A comparison of radioprotection from three neutron sources and 60Co by WR-2721 and WR-151327

Two thiophosphoroate radiation protectors (WR-2721 and WR-151327) were assessed for their ability to modify the effects of neutron or gamma irradiation on the gastrointestinal tract. Three neutron sources (DOSAR, JANUS, and FERMILAB) were compared to the response obtained after 60Co irradiation. The end points studied were intestinal stem cell survival and LD50(6). DOSAR and JANUS, both fission-spectrum neutrons, showed somewhat different gut sensitivities [LD50(6)] of about 240 and 400 cGy respectively. The intestinal LD50 obtained with FERMILAB neutrons (25 meV) was closer (875 cGy) to that obtained after 60Co (1068 cGy) irradiation. WR-151327 protected against the lethal effects of fission neutron (DOSAR and JANUS) to a greater degree (DMF = 2.2) than with lower LET sources such as FERMILAB neutrons (DMF = 1.7) or 60Co (DMF = 1.7). The results did not correlate with the intestinal stem cell assays where WR-2721 when compared to WR-151327 showed either similar (DOSAR; fission spectrum neutrons) or somewhat better (60Co and FERMILAB neutrons) protection. Possible explanations for the differing results are discussed.     

Cytogenetic effects of low-dose radiation with different LET in human peripheral blood lymphocytes

Chromosome damage and the spectrum of aberrations induced by low doses of γ-irradiation, X-rays and accelerated carbon ions (195 MeV/u, LET 16.6 keV/μm) in peripheral blood lymphocytes of four donors were studied. G₀-lymphocytes were exposed to 1–100 cGy, stimulated by PHA, and analyzed for chromosome aberrations at 48 h post-irradiation by the metaphase method. A complex nonlinear dose–effect dependence was observed over the range of 1 to 50 cGy. At 1–7 cGy, the cells showed the highest radiosensitivity per unit dose (hypersensitivity, HRS), which was mainly due to chromatid-type aberration. According to the classical theory of aberration formation, chromatid-type aberrations should not be induced by irradiation of unstimulated lymphocytes. With increasing dose, the frequency of aberrations decreased significantly, and in some cases it even reached the control level. At above 50 cGy the dose–effect curves became linear. In this dose range, the frequency of chromatid aberrations remained at a low constant level, while the chromosome-type aberrations increased linearly with dose. The high yield of chromatid-type aberrations observed in our experiments at low doses confirms the idea that the molecular mechanisms which underlie the HRS phenotype may differ from the classical mechanisms of radiation-induced aberration formation. The data presented, as well as recent literature data on bystander effects and genetic instability expressed as chromatid-type aberrations on a chromosomal level, are discussed with respect to possible common mechanisms underlying all low-dose phenomena.     

Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. II. Relationship between cell survival and the induction of chromosomal damage with low doses of alpha particles

The induction of cytotoxicity, chromosomal aberrations, and sister chromatid exchanges (SCEs) was measured in CHO K-1c cells and in isogenic X-ray-sensitive mutant xrs-6c cells that had been irradiated with X rays and alpha particles in isoleucine-deficient alpha-minimal essential medium in G1 phase of the cell cycle. There was a noticeable shoulder region on the survival curve for CHO K-1c cells irradiated with very low doses of alpha particles, whereas this feature was absent for xrs-6c cells with alpha-particle doses as low as 0.5 cGy. Higher frequencies of chromatid-type aberrations were induced in G1-phase xrs-6c cells than in G1-phase CHO K-1c cells by both gamma- and alpha-particle irradiation. Induction of nonlethal chromosomal aberrations was observed following exposure to 2-6 cGy of alpha particles, doses yielding 97-100% cell survival. Irradiation with 0.5 cGy of alpha particles induced SCE; nearly 60% of irradiated cells contained significantly increased levels of SCE. However, only 3% of the nuclei of cells exposed to 0.5 cGy of alpha-particle radiation were actually traversed by an alpha particle. The observation that a large fraction of cells apparently survive exposure to very low doses of alpha-particle radiation with persistent genetic damage manifested by both chromosomal aberrations and SCEs may have important implications for the carcinogenic hazards of high-LET radiation.     

