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

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

The induction of reciprocal translocations in rhesus monkey stem-cell spermatogonia: effects of low doses and low dose rates

The induction of reciprocal translocation in rhesus monkey spermatogonial stem cells was studied following exposure to low doses of acute X rays (0.25 Gy, 300 mGy/min) or to low-dose-rate X rays (1 Gy, 2 mGy/min) and gamma rays (1 Gy, 0.2 mGy/min). The results obtained at 0.25 Gy of X rays fitted exactly the linear extrapolation down from the 0.5 and 1.0 Gy points obtained earlier. Extension of X-ray exposure reduced the yield of translocations similar to that in the mouse by about 50%. The reduction to 40% of translocation rate after chronic gamma exposure was clearly less than the value of about 80% reported for the mouse over the same range of dose rates. Differential cell killing with ensuing differential elimination of aberration-carrying cells is the most likely explanation for the differences between mouse and monkey.     

Induction and assessment of persistent radioresistance in murine leukocytes in vivo

The aim of the present study was to investigate whether weekly exposure to gamma rays causes a persistent increase in the number of radioresistant leukocytes in mice in vivo. Using the comet assay, 1 Gy radiation exposure decreased the percentage of leukocytes with less than 5% DNA in the tail (<5% DNAT), and we propose that radioresistance induction might increase the number of cells with <5% DNAT after radiation exposure. We exposed mice to 1 Gy gamma rays weekly for four weeks or 2 Gy per week for nine weeks. We observed a significant increase in cells with <5% DNAT after the third week and up to nine weeks of exposure. We exposed animals to gradually increasing radiation doses and finally challenged the lymphocytes with 1 Gy radiation both in vivo and in vitro. We observed increased radioresistance in vitro, providing evidence that a cellular process is involved. However, more radioresistance was observed in vivo than in vitro, suggesting a physiological effect. Cells challenged in vitro were maintained on ice during and after exposure, which likely caused a reduction in DNA repair. Radioresistance induction likely arose from mutation selection in stem cells because leukocytes are unable to proliferate in peripheral blood.     

Evidence for differences among the sectors of potentially lethal damage expressed by hypertonic treatment in plateau-phase V79 cells after exposure to neutrons or gamma rays: the importance of distinction between alpha and beta-PLD forms

Expotentially growing and plateau-phase V79 cells were exposed to various doses of neutrons and plated either immediately or after treatment in hypertonic medium (250-500 mM NaCl) to express radiation-induced potentially lethal damage (PLD). Postirradiation treatment of exponentially growing cells in hypertonic medium (500 mM) resulted in a decrease in both Dq and D0, whereas postirradiation treatment of plateau-phase cells in hypertonic medium (in the range between 200 to 1,500 mM) resulted mainly in a reduction of Dq. This difference in response between exponentially growing and plateau-phase cells may reflect differences in the chromatin structure in cells at various stages of the cell cycle, affecting fixation of radiation-induced damage. Exposure of plateau-phase cells to gamma rays, on the other hand, resulted in a treatment time and salt concentration-dependent decrease in Dq along with a decrease in D0. Repair of neutron-induced, hypertonic treatment-sensitive PLD, measured by delaying treatment for various periods after irradiation, was found to proceed with a t1/2 of about 1 h. This is similar to the repair kinetics obtained by delaying treatment of plateau-phase cells with 150 microM beta-D-arabinofuranosyladenine (araA) after exposure to gamma rays or neutrons and contrasts the repair kinetics observed after exposure of cells to gamma rays. In this case, hypertonic treatment was found to affect a form of PLD repaired with a t1/2 of 10-15 min (beta-PLD) and araA, a different form of PLD, repaired with a t1/2 of about 1 h (alpha-PLD). Based on these results it is hypothesized that the sector of lesions affected by hypertonic treatment and araA coincides after exposure to neutrons (effect on alpha-PLD) but only partly overlaps after exposure to gamma rays (due to the effect on beta-PLD of hypertonic treatment). The results presented, together with previously published observations, suggest a differential induction and/or fixation by hypertonic medium of the alpha- and beta-PLD forms as the LET of the radiation increases. Furthermore, they indicate that direct comparison of the effects of a postirradiation treatment, as well as of the repair kinetics obtained by its delayed application after exposure to radiations of various LET, should be made with caution.     

