Lack of adaptive response of gamma radiation for protection against neutron-induced teratogenesis

BACKGROUND: Although there are some reports on neutron teratology, there is little information on the adaptive response of gamma radiation for protection against neutron‐induced teratogenesis. This study examined whether or not a low dose of gamma radiation can induce an adaptive response in mouse fetuses exposed to a subsequent dose of neutrons in vivo. METHODS: Pregnant ICR mice were exposed to a priming dose of 0.3 Gy (0.9 Gy/min) of gamma rays on day 10.5 of gestation and challenged with 0.8 Gy (0.94 Gy/minute) of neutrons 24 hlater. The mice were sacrificed on day 18.5 of gestation. The fetuses were examined for mortality, growth retardation, and other morphologic abnormalities. RESULTS: The tail length in the 0.3 Gy of gamma rays + 0.8 Gy of neutrons group was significantly shorter than in the 0.8 Gy of neutrons group. Although there was no significant difference compared with the 0.8 Gy of neutrons group, the number of live fetuses in the 0.3 Gy of gamma rays +0.8 Gy of neutrons group was lower. There was no evidence of primed exposure‐related reductions in the malformed fetuses. Although there was no significant difference compared with the unprimed group, the number of malformed offspring in the primed group was higher. Furthermore, the incidence of kinked tail and adactyly was significantly higher in the primed mice than in the unprimed mice. CONCLUSIONS: Overall, this study shows that exposure to 0.3 Gy of gamma rays failed to induce an adaptive response of fetogenesis to a neutron challenge dose. Birth Defects Res (Part B) 83:502‐506, 2008. © 2008 Wiley‐Liss, Inc.     

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.     

Activation of antioxidative enzymes induced by low-dose-rate whole-body gamma irradiation: adaptive response in terms of initial DNA damage

An adaptive response induced by long-term low-dose-rate irradiation in mice was evaluated in terms of the amount of DNA damage in the spleen analyzed by a comet assay. C57BL/ 6N female mice were irradiated with 0.5 Gy of (137)Cs gamma rays at 1.2 mGy/h; thereafter, a challenge dose (0.4, 0.8 or 1.6 Gy) at a high dose rate was given. Less DNA damage was observed in the spleen cells of preirradiated mice than in those of mice that received the challenge dose only; an adaptive response in terms of DNA damage was induced by long-term low-dose-rate irradiation in mice. The gene expression of catalase and Mn-SOD was significantly increased in the spleen after 23 days of the low-dose-rate radiation (0.5 Gy). In addition, the enzymatic activity of catalase corresponded to the gene expression level; the increase in the activity was observed at day 23 (0.5 Gy). These results suggested that an enhancement of the antioxidative capacities played an important role in the reduction of initial DNA damage by low-dose-rate radiation.     

Repair kinetics of gamma-ray induced DNA damage determined by the single cell gel electrophoresis assay in murine leukocytes in vivo

The repair kinetics of the gamma rays induced DNA damage was determined in murine peripheral blood leukocytes in vivo by the comet assay. Mice were exposed to 1.0 Gy of gamma rays in a 137Cs source and samples of peripheral blood were taken from their tails at different times. The repair was evaluated per mice in separate experiments by measuring the proportion of cells with tail (comets) in each sample. An average of nearly 80% of comets was obtained at the initial time after the exposure; 2 min later the frequency decreased to 45% and continued diminishing to 22% at 15 min. This evidences the presence of a rapid repair mechanism. For a period of 25 to 40 min after exposure there was a slight but consistent increase of comets from 22 to 38% followed by a second reduction, which could be due to a late repair process that causes strand breaks and then joined them. In summary our results indicated that this system seems to be appropriate for the study of the repair capacity of cells following exposure to ionizing radiation.     

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.     

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.     