Effect of novobiocin on the frequencies of chromatid-type aberrations and sister-chromatid exchanges following gamma-irradiation

The effect of novobiocin on the frequencies of chromatid-type aberrations and SCEs was examined in Chinese hamster V79 cells which were exposed to gamma-rays and post-treated with novobiocin. While no chromatid aberrations were induced in the unirradiated cells by novobiocin, the frequency of SCEs was slightly increased by treatment with novobiocin alone. Irradiation of G2 cells produced multiple chromatid-type aberrations and post-treatment of the irradiated cells with novobiocin resulted in a significant increase of the aberrations, including chromatid gaps and breaks. In contrast, novobiocin failed to increase the frequency of SCEs induced by gamma-rays when the irradiated cells were post-treated with novobiocin.     

Protection against radiation-induced mutagenesis in V79 cells by 2-[(aminopropyl)amino] ethanethiol under conditions of acute hypoxia

The effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells under hypoxic or aerobic conditions were examined. Conditions of acute hypoxia were attained by gassing 10(6) cells in 1-ml volumes in individual glass ampoules for 2 min with nitrogen. Ampoules were then sealed and incubated at 37 degrees C for 60 min. Following this treatment, cell survival after irradiation as expected was significantly enhanced. The effect of acute hypoxia on the formation of HGPRT mutants by irradiation was also investigated. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was approximately linear as a function of radiation dose under most conditions tested, it was significantly reduced in cell populations made acutely hypoxic prior to irradiation. Protection against mutation induction was apparent and similar when cells were irradiated in the presence of the radioprotector, regardless of whether they were also hypoxic or aerated. If cells were irradiated in air and then made hypoxic, no significant protection was still observed. These results suggest that the antimutagenic effect of WR-1065 is not due solely to its ability to scavenge radiation-induced oxygen-free radicals, but rather that it may also modulate these effects through the scavenging of metabolically induced free radicals and/or the chemical repair of radiation-induced DNA lesions.     

Studies on radiation-induced chromosome aberrations in mouse spermatocytes. II. Dose-response relationships of chromosome aberrations induced at zygotene stage in mouse primary spermatocytes following fast neutron- and 60Co gamma-irradiations

The chromosome aberrations induced at zygotene stage in mouse spermatocytes following exposures to fast neutrons and 60Co gamma-rays were examined at diakinesis-metaphase I. The dose-response relationships were well fitted to linear equation for deletion-type aberrations and to linear-quadratic equation for exchange-type aberrations in 60Co gamma-irradiation group. In fast neutron-irradiation group, the dose-response relationships were well fitted to linear equations for deletion- and exchange-type aberrations. The rate of deletion-type aberrations was remarkably high for fast neutrons, about 6 times higher than that after 60Co gamma-irradiation. The main types of chromosome aberrations observed were iso-chromatid breaks or fragments and chromatid exchanges in both irradiation groups as well as X-irradiation. These results indicate that there is a possibility that two double-strand breaks are induced simultaneously at iso-locus position in sister chromatids by a single track of radiations. Production of such single-track-induced two double-strand breaks in iso-chromatids may be very frequently expressed as iso-chromatid-type deletions in the high LET fast neutron-irradiation group. On the contrary, in the low LET 60Co gamma- or X-irradiation group, the above-mentioned mechanism may not be so effective for contribution to chromosome aberration induction in mouse spermatocytes. This mechanism was discussed in detail.     

