assumption that strand breakage causes the accelerating effect, is proposed and found to describe the experimental data adequately.

The lethal response of Chinese hamster ovary cells to hyperthermia was determined at selected extracellular pH. Decreasing pH from 7.6 to 6.7 increased the lethal response of cells over the temperature range of 41 to 44°C. Cell viability was not effected over this pH range at 37°C. The pH sensitizing affect was most prominent at temperatures which were marginally lethal at normal pH (7.4). Four hours of exposure to 42°C decreased survival to 10% at pH 7.4 and 0.01% at pH 6.7. Enhanced cell killing was observed when the cells were exposed to reduced pH and elevated temperatures simultaneously. Prolonging the time of pH exposure before and after hyperthermia did not influence survival. High-density culturing increased the sensitivity of cells to hyperthermia. This affect was due to metabolic acidification of the medium and could be reversed by adjusting the pH.

Dual radiation action is a process in which cellular lesions are produced as a result of the interaction of pairs of sublesions that are molecular alterations produced by ionizing radiation. Previous formulations of this process have employed a number of simplifying assumptions that limit the accuracy and the range of application of theoretical analysis. The formulation presented here removes some of these restrictions by introducing three functions that describe the geometry of the sensitive material in the cell, the geometry of the pattern of energy deposition, and the interaction probability of sublesions as a function of their separation. The relation derived is similar to that obtained previously, in that lesion production is found to depend on two terms that are proportional to the first and the second power of the absorbed dose. However, the coefficients of these terms are now derived on the basis of a more realistic treatment.

The radiosensitizing and radioprotective effects of various compounds have been characterized in Chinese hamster fibroblasts growing in vitro and in a model chemical system utilizing DNA as target. The contribution to the lethal action of ionizing radiation in mammalian cells from the indirect effect of OH has been measured. The data are consistent with the “oxygen fixation hypothesis,” whereby target free radicals react either with radical-reducing species, resulting in “chemical repair,” or with radical-oxidizing species, resulting in “fixation” of radical damage to a potentially lethal form. Radical repair–fixation competition has been demonstrated in the in vitro chemical system with a sensitizer other than O₂.

Dimethyl sulfoxide is shown to radioprotect in both the cellular and chemical systems by scavenging OH. Cysteamine, on the other hand, is shown to protect primarily by adding to the pool of radical-reducing species, resulting in enhanced repair of free-radical damage in the targets. Electron-affinic compounds are shown to radiosensitize, predominantly, by adding to the pool of radical-oxidizing species, resulting in enhanced fixation of free-radical damage in the targets. Diamide is shown to radiosensitize mammalian cells by a mechanism which is additive to that of the electron-affinic compounds. N-Ethylmaleimide shows radiosensitizing characteristics, in mammalian cells, which, in some respects, are qualitatively similar to those of Diamide. These compounds may radiosensitize cells by chemically and/or biochemically altering the pool of radical-reducing species in the vicinity of the cellular targets, resulting in enhanced fixation of radical damage in the targets by endogenous radical-oxidizing species. The oxygen enhancement ratio for hamster cells is shown to depend upon the intracellular target environment, and can be made to vary between near 1 and 3.3.

A long-standing dogma in the radiation sciences is that energy from radiation must be deposited in the cell nucleus to elicit a biological effect. A number of non-targeted, delayed effects of ionizing radiation have been described that challenge this dogma and pose new challenges to evaluating potential hazards associated with radiation exposure. These effects include induced genomic instability and non-targeted bystander effects. The in vitro evidence for non-targeted effects in radiation biology will be reviewed, but the question as to how one extrapolates from these in vitro observations to the risk of radiation-induced adverse health effects such as cancer remains open.

The mean inactivation dose (D̄) is calculated for published in vitro survival curves obtained from cell lines of both normal and neoplastic human tissues. Cells belonging to different histological categories (melanomas, carcinomas, etc.) are shown to be characterized by distinct values of D̄ which are related to the clinical radiosensitivity of tumors from these categories. Compared to other ways of representing in vitro radiosensitivity, e.g., by the multitarget parameters D₀ and n, the parameter D̄ has several specific advantages: (i) D̄ is representative for the whole cell population rather than for a fraction of it; (ii) it minimizes the fluctuations of the survival curves of a given cell line investigated by different authors; (iii) there is low variability of D̄ within each histological category; (iv) significant differences in radiosensitivity between the categories emerge when using D̄. D̄ appears to be a useful concept for specifying intrinsic radiosensitivity of human cell lines.