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.     

mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (<Superscript>56</Superscript>Fe ions)

To date, there is scant information on in vivo induction of chromosomal damage by heavy ions found in space (i.e. ⁵⁶Fe ions). For radiation-induced response to be useful for risk assessment, it must be established in in vivo systems especially in cells that are known to be at risk for health problems associated with radiation exposure (such as hematopoietic cells, the known target tissue for radiation-induced leukemia). In this study, the whole genome multicolor fluorescence in situ hybridization (mFISH) technique was used to examine the in vivo induction of chromosomal damage in hematopoietic tissues, i.e. bone marrow cells. These cells were collected from CBA/CaJ mice at day 7 following whole-body exposure to different doses of 1 GeV/amu ⁵⁶Fe ions (0, 0.1, 0.5 and 1.0 Gy) or ¹³⁷Cs γ rays as the reference radiation (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min using a GammaCell40). These radiation doses were the average total-body doses. For each radiation type, there were four mice per dose. Several types of aberrations in bone marrow cells collected from mice exposed to either type of radiation were found. These were exchanges and breaks (both chromatid- and chromosome-types). Chromosomal exchanges included translocations (Robertsonian or centric fusion, reciprocal and incomplete types), and dicentrics. No evidence of a non-random involvement of specific chromosomes in any type of aberrations observed in mice exposed to ⁵⁶Fe ions or ¹³⁷Cs γ rays was found. At the radiation dose range used in our in vivo study, the majority of exchanges were simple. Complex exchanges were detected in bone marrow cells collected from mice exposed to 1 Gy of ⁵⁶Fe ions or 3 Gy of ¹³⁷Cs γ rays only, but their frequencies were low. Overall, our in vivo data indicate that the frequency of complex chromosome exchanges was not significantly different between bone marrow cells collected from mice exposed to ⁵⁶Fe ions or ¹³⁷Cs γ rays. Each type of radiation induced significant dose-dependent increases (ANOVA, P < 0.01) in the frequencies of chromosomal damage, including the numbers of abnormal cells. Based upon the linear-terms of dose-response curves, ⁵⁶Fe ions were 1.6 (all types of exchanges), 4.3 (abnormal cells) and 4.2 (breaks, both chromatid- and chromosome-types) times more effective than ¹³⁷Cs γ rays in inducing chromosomal damage.     

Characterization of the adaptive response to ionizing radiation induced by low doses of X rays to human lymphocytes

In previous studies we have shown that low doses of radiation from incorporated tritiated thymidine can make human lymphocytes less susceptible to the genetic damage manifested as chromatid breakage induced by a subsequent high dose of X rays. We have also shown that this adaptive response to ionizing radiation can be induced by very low doses of X rays (0.01 Gy; i.e., 1 rad) delivered during S phase of the cell cycle. To see if a low dose of X rays could induce this response in cells at other phases of the cell cycle, human lymphocytes were irradiated with 0.01 or 0.05 Gy before stimulation by phytohemagglutinin (G0) or with 0.01 Gy at various times after stimulation (G1), followed by 1.5 Gy (150 rad) at G2 phase. Although G0 lymphocytes failed to exhibit an adaptive response, G1 cells irradiated as early as 4 h after stimulation did show the response. Experiments were also carried out to determine how long the adaptive response induced by 0.01 Gy could persist. A 0.01-Gy dose was delivered to lymphocytes in the first S phase, followed by 1.5 Gy in the same or subsequent cell cycles. Lymphocytes receiving a 1.5-Gy dose at 40, 48, or 66 h after stimulation exhibited an adaptive response, whereas those receiving a 1.5-Gy dose at 90 or 114 h did not. Duplicate cultures containing bromodeoxyuridine showed that at 40 h all the lymphocytes were in their first cell cycle after stimulation, at 48 h half of the lymphocytes were in their first cell cycle and half in their second, and at 66 h 80% of the lymphocytes were in their third cell cycle. Thus the adaptive response persists for at least three cell cycles after it is induced by 0.01 Gy of X rays. In other experiments, the time necessary for maximal expression of the adaptive response was determined by delivering 0.01 Gy at hourly intervals 1-6 h before the 1.5-Gy dose. While a 4-h interval was enough for expression of the adaptive response, shorter intervals were not.     