Increase in the frequency of gamma-ray induced chromosomal aberrations in mammalian cells by post-treatment with novobiocin

The effect of novobiocin (an inhibitor of DNA topoisomerase and polymerase) on the frequency of chromosomal aberrations was examined in Chinese hamster V79 cells irradiated with gamma-rays in the plateau phase of growth and subcultured in the presence of novobiocin until the first mitosis after irradiation. Novobiocin alone affected cell survival, DNA synthesis and the mitotic frequency of unirradiated cells in a dose-dependent manner, without causing any significant increase in the frequency of chromosome- or chromatid-type aberrations. The frequency of chromosome-type aberrations induced by gamma-radiation was not influenced by novobiocin at 200 microM, but the frequency of chromosome deletions (but not rings and dicentrics) showed a two-fold increase when 300 microM novobiocin was present. Irradiation produced a low level of chromatid-type aberrations and post-treatment with novobiocin at concentrations greater than 100 microM significantly increased the frequency of chromatid gaps and breaks. The results support the idea that different radiation-induced lesions lead to chromosome- as opposed to chromatid-type aberrations.     

Dose-response modeling of life shortening in a retrospective analysis of the combined data from the JANUS program at Argonne National Laboratory

Life shortening was investigated in both sexes of the B6CF1 (C57BL/6 x BALB/c) mouse exposed to fission neutrons and 60Co gamma rays. Three basic exposure patterns for both neutrons and gamma rays were compared: single exposures, 24 equal once-weekly exposures, and 60 equal once-weekly exposures. Ten different dose-response models were fitted to the data for animals exposed to neutrons. The response variable used for all dose-response modeling was mean after-survival. A simple linear model adequately described the response to neutrons for females and males at doses less than or equal to 80 cGy. At higher neutron dose levels a linear-quadratic equation was required to describe the life-shortening response. An effect of exposure pattern was observed prior to the detection of curvature in the dose response for neutrons and emerged as a potentially significant factor at neutron doses in the range of 40-60 cGy. Augmentation of neutron injury with dose protraction was observed in both sexes and began at doses as low as 60 cGy. The life-shortening response for all animals exposed to gamma rays (22-1918 cGy) was linear and inversely dependent upon the protraction period (1 day, 24 weeks, 60 weeks). Depending on the exposure pattern used for the gamma-ray baseline, relative biological effectiveness (RBE) values ranged from 6 to 43. Augmentation, because it occurred only at higher levels of neutron exposure, had no influence on the estimation of RBEm.     

Genotoxic effects in M5 cells and Chinese hamster V79 cells after exposure to 7Li-beam (LET=60 keV/microm) and correlation of their survival dynamics to nuclear damages and cell death

Chinese hamster V79 cell and a cell strain M5, derived from V79 cells and reported to be relatively resistant to gamma-ray, hydrogen peroxide, and N-methyl-N-nitro-N-nitrosoguanidine (MNNG; a potent human carcinogen), were exposed to high LET (7)Li-beam (LET=60 keV/microm) at approximately 90% confluent state in the dose range of 0-1 Gy. Effects of (7)Li-beam exposure on cell survival, micronuclei induction (MN), chromosomal aberrations (CA) and apoptosis were compared in both the cell lines. A dose-dependent decline in survival for both the cell lines was noted, relatively less in M5 cells (mostly p<0.01) indicating greater radio-resistance in this strain. The MN, CA and apoptosis increased in a dose-dependent manner in both V79 and M5 cells. Significant differences in various other parameters between these two cell lines were also noted. The relative intensity of DNA ladder, which is a useful marker for the determination of the extent of apoptosis induction, was much higher in V79 cells. A good correlation between the reduction of the surviving fractions and the increase in frequencies of MN or CA or apoptosis was noted for both the cell lines.     

Relationship between phosphorylated histone H2AX formation and cell survival in human microvascular endothelial cells (HMEC) as a function of ionizing radiation exposure in the presence or absence of thiol-containing drugs

