Radioadaptive response for protection against radiation-induced teratogenesis

To clarify the characteristics of the radioadaptive response in mice, we compared the incidence of radiation-induced malformations in ICR mice. Pregnant ICR mice were exposed to a priming dose of 2 cGy (667 muGy/min) on day 9.5 of gestation and to a challenging dose of 2 Gy (1.04 Gy/min) 4 h later and were killed on day 18.5 of gestation. The incidence of malformations and prenatal death and fetal body weights were studied. The incidence of external malformations was significantly lower (by approximately 10%) in the primed (2 cGy + 2 Gy) mice compared to the unprimed (2 Gy alone) mice. However, there were no differences in the incidence of prenatal death or the skeletal malformations or the body weights between primed and unprimed mice. These results suggest that primary conditioning with low doses of radiation suppresses radiation-induced teratogenesis.     

Adaptive response in embryogenesis. III. Relationship to radiation-induced apoptosis and Trp53 gene status

We reported previously that a radiation-induced adaptive response existed in the late period of embryogenesis, and that radiation-induced apoptosis in the predigital regions was responsible for digital defects in embryonic ICR mice. To investigate the possible involvement of the Trp53 gene and radiation-induced apoptosis in radiation-induced adaptive responses in embryogenesis, the present study was conducted using Trp53 wild-type (Trp53(+/+)) and Trp53 heterozygous (Trp53(+/-)) embryonic mice of the C57BL/6 strain. The existence of a radioadaptive response in the Trp53(+/+) embryonic mice was demonstrated by irradiating the embryos with 5 or 30 cGy on embryonic day 11 prior to a challenging irradiation at 3 Gy on embryonic day 12. The two conditioning doses at 5 and 30 cGy significantly suppressed the induction of apoptosis by the challenging dose in the predigital regions of limb buds in the Trp53(+/+) embryonic mice, while no such effect was found in the Trp53(+/-) embryonic mice. These findings indicate that induction of a radioadaptive response in embryogenesis is related to Trp53 gene status and the occurrence of radiation-induced apoptosis.     

The biological effects in animals in relation to the accident at the Chernobyl Atomic Electric Power Station. 9. The morphofunctional indices of the immunocompetent organs in mice]

The permanent action of small doses of low-intensity radiation on the immune status of 2.5-3.5 month CC57W mice has been investigated. Total doses of internal and external irradiation were about few cGy. The permanent action of low-level radiation on the experimental animals of the first and fourth generations was shown to change spleen and lymph nodes weights and the count of lymphocytes isolated from these organs. Cellularity and DNA synthesis in the lymph-node lymphocytes and their proliferative response to polyclonal mitogens also changed. The alterations in the parameters that characterized T-lymphocyte population were statistically significant.     

Characterization of radiation-induced emesis in the ferret

Forty-eight ferrets (Mustela putorius furo) were individually head-shielded and radiated with bilateral 60Co gamma radiation at 100 cGy min-1 at doses ranging between 49 and 601 cGy. The emetic threshold was observed at 69 cGy, the ED50 was calculated at 77 cGy, and 100% incidence of emesis occurred at 201 cGy. With increasing doses of radiation, the latency to first emesis after radiation decreased dramatically, whereas the duration of the prodromal period increased. Two other sets of experiments suggest that dopaminergic mechanisms play a minor role in radiation-induced emesis in the ferret. Twenty-two animals were injected either intravenously or subcutaneously with 30 to 300 micrograms/kg of apomorphine. Fewer than 50% of the animals vomited to 300 micrograms/kg apomorphine; central dopaminergic receptor activation was apparent at all doses. Another eight animals received 1 mg/kg domperidone prior to either 201 (n = 4) or 401 (n = 4) cGy radiation and their emetic responses were compared with NaCl-injected-irradiated controls (n = 8). At 201 cGy, domperidone significantly reduced only the total time in emetic behavior. At 401 cGy, domperidone had no salutary effect on radiation-induced emesis. The emetic responses of the ferret to radiation and apomorphine are compared with these responses in other vomiting species.     

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.     

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.     