低水平辐射诱导的细胞遗传学适应性反应

先用0.01GY x-射线(剂量率:0.01GY/分)体外照射人、兔外周血,经不同时间后再用1.5GY X-射线(0.44GY/分)照射,发现在G_0、G_1、S和G_2期受0.01GY X-射线照射后再给大剂量照射者,其染色体畸变率明显低于单纯受1.5GY X-射线照射组(P<0.01)。这一适应性反应能持续3个细胞周期,在接受小剂量照射后超过3个细胞周期再受大剂量照射者,染色体畸变率未见减少。若在第三细胞周期以后再次给予小剂量照射,可再次诱导适应性反应。用小鼠整体小剂量照射后骨髓细胞和生殖细胞亦出现这种适应性反应。另外也探讨了不同剂量和不同剂量率的预先照射对适应性反应的影响。     

Suppression of thymic lymphoma induction by life-long low-dose-rate irradiation accompanied by immune activation in C57BL/6 mice

The induction of thymic lymphomas by whole-body X irradiation with four doses of 1.8 Gy (total dose: 7.2 Gy) in C57BL/6 mice was suppressed from a high frequency (90%) to 63% by preirradiation with 0.075 Gy X rays given 6 h before each 1.8-Gy irradiation. This level was further suppressed to 43% by continuous whole-body irradiation with 137Cs gamma rays at a low dose rate of 1.2 mGy/h for 450 days, starting 35 days before the challenging irradiation. Continuous irradiation at 1.2 mGy/h resulting in a total dose of 7.2 Gy over 258 days yielded no thymic lymphomas, indicating that this low-dose-rate radiation does not induce these tumors. Further continuous irradiation up to 450 days (total dose: 12.6 Gy) produced no tumors. Continuously irradiated mice showed no loss of hair and a greater body weight than unirradiated controls. Immune activities of the mice, as measured by the numbers of CD4+ T cells, CD40+ B cells, and antibody-producing cells in the spleen after immunization with sheep red blood cells, were significantly increased by continuous 1.2-mGy/h irradiation alone. These results indicate the presence of an adaptive response in tumor induction, the involvement of radiation-induced immune activation in tumor suppression, and a large dose and dose-rate effectiveness factor (DDREF) for tumor induction with extremely low-dose-rate radiation.     

Micronucleus formation in the bone marrow cells of chronically irradiated mice with subsequent acute gamma irradiation]

With the use of the micronuclear test method it has been shown that mice preirradiated with gamma rays at a low dose rate exhibit a decreased frequency of chromosome aberrations induced in bone marrow cells by subsequent acute exposure to gamma radiation as compared to mice not subjected to preliminary irradiation. Such animals have a higher radioresistance with respect to the survival rate. The results obtained suggest the possibility of induction by ionizing radiation, at a low dose rate, of adaptive repair response at the organism level.     

Lung cancer risk in mice: analysis of fractionation effects and neutron RBE with a biologically motivated model

Data from Argonne National Laboratory on lung cancer in 15,975 mice with acute and fractionated exposures to gamma rays and neutrons are analyzed with a biologically motivated model with two rate-limiting steps and clonal expansion. Fractionation effects and effects of radiation quality can be explained well by the estimated kinetic parameters. Both an initiating and a promoting action of neutrons and gamma rays are suggested. While for gamma rays the initiating event is described well with a linear dose-rate dependence, for neutrons a nonlinear term is needed, with less effectiveness at higher dose rates. For the initiating event, the neutron RBE compared to gamma rays is about 10 when the dose rate during each fraction is low. For higher dose rates this RBE decreases strongly. The estimated lifetime relative risk for radiation-induced lung cancers from 1 Gy of acute gamma-ray exposure at an age of 110 days is 1.27 for male mice and 1.53 for female mice. For doses less than 1 Gy, the effectiveness of fractionated exposure to gamma rays compared to acute exposure is between 0.4 and 0.7 in both sexes. For lifetime relative risk, the RBE from acute neutrons at low doses is estimated at about 10 relative to acute gamma-ray exposure. It decreases strongly with dose. For fractionated neutrons, it is lower, down to about 4 for male mice.     