Human microvascular endothelial cells (HMEC) were exposed to ionizing radiation at doses ranging from 0 to 16 Gy in either the presence or absence of the active thiol forms of amifostine (WR1065), phosphonol (WR255591), N-acetyl-l-cysteine (NAC), captopril or mesna. Each of these clinically relevant thiols, administered to HMEC at a dose of 4 mM for 30 min prior to irradiation, is known to exhibit antioxidant properties. The purpose of this investigation was to determine the relationship(s), if any, between the frequency of radiation-induced histone H2AX phosphorylation at serine 139 (gamma-H2AX) in cells and subsequent survival, as assessed by colony-forming ability, in exposed cell populations as a function of the presence or absence of each of the five thiol compounds during irradiation. gamma-H2AX formation in irradiated cells, as a function of relative DNA content, was quantified by bivariant flow cytometry analysis with FITC-conjugated gamma-H2AX antibody and nuclear DAPI staining. gamma-H2AX formation in cells was measured as the relative fold increase as a function of the treatment conditions. The frequency of gamma-H2AX-positive cells increased with increasing dose of radiation followed by a dose- and time-dependent decay. The most robust response for gamma-H2AX formation occurred 1 h after irradiation with their relative frequencies decreasing as a function of time 4 and 24 h later. To assess the effects of the various thiols on gamma-H2AX formation, all measurements were made 1 h after irradiation. WR1065 was not only effective in protecting HMEC against gamma-H2AX formation across the entire dose range of radiation exposures used, but it was also significantly more cytoprotective than either its prodrug (WR2721) or disulfide (WR33278) analogue. WR1065 had no significant effect on gamma-H2AX formation when administered immediately or up to 30 min after radiation exposure. An inhibitory effect against gamma-H2AX formation induced by 8 Gy of radiation was expressed by each of the thiols tested. NAC, captopril and mesna were equally effective in reducing the frequency of gamma-H2AX formation, with both WR1065 and WR255591 exhibiting a slightly more robust protective effect. Each of the five thiols was effective in reducing the frequency of gamma-H2AX-positive cells across all phases of the cell cycle. In contrast to the relative ability of each of these thiols to inhibit gamma-H2AX formation after irradiation, NAC, captopril and mesna afforded no protection to HMEC as determined using a colony-forming survival assay. Only WR1065 and WR255591 were effective in reducing the frequencies of radiation-induced gamma-H2AX-positive cells as well as protecting against cell death. These results suggest that the use of gamma-H2AX as a biomarker for screening the efficacy of novel antioxidant radioprotective compounds is highly problematic since their formation and disappearance may be linked to processes beyond simply the formation and repair of radiation-induced DSBs.     

The effect of wortmannin on radiation-induced chromosome aberration formation in the radioresistant tumor cell line WiDr

We analyzed the formation of radiation-induced chromosome aberrations in the cells of the radioresistant colon carcinoma cell line WiDr after treatment with wortmannin, an inhibitor of PI-3 kinases, including DNA-PK. Cells irradiated in G0/G1 phase with 200 kV X rays were treated with wortmannin before or after irradiation. Chromosome-type and chromatid-type aberrations were scored in metaphase cells by either Giemsa staining or FISH. Moreover, DNA-PK activity was measured in the absence and presence of wortmannin. In irradiated G0/G1-phase WiDr cells, only chromosome-type aberrations, including simple and complex exchanges and excess acentrics, were observed. After addition of 1 to 20 microM wortmannin, the formation of chromosome-type exchange aberrations was completely suppressed. The irradiated cells displayed exclusively chromatid-type aberrations including simple and complex chromatid exchanges and chromatid/isochromatid breaks. Whether the chromatid-type aberrations arise during G0/G1 as a result of homologous recombination processes coping with damaged DNA or whether DNA damage induced during G0/G1 phase persists until S and G2 phase and is then processed by homologous recombination pathways must be investigated further.     

An association between the radiation-induced arrest of G2-phase cells and low-dose hyper-radiosensitivity: a plausible underlying mechanism?