Radiation damage repair capacity of a human germ-cell tumour cell line: inhibition by 3-aminobenzamide

The capacity of a human germ-cell tumour line to repair radiation damage has been investigated by means of a clonogenic assay. Dose-rate dependence studies, split-dose experiments and experiments designed to measure repair of potentially lethal damage have been performed. The cells showed some ability to repair radiation-induced damage in all three types of experiment. An attempt has been made to understand the possible cellular mechanisms of these repair processes by the use of 3-aminobenzamide (3-AB), an agent thought to act by inhibition of ADP-ribosylation. 3-AB added 2 h prior to and removed 18 h after irradiation at a non-toxic dose to unirradiated cells caused a small but consistent increase in cell kill with acute (150 cGy min-1) irradiation, largely involving a reduction in the shoulder region of the survival curve, but had a greater effect in increasing cell kill at a dose rate of 7.6 cGy min-1 and an even greater effect at a dose rate of 1.6 cGy min-1. When 3-Ab was present 2 h prior to the first dose and between two equal doses in a split-dose experiment, inhibition of split-dose recovery was observed. In addition, some inhibition of potentially lethal damage recovery was observed with 3-AB. A possible role for poly(ADP-ribosylation) is thus implicated in the repair of radiation-induced damage of this human tumour cell line during continuous low dose rate or fractionated radiation schedules, although other effects of 3-AB on respiratory metabolism and/or purine synthesis cannot be eliminated as the cause of the observed inhibitory effects.     

The effect of exogenous whole-body hyperthermia on humoral immunity]

We investigated the proliferative responses of spleen cells (SC) to polyclonal mitogens lipopolysaccharide (LPS) and pokeweed mitogen (PWM), immune responses to sheep red cells (SRC) in mice undergoing hyperthermia. There were increased proliferative responses of lymphocytes to PWM if we used mice having rectal temperature 42 degrees C. Thermal shock in mice was accompanied by suppression of immune response. If we used mice suffering from hyperthermia (43-44 degrees C) for 20 minutes; there were decreased proliferative responses of lymphocytes to PWM or LPS for 10-30 days. We observed low immune response to sheep red cells in mice for 5-20 days. The changes of immune response were not revealed on the 40th day after induction of hyperthermia in mice.     

A model of cytokine shedding induced by low doses of gamma radiation

A model for sustained shedding of epidermal growth factor (EGF) in response to low doses of gamma radiation was developed based on a time delay in the feedback from mitogen-activated protein kinase (MAPK) activation to metalloprotease activity in an autocrine signaling process. We determined the kinetic parameters of our model using the data available for MAPK activation by gamma irradiation in the 1-2-Gy dose range and then showed that predictions of the model were consistent with experimental results for the kinetics of EGF shedding into the growth medium after exposure of human mammary epithelial cells to 1-5 cGy of gamma radiation in the presence of antibodies that block ligand binding to EGF receptors. The model allowed us to estimate the rate of radiation-induced cytokine release per cell from measurements of EGF concentration in the growth medium and to assess the effectiveness of EGF shedding and subsequent diffusion through the medium as a mechanism for signal transmission between hit cells and bystanders.     

Promoter-enhanced neoplastic transformation after gamma-ray exposure at 10 cGy/day

We have measured gamma-ray-induced neoplastic transformation in C3H10T1/2 mouse embryo cells irradiated at an average 10 cGy/day throughout the useful life span of these cells for transformation studies. At cumulative total doses of 50, 150, 300, and 450 cGy, samples of cells were assayed for cell survival and neoplastic transformation with or without the administration of 0.1 micrograms/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) starting 24 h after the irradiation. The results indicate that, at a dose rate of 10 cGy/day, the rate of induction of neoplastic transformation is reduced by a factor of thirteen compared to that at 100 cGy/min. Still, frequencies above the background level are observed. These results are consistent with previous data which, at 144 cGy/day (0.1 cGy/min), showed that radiation-induced initiation events could be repaired during exposure, thus reducing the frequency of transformation from that observed at 100 cGy/min [A. Han et al., Cancer Res. 40, 3328-3332 (1980)]. Although the addition of TPA after the delivery of a particular dose at 10 cGy/day produced a significant increase in the frequency of neoplastic transformation, the degree of enhancement was less than after higher-dose-rate exposures [C.K. Hill et al., Radiat. Res. 109, 347-351 (1987)]. These results indicate that during 7 weeks of exposure, the repair of radiation-induced initiation was extensive but not complete, and suggest that a significant part of the damage persists which can be promoted by TPA. These observations support the inference that initiation and promotion are not tightly coupled and are probably independent processes.     

Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts

In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities.     

Suppression of antigen-specific lymphocyte activation in modeled microgravity

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.     

Effect of low doses of low-intensity irradiation on the incidence and progress of spontaneous leukosis in AKR mice

Development of spontaneous leukosis in AKR mice is accelerated by irradiation with low doses (1.2-2.4 cGy, dose-rate 0.6 cGy/day): the leukoses incidence rate increases, deaths of the animals from leukosis occurs earlier, shortening the average and maximum life-spans of the animals. The dynamics of changes in the mass of organs of the immune systems (thymus and spleen) shown extrema. The moment of reaching the extremum correlates with the maximum rate of animals' deaths.     

Polyclonal activation of murine B lymphocytes by immune complexes

Murine splenic B lymphocytes are stimulated by homologous immune complexes to proliferate and secrete polyclonal antibody. The use of antibody from whole serum or monoclonal antibodies to form complexes resulted in the stimulation of mouse B lymphocytes. The ratio of antibody to antigen appears to be critical for the generation of the polyclonal antibody response. Because antigen and antibody are added independently at culture initiation, the exact nature of the complex is unknown, but optimal polyclonal antibody formation occurs in slight antigen excess. Immune complex-induced polyclonal antibody production requires the presence of both macrophages and T cells, whereas B cell proliferation requires only macrophages. The role of the macrophage appears to be to cleave a low m.w. (17,000) fragment from the complex, which is responsible for lymphocyte activation.     

Pathology effects at radiation doses below those causing increased mortality

Mortality data from experiments conducted at the Argonne National Laboratory (ANL) on the long-term effects of external whole-body irradiation on B6CF(1) mice were used to investigate radiation-induced effects at intermediate doses of (60)Co gamma rays or fission-spectrum neutrons either delivered as a single exposure or protracted over 60 once-weekly exposures. Kaplan-Meier analyses were used to identify the lowest dose in the ANL data (within radiation quality, pattern of exposure, and sex) at which radiation-induced mortality caused by primary tumors could be detected (approximately 1-2 Gy for gamma rays and 10-15 cGy for neutrons). Doses at and below these levels were then examined for radiation-induced shifts in the spectrum of pathology detected at death. To do this, specific pathology events were pooled into larger assemblages based on whether they were cancer, cardiovascular disease or non-neoplastic diseases detected within the lungs and pleura, liver and biliary tract, reproductive organs, or urinary tract. Cancer and cardiovascular disease were further subdivided into categories based on whether they caused death, contributed to death, or were simply observed at death. Counts of how often events falling within each of these combined pathology categories occurred within a mouse were then used as predictor variables in logistic regression to determine whether irradiated mice could be distinguished from control mice. Increased pathology burdens were detected in irradiated mice at doses lower than those causing detectable shifts in mortality-22 cGy for gamma rays and 2 cGy for neutrons. These findings suggest that (1) models based on mortality data alone may underestimate radiation effects, (2) radiation may have adverse health consequences (i.e. elevated health risks) even when mortality risks are not detected, and (3) radiation-induced pathologies other than cancer do occur, and they involve multiple organ systems.     