Response of X-ray-sensitive CHO mutant cells to gamma radiation. I. Effects of low dose rates and the process of repair of potentially lethal damage in G1 phase

X-ray-sensitive CHO mutants (xrs-5 and xrs-6) were exposed to isoleucine-deficient (IL-) medium for 24-36 h to accumulate G1-phase cells. Cells exposed to IL- medium for up to 5 days did not show significant changes in plating efficiency when returned to normal medium. Nearly confluent cultures of IL- -treated cells were irradiated with either 60Co gamma rays (75 cGy/min) or 137Cs gamma rays (2.7, 6.0, or 15.3 cGy/h). A significant reduction (approximately 2.5-fold) in the radiation sensitivity of the parental CHO K-1 cells was observed for chronic low-dose-rate radiation exposure compared to the results obtained for acute high-dose-rate exposure. However, no noticeable differences were observed in the survival curves of either xrs-5 or xrs-6 cells when low-dose-rate and acute exposures were compared. CHO K-1 cells exhibited potentially lethal damage repair while held in IL- medium after gamma irradiation, whereas no repair was observed in either of the radiation-sensitive mutant lines examined at similar survival levels.     

Quantification of DNA repair capacity in whole blood of patients with head and neck cancer and healthy donors by comet assay

Comet assay has been used to estimate cancer risk by quantification of DNA damage and repair in response to mutagen challenge. Our goal was to adopt best practices for the alkaline comet assay to measure DNA repair capacity of white blood cells in whole blood of patients with squamous cell carcinoma of the head and neck (HNSCC). The results show that initial damage by 10 Gy of gamma radiation expressed as percent DNA in comet tail was higher in stimulated lymphocytes (61.1+/-11.8) compared to whole blood (43.0+/-12.1) but subsequent repair was similar with comet tail of approximately 20% at 15 min and 13% at 45 min after exposure. Exposure of whole blood embedded in agarose from 5 to 10 Gy gamma radiation was followed by an approximately 70% repair of the DNA damage within 45 min with a faster repair phase in the first 15 min. Variability of the measurement was lower within repeated measurements of the same person compared to measurement of different healthy individuals. The repair during first 15 min was slower (p=0.01) in ex-/non-smokers (41.0+/-2.1%) compared to smokers (50.3+/-2.7%). This phase of repair was also slower (p=0.02) in HNSCC patients (36.8+/-2.1%) compared to controls matched on age and smoking (46.4+/-3.0%). The results of this pilot study suggest that quantification of repair in whole blood following a gamma radiation challenge is feasible. Additional method optimization would be helpful to improve the assay for a large population screening.     

Effect of chlorophyllin on gamma ray induced micronuclei in polychromatic erythrocytes of murine peripheral blood determined by the ABC strategy

The effect of chlorophyllin on micronucleated polychromatic erythrocyte (MN-PCE) induction by gamma ray exposure in peripheral blood of mice was studied. The area beneath the curve (ABC) of MN-PCE frequency versus time was used as an index of total MN-PCE induction. The dose of 200 mg chlorophyllin per kg of body weight caused a slight, but not significant, reduction of the MN-PCE caused by 1.0 Gy exposure. This result indicates that chlorophyllin did not protect the cells against MN induction. In previous studies it was observed that the same chlorophyllin dose was able to protect 100% against sister chromatid exchange (SCE) induction by 1.0 gamma rays in both murine spermatogonia and bone marrow cells. These contradictory results indicate that chlorophyllin did not protect cells by scavenging free radicals, but by other mechanism, i.e. stimulating repair of lesions involved in SCE induction.     

Evidence for similarities between radiation damage expressed by beta-araA and damage involved in the interaction effect observed after exposure of V79 cells to mixed neutrons and gamma radiation

Plateau-phase V79 cells were exposed sequentially to fast neutrons and gamma rays. A dose-dependent reduction in the shoulder width of the gamma-ray survival curve was observed after preexposure of cells to neutrons. A similar effect was demonstrated on the neutron survival curve when cells were preirradiated with gamma rays. Treatment of cells with 150 microM beta-araA after either gamma or neutron irradiation reduced primarily the shoulder of the survival curve. When beta-araA was given to the cells after exposure to mixed radiation modalities, survival curves similar to those observed after exposure to a single radiation modality and treatment with beta-araA were obtained. The kinetics of loss of the interaction observed after exposure of cells to gamma rays following neutron irradiation was similar to the kinetics of loss of sensitivity to beta-araA (T1/2 = 1 h) measured by delaying drug administration after exposure to gamma rays. The results suggest that the PLD expressed by beta-araA is at least partly involved in the interactive effect observed after combined exposure of plateau-phase V79 cells to neutrons and gamma rays.     