The survival of asynchronous and highly enriched G1-, S- and G2-phase populations of Chinese hamster V79 cells was measured after irradiation with 60Co gamma rays (0.1-10 Gy) using a precise flow cytometry-based clonogenic survival assay. The high-dose survival responses demonstrated a conventional relationship, with G2-phase cells being the most radiosensitive and S-phase cells the most radioresistant. Below 1 Gy, distinct low-dose hyper-radiosensitivity (HRS) responses were observed for the asynchronous and G2-phase enriched cell populations, with no evidence of HRS in the G1- and S-phase populations. Modeling supports the conclusion that HRS in asynchronous V79 populations is explained entirely by the HRS response of G2-phase cells. An association was discovered between the occurrence of HRS and the induction of a novel G2-phase arrest checkpoint that is specific for cells that are in the G2 phase of the cell cycle at the time of irradiation. Human T98G cells and hamster V79 cells, which both exhibit HRS in asynchronous cultures, failed to arrest the entry into mitosis of damaged G2-phase cells at doses less than 30 cGy, as determined by the flow cytometric assessment of the phosphorylation of histone H3, an established indicator of mitosis. In contrast, human U373 cells that do not show HRS induced this G2-phase checkpoint in a dose-independent manner. These data suggest that HRS may be a consequence of radiation-damaged G2-phase cells prematurely entering mitosis.     

Lack of synergistic effect between X-ray and UV irradiation on the frequency of chromosome aberrations in PHA-stimulated human lymphocytes in the G1 stage.

PHA-stimulated human lymphocytes in the G1 stage were irradiated with UV radiation and X-rays, and the cells were analyzed for chromosomal aberrations in the first mitotic division. The frequency of dicentric chromosomes after single X-irradiation in the G1 stage was about twice the yield in the G0 stage. No increase in the yield of dicentrics was observed after combined irradiation with UV and X-rays. This is contrary to the finding for G0 lymphocytes, where a 2-fold increase of chromosome aberrations was observed. UV irradiation of G1 lymphocytes induced chromatid-type aberrations whereas no significant yield of dicentric chromosomes was observed. This is in agreement with previous findings in Chinese hamster cells in the G1 stage [7]. Irradiation of G0 lymphocytes with UV radiation induce a low frequency of dicentric chromosomes. Thus, the present data indicate that the ratio between chromosome-type and chromatid-type aberrations is different in the G1 and G0 stages in human lymphocytes irradiated with UV radiation.     

Life shortening in mice exposed to fission neutrons and gamma rays. VII. Effects of 60 once-weekly exposures

A total of 6316 B6CF1 mice were exposed to 60 equal once-weekly doses of 0.85-MeV fission neutrons (0.033 to 0.67 cGy per weekly fraction) or 60Co gamma rays (1.67 to 10 cGy per weekly fraction) and were observed until they died. The mean aftersurvival times showed that the dose-response curves for both neutron and gamma-ray exposures were indistinguishable from linear over all doses except the highest neutron dose. The relative biological effectiveness (RBE) for neutrons, calculated as the ratio of the initial slopes of the dose-response curves, was about 20 for both males and females. Essentially the same value was obtained by a number of other analyses of the data. Virtually all of the radiation-specific excess mortality could be attributed to tumors; after decrementation of the population for nontumor deaths, the value of the RBE was not significantly changed.     

Cytogenetic effects of low dose radiation with different LET in human peripheral blood lymphocytes and possible mechanisms of their realization

The induction of chromosome damage by the exposure to low doses of gamma-(60)Co and accelerated carbon ions 12C in peripheral blood lymphocytes of different donors was investigated. The complex nonlinear dose-effect dependence at the range from 1 to 50-70 cGy was observed. At the doses of 1-5 cGy the cells show the highest radiosensitivity (hypersensitivity), mainly due to the chromatid-type aberration, which is typical to those spontaneously generated in the cell and believed not to be induced by the irradiation of unstimulated lymphocytes according to the classical theory of aberration formation. With the increasing dose the frequency of the aberrations decreases significantly, in some cases up to the control level. At the doses over 50-70 cGy the dose-effect curve becomes linear. The possible role of the oxidative stress, caused by radiation-induced increase in mitochondrial reactive oxigen species (ROS) release in the phenomenon of hypersensitivity (HS) at low doses is discussed as well as cytoprotective mechanisms causing the increased radioresistance at higher doses.     