Adaptive responses to low-dose/low-dose-rate gamma rays in normal human fibroblasts: the role of growth architecture and oxidative metabolism

To investigate low-dose/low-dose-rate effects of low-linear energy transfer (LET) ionizing radiation, we used gamma-irradiated cells adapted to grow in a three-dimensional architecture that mimics cell growth in vivo. We determined the cellular, molecular and biochemical changes in these cells. Quiescent normal human fibroblasts were irradiated with single acute or chronic doses (1-10 cGy) of (137)Cs gamma rays. Whereas exposure to an acute dose of 10 cGy increased micronucleus formation, protraction of the dose over 48 h reduced micronucleus frequency to a level similar to or lower than what occurs spontaneously. The protracted treatment also up-regulated the cellular content of the antioxidant glutathione. These changes correlated with modulation of phospho-TP53 (serine 15), a stress marker that was regulated by doses as low as 1 cGy. The DNA damage that occurred after exposure to an acute dose of 10 cGy was protected against in two ways: (1) up-regulation of cellular antioxidant enzyme activity by ectopic overexpression of MnSOD, catalase or glutathione peroxidase, and (2) inhibition of superoxide anion generation by flavin-containing oxidases. These results support a significant role for oxidative metabolism in mediating low-dose radiation effects and demonstrate that cell culture in three dimensions is ideal to investigate radiation-induced adaptive responses. Expression of connexin 43, a constitutive protein of gap junctions, and the G(1) checkpoint were more sensitive to regulation by gamma rays in cells maintained in a three-dimensional than in a two-dimensional configuration.     

Serum-mediated suppression of nonspecific B-cell activation. III. Selective inhibition of the polyclonal B-cell response by normal mouse serum

Normal, nonimmune adult serum is known to inhibit in vitro immune responses when present in sufficient amounts. The significance of inhibition of the immune response by serum, however, is not known. Previous work suggested that normal mouse plasma or serum (NMS) was selectively more inhibitory to nonantigen-specific (e.g., polyclonal) as compared to antigen-specific responses. This led to the hypothesis that constituents of serum (or plasma) may serve naturally to minimize the polyclonal type of antibody response, preserving immune specificity. The present study further examined the effect of NMS on polyclonal versus antigen-specific antibody responses. Under the in vitro assay conditions used, 0.5% NMS supported bacterial endotoxin (ET)-induced mitogenic and polyclonal B lymphocyte responses, antigen (SRBC, TNP-KLH)-specific antibody (IgM, IgG) responses, and antigen-induced or -specific T-lymphocyte proliferative responses, while 5% NMS inhibited all of these responses. However, antigen-specific T-lymphocyte responses could be restored by a 10-fold increase in the antigen concentration and antigen-specific antibody responses could be restored by the addition of ET (10 micrograms/ml) as adjuvant. On the other hand, the mitogenic response to ET remained suppressed regardless of ET concentration. Thus, despite significant reduction of the mitogenic and polyclonal properties of ET in 5% NMS (greater than 70% suppression), sufficient antigenic stimuli permitted optimal specific T- and B-cell responses. Many naturally occurring antigens, e.g., bacterial, fungal, and viral, have inherent B-cell mitogenic and polyclonal activity in addition to adjuvanticity and the presence of the serum inhibitory factor may serve to minimize their indiscriminate polyclonal stimulation of antibody.     

Standardization of a fluorometric assay for measuring oxidative stress in irradiated cells

The present study was undertaken to standardize a dichlorofluorescein (DCF) assay for measurement of radiation-induced oxidation of dichlorofluorescin (DCFH) substrate in MCF-10 cells. This assay was highly sensitive and capable of detecting increased DCFH oxidation in the cells exposed to gamma radiation at doses as low as 1.5 cGy with linear dose-response curves. However, the slope of the dose-response curves varied considerably from one experiment to another and was influenced by the fluorescent substrate concentration and cell density. To make the assay reproducible so that results obtained from different experiments could be compared, a series of conversion factors and equations have been established to normalize the data for these variables. The results demonstrate that the DCF assay, as standardized in the present study, is highly reproducible with acceptable assay precision. The normalized results can be compared from one experiment to another even when the experiments were performed using different fluorescent substrate concentrations and/or cell densities. Since changes in DCFH oxidation may be related to changes that are indicative of oxidative stress in cells, this assay can be useful to quantify radiation-induced oxidative stress and evaluate the efficacy of antioxidant agents in protection against radiation-induced oxidative stress.