Reciprocal adaptive response of human peripheral lymphocytes induced by bleomycine or gamma rays.

The adaptive response and reciprocal adaptive response induced in vitro by exposure to low doses of gamma rays (0.05 Gy) or bleomycin (0.05 microg/ml) in human peripheral blood lymphocytes were assessed by the frequency of chromosome aberrations. Gamma rays (1.5 Gy) or bleomycin (1.5 microg/ml) were used as the challenge doses. In the experiments, blood samples from 5 healthy donors were investigated. It has been found that low doses of bleomycin and gamma rays induced a reciprocal adaptive response to high doses of gamma rays or bleomycin. Moreover, the results confirmed that the adaptive response did not correlate with the radiosensitivity of the peripheral blood lymphocytes.     

Mutation induction by tritiated water and effects of deuterium oxide in cultured mouse leukemia cells

Induction of mutation to 6-thioguanine resistance was studied in L5178Y mouse leukemia cells after exposure to low-dose-rate gamma rays or tritiated water at dose rates of approximately 0.025 to 0.4 Gy/hr for 20 hr in the presence or absence of 45% (v/v) deuterium oxide. The effect of acute gamma-ray exposure was also examined. A higher frequency of induced mutations was observed after tritium beta rays than after gamma rays, both at equivalent doses and cell survival. Deuterium oxide enhanced the mutation induced by gamma rays and tritium beta rays but did not affect the survival-mutation correlation of the two radiations.     

Nonlinear survival curves for cells of solid tumors after large doses of fast neutrons and gamma rays.

We have previously proposed that survival curves for cells of murine NFSa fibrosarcomas after exposure to fast neutrons might demonstrate curvature when the neutron doses reach a level high enough to cure the fibrosarcomas. We report here that this is the case. Murine NFSa fibrosarcomas growing in the hind legs of syngeneic mice were exposed to either gamma rays or fast neutrons. The tumors were removed and retransplanted into fresh recipients to obtain 50% tumor cell doses, from which the dose-cell survival relationship was constructed. Survival curves showed continuous bending down to 10(-7), and were well fitted using the linear-quadratic model. The alpha and beta values for neutrons were larger than those for gamma rays. When the surviving fractions at experimental TCD50 doses were calculated using these values, comparable figures were obtained for neutrons and gamma rays. The RBEs for neutrons were comparable for the TCD50 and TD50 assays. Neutron RBE was independent of dose within a range of 5-28 Gy. The capacity of the tumors to repair the damage caused by large doses of neutrons was identical to that for small doses of neutrons, indicating that cells retained the capacity to repair neutron damage irrespective of the size of the dose.     

A comparison of gamma and neutron irradiation on Raji cells: effects on DNA damage, repair, cell cycle distribution and lethality.

The Comet assay (microgel electrophoresis) was used to study DNA damage in Raji cells, a B-lymphoblastoid cell line, after treatment with different doses of neutrons (0.5 to 16 Gy) or gamma rays (1.4 to 44.8 Gy). A better growth recovery was observed in cells after gamma-ray treatments compared with neutron treatments. The relative biological effectiveness (RBE) of neutron in cell killing was determined to be 2.5. Initially, the number of damaged cells per unit dose was approximately the same after neutron and gamma-ray irradiation. One hour after treatment, however, the number of normal cells per unit dose was much lower for neutrons than for gamma rays, suggesting a more efficient initial repair for gamma rays. Twenty-four hours after treatment, the numbers of damaged cells per unit dose of neutrons or gamma rays were again at comparable level. Cell cycle kinetic studies showed a strong G2/M arrest at equivalent unit dose (neutrons up to 8 Gy; gamma rays up to 5.6 Gy), suggesting a period in cell cycle for DNA repair. However, only cells treated with low doses (up to 2 Gy) seemed to be capable of returning into normal cell cycle within 4 days. For the highest dose of neutrons, decline in the number of normal cells seen at already 3 days after treatment was deeper compared with equivalent unit doses of gamma rays. Our present results support different mechanisms of action by these two irradiations and suggest the generation of locally multiply damaged sites (LMDS) for high linear energy transfer (LET) radiation which are known to be repaired at lower efficiency.     