The effect of chemical radiation protectors on cell cycle progression after gamma or neutron irradiation

Abstract. The effects of two chemical radiation protectors, WR-1065 and WR-151326, were characterized in V79 Chinese hamster cells after either cobalt-60 (⁶⁰Co) gamma or fission spectrum neutron irradiation. Each protector was administered at a concentration of 4 mM to exponentially growing cultures for 30 min prior to and during irradiation with either ⁶⁰Co gamma or JANUS fission spectrum neutrons. After irradiation the cells were either plated immediately for survival or returned to the incubator and assayed for cell progression. Aliquots of cells were removed at selected times, counted, fixed and stained with 4'6-diamidino-2-phenylindole (DAPI). Analysis of DNA histograms indicate that the presence of the protector during irradiation reduced the division delay experienced at the G₂-M interface. Implications of these effects are discussed.     

Chromosome aberrations induced in human lymphocytes by neutron irradiation.

In vitro dose--response curves of unstable chromosome aberrations in human lymphocytes have been obtained for neutron spectra of mean energies 0-7, 0-9, 7-6 and 14-7 MeV. The aberration yields have been fitted to the quadratic function Y = alphaD + betaD2, which is consistent with the single-track and two-track model of aberration formation. However with high-LET radiation, the linear component of yield, corresponding to damage caused by single tracks, predominants, and this term becomes more dominant with increasing LET, so that for fission spectrum neutrons the relationship is linear, Y = alphaD. At low doses, such as those recieved by radiation workers, limiting r.b.e. values between 13 and 47 are obtained relative to 60Co gamma-radiation. At higher doses, as used in radiotherapy, the values are much lower; ranging from 2-7 to 8 at 200 rad of equivalent gamma-radiation. Both sets of r.b.e. values correlate well with track-averaged LET but not with dose-averaged LET. When the numbers of cells without aberrations are plotted against radiation dose, curves are obtained which are similar in shape to those for conventional cell-survival experiments with comparable neutron spectra. The Do values obtained in the present study are close to those from other cell system.     

Chromosomal aberrations induced by (12)C6+ ions and 60Co gamma-rays in mouse immature oocytes

The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of (12)C6+ ion or (60)Co gamma-ray. Chromosomal aberrations were analyzed in metaphase II oocytes at 7 weeks after irradiation. The relative biological effectiveness (RBE) of (12)C6+ ion was calculated with respect to 60Co gamma-ray for the induction of chromosomal aberrations. The (12)C6+ ion and 60Co gamma-ray dose-response relationships for chromosomal aberrations were plotted by linear quadratic models. The data showed that there was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values for (12)C6+ ions relative to 60Co gamma-rays were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses of 0.5, 1.0, 2.0, 4.0 or 6.0 Gy, respectively. Moreover, a different distribution of the various types of aberrations has been found for (12)C6+ ion and 60Co gamma-ray irradiations. The dose-response relationships for (12)C6+ ion and 60Co gamma-ray exhibited positive correlations. The results from the present study may be helpful for assessing genetic damage following exposure of immature oocytes to ionizing radiation.     

Radioprotection of mouse colony forming units-spleen against heavy-charged particle damage by WR 2721

The effectiveness of S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR 2721) to protect against the heavy-charged particle beams with dose-averaged LET infinity's ranging from 26 to 260 keV/micron was studied using the marrow colony forming units-spleen as a model system. WR 2721 (400 mg/kg) was injected ip 30 min before whole-body irradiation in the plateau ionization region of the Bragg curve. Significant protection was observed at 26, 51, and 135 keV/micron LET values where the data were collected with 20Ne, 28Si, and 40Ar ions, respectively. The largest component of protection was the slope change, where at LET values of 26 and 51 keV/micron the DMFs (slope) were 2.1 and 2.3, respectively, which are very close to the gamma-ray value of 2.4 (gamma LET approximately equal to 0.2 keV/micron). Protection, however, decreased with increase in LET from 51 to 135 keV/micron to the DMF value of 1.2 and no significant protection was observed against 56Fe ions at 260 keV/micron. Significant increases in extrapolation number occurred with gamma rays and neon particles. The results are discussed in terms of charged particle track structure, radiation chemistry, and potential clinical applications.