Effect of dose rate on the survival of irradiated human skin fibroblasts.

The survival of cells in density-inhibited, confluent cultures maintained at 37 degrees C was examined following exposure to 137Cs gamma rays at low dose rates (0.023 or 0.153 Gy/h) or to 60Co gamma rays at a single high dose rate (0.70-0.75 Gy/min). Cells from an ataxia telangiectasia (AT) homozygote showed no dose-rate effect, whereas a three- to fivefold increase in D0 was observed for all other cell strains exposed at low dose rates. The magnitude of the dose-rate effect did not differ significantly among cells from persons with hereditary retinoblastoma, basal cell nevus syndrome, or AT-heterozygote compared with normal cell strains, and was not related to the size of the shoulder (extrapolation number) of the survival curve. Furthermore, no differences in the capacity for the repair of potentially lethal damage during confluent holding were observed among these latter cell strains.     

Kinetics of the Early Adaptive Response to Gamma Rays: Induction of a Cellular Radioprotective Mechanism in Murine Leukocytes <Emphasis Type="Italic">In vivo</Emphasis>

The aims of the study were to establish the kinetics of the early adaptive response and to determine the minimum adaptive dose of gamma rays capable of inducing this response. The minimum adaptive dose was determined by exposing groups of five BALB/c male mice to an adaptive dose of 0.005 or of 0.02 Gy γ rays from a ¹³⁷Cs source and challenge with 1.0 Gy 60 min later. The kinetics of adaptive response induction was established by exposing mice to an adaptive dose of 0.01 Gy, and subsequently to a challenge dose of 1.0 Gy at different times. Blood samples were collected from the tail immediately after exposure to the challenge dose, and the percentage of DNA-damaged cells and the extent of damaged were determined by single cell gel electrophoresis in 300 leukocytes per animal in five mice. The results confirms the capability of an in vivo induction of an early radioprotective process against the DNA-damage produced by gamma rays in murine leukocytes, and allows us to conclude that the minimum adaptive dose lies between 0.005 and 0.01Gy of gamma rays, and the early adaptive response is induced as early as 30 min after the exposure and persists for at least 18 hr.     

Chromosomes lacking telomeres are present in the progeny of human lymphocytes exposed to heavy ions

High-charge and energy (HZE) nuclei represent one of the main health risks for human space exploration, yet little is known about the mechanisms responsible for the high biological effectiveness of these particles. We have used in situ hybridization probes for cross-species multicolor banding (RxFISH) in combination with telomere detection to compare yields of different types of chromosomal aberrations in the progeny of human peripheral blood lymphocytes exposed to either high-energy iron ions or gamma rays. Terminal deletions showed the greatest relative variation, with many more of these types of aberrations induced after exposure to accelerated iron ions (energy 1 GeV/nucleon) compared with the same dose of gamma rays. We found that truncated chromosomes without telomeres could be transmitted for at least three cell cycles after exposure and represented about 10% of all aberrations observed in the progeny of cells exposed to iron ions. On the other hand, the fraction of cells carrying stable, transmissible chromosomal aberrations was similar in the progeny of cells exposed to the same dose of densely or sparsely ionizing radiation. The results demonstrate that unrejoined chromosome breaks are an important component of aberration spectra produced by the exposure to HZE nuclei. This finding may well be related to the ability of such energetic particles to produce untoward late effects in irradiated organisms.     

Measurement of the initial levels of DNA damage in human lymphocytes induced by 29 kV X rays (mammography X rays) relative to 220 kV X rays and gamma rays

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137Cs gamma rays and 60Co gamma rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F(d) = A/(t + A), where F(d) is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the gamma rays of 137Cs and 60Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150-250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.     

Differential modulation of specific gene expression following high- and low-LET radiations

Experiments were designed to examine the effects of radiation quality on specific gene expression within the first 3 h following radiation exposure in Syrian hamster embryo (SHE) cells. Preliminary work demonstrated the induction of c-fos and alpha-interferon genes following exposure to low-linear-energy-transfer (low-LET) radiations (X rays or gamma rays). More detailed experiments revealed induction of c-fos mRNA within the first 3 h following exposure to either X rays (75 cGy) or gamma rays (90 cGy). We could not detect induction of c-fos following exposure of SHE cells to fission-spectrum neutrons (high-LET) from the JANUS reactor administered at either high (12 cGy/min) or low (0.5 cGy/min) dose rates. Expression of alpha-interferon mRNA was similarly induced by low-LET radiations but only modestly by JANUS neutrons. The induction by gamma rays was dose-dependent, while induction by neutrons was specific for low doses and low dose rates. These experiments demonstrate the differential gene inductive response of cells following exposure to high- and low-LET radiations. These experiments suggest that these different qualities of ionizing radiation may have different mechanisms for inducing many of the cellular consequences of radiation exposure, such as cell survival and cell transformation.     

Relative effectiveness of HZE iron-56 particles for the induction of cytogenetic damage in vivo

One of the risks of prolonged manned space flight is the exposure of astronauts to radiation from galactic cosmic rays, which contain heavy ions such as (56)Fe. To study the effects of such exposures, experiments were conducted at the Brookhaven National Laboratory by exposing Wistar rats to high-mass, high-Z, high-energy (HZE) particles using the Alternating Gradient Synchrotron (AGS). The biological effectiveness of (56)Fe ions (1000 MeV/nucleon) relative to low-LET gamma rays and high-LET alpha particles for the induction of chromosome damage and micronuclei was determined. The mitotic index and the frequency of chromosome aberrations were evaluated in bone marrow cells, and the frequency of micronuclei was measured in cells isolated from the trachea and the deep lung. A marked delay in the entry of cells into mitosis was induced in the bone marrow cells that decreased as a function of time after the exposure. The frequencies of chromatid aberrations and micronuclei increased as linear functions of dose. The frequency of chromosome aberrations induced by HZE particles was about 3.2 times higher than that observed after exposure to (60)Co gamma rays. The frequency of micronuclei in rat lung fibroblasts, lung epithelial cells, and tracheal epithelial cells increased linearly, with slopes of 7 x 10(-4), 12 x 10(-4), and 11 x 10(-4) micronuclei/binucleated cell cGy(-1), respectively. When genetic damage induced by radiation from (56)Fe ions was compared to that from exposure to (60)Co gamma rays, (56)Fe-ion radiation was between 0.9 and 3.3 times more effective than (60)Co gamma rays. However, the HZE-particle exposures were only 10-20% as effective as radon in producing micronuclei in either deep lung or tracheal epithelial cells. Using microdosimetric techniques, we estimated that 32 cells were hit by delta rays for each cell that was traversed by the primary HZE (56)Fe particle. These calculations and the observed low relative effectiveness of the exposure to HZE particles suggest that at least part of the cytogenetic damage measured was caused by the delta rays. Much of the energy deposited by the primary HZE particles may result in cell killing and may therefore be "wasted" as far as production of detectable micronuclei is concerned. The role of wasted energy in studies of cancer induction may be important in risk estimates for exposure to HZE particles.     

Karyotypes of human lymphocytes exposed to high-energy iron ions

Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.     

Inhibition of repair of radiation-induced DNA double-strand breaks by nickel and arsenite

The effect of arsenite or nickel on the repair of DNA double-strand breaks (DSBs) was studied in gamma-irradiated Chinese hamster ovary cells using pulsed-field gel electrophoresis. After treatment with nickel chloride or arsenite for 2 h, cells were irradiated with gamma rays at a dose of 40 Gy, and the numbers of DNA DSBs were measured immediately after irradiation as well as at 30 min postirradiation. Both arsenite and nickel(II) inhibited repair of DNA DSBs in a concentration-dependent manner; 0.08 mM arsenite significantly inhibited the rejoining of DSBs, while 76 mM nickel was necessary to observe a clear inhibition. The mean lethal concentrations for the arsenite and nickel(II) treatments were approximately 0.12 and 13 mM, respectively. This indicates that the inhibition of repair by arsenite occurred at a concentration at which appreciable cell survival occurred, but that nickel(II) inhibited repair only at cytotoxic concentrations at which the cells lost their proliferative ability. These novel observations provide insight into the mechanisms underlying the effects of combined exposure to arsenite and ionizing radiation in our environment.