Cellular proto-oncogene expression following exposure of mice to gamma rays.

Many studies have shown the importance of altered cellular proto-oncogene expression in contributing to changes in cell survival, cell transformation, and cell cycle progression. In these experiments we examined the effects of total-body exposure of BCF1 mice to gamma rays (3 Gy) in modulating expression of cellular oncogenes in both gut and liver tissues. We selected specific cellular oncogenes (c-fos, c-myc, c-src, and c-H-ras), based on their normal expression in liver and gut tissues from untreated mice. As early as 5 min following whole-body exposure of BCF1 mice to gamma rays we detected induction of mRNA specific for c-src and c-H-ras in both liver and gut tissues. Accumulation of c-fos-RNA was slightly decreased in gut but was unaffected in liver tissue from irradiated mice relative to untreated controls. Accumulation of c-myc mRNA was unaffected in all tissues examined. These experiments document that modulation of cellular proto-oncogene expression can occur as an early event in tissues following irradiation and suggest that this modulation may play a role in radiation-induced cellular changes.     

Adaptive response in embryogenesis: V. Existence of two efficient dose-rate ranges for 0.3 Gy of priming irradiation to adapt mouse fetuses

The adaptive response is an important phenomenon in radiobiology. A study of the conditions essential for the induction of an adaptive response is of critical importance to understanding the novel biological defense mechanisms against the hazardous effects of radiation. In our previous studies, the specific dose and timing of radiation for induction of an adaptive response were studied in ICR mouse fetuses. We found that exposure of the fetuses on embryonic day 11 to a priming dose of 0.3 Gy significantly suppressed prenatal death and malformation induced by a challenging dose of radiation on embryonic day 12. Since a significant dose-rate effect has been observed in a variety of radiobiological phenomena, the effect of dose rate on the effectiveness of induction of an adaptive response by a priming dose of 0.3 Gy administered to fetuses on embryonic day 11 was investigated over the range from 0.06 to 5.0 Gy/min. The occurrence of apoptosis in limb buds, incidences of prenatal death and digital defects, and postnatal mortality induced by a challenging dose of 3.5 Gy given at 1.8 Gy/min to the fetuses on embryonic day 12 were the biological end points examined. Unexpectedly, effective induction of an adaptive response was observed within two dose-rate ranges for the same dose of priming radiation, from 0.18 to 0.98 Gy/ min and from 3.5 to 4.6 Gy/min, for reduction of the detrimental effect induced by a challenging dose of 3.5 Gy. In contrast, when the priming irradiation was delivered at a dose rate outside these two ranges, no protective effect was observed, and at some dose rates elevation of detrimental effects was observed. In general, neither a normal nor a reverse dose- rate effect was found in the dose-rate range tested. These results clearly indicated that the dose rate at which the priming irradiation was delivered played a crucial role in the induction of an adaptive response. This paper provides the first evidence for the existence of two dose-rate ranges for the same dose of priming radiation to successfully induce an adaptive response in mouse fetuses.     

Critical target and dose and dose-rate responses for the induction of chromosomal instability by ionizing radiation.

To investigate the critical target, dose response and dose-rate response for the induction of chromosomal instability by ionizing radiation, bromodeoxyuridine (BrdU)-substituted and unsubstituted GM10115 cells were exposed to a range of doses (0.1-10 Gy) and different dose rates (0.092-17.45 Gy min(-1)). The status of chromosomal stability was determined by fluorescence in situ hybridization approximately 20 generations after irradiation in clonal populations derived from single progenitor cells surviving acute exposure. Overall, nearly 700 individual clones representing over 140,000 metaphases were analyzed. In cells unsubstituted with BrdU, a dose response was found, where the probability of observing delayed chromosomal instability in any given clone was 3% per gray of X rays. For cells substituted with 25-66% BrdU, however, a dose response was observed only at low doses (<1.0 Gy); at higher doses (>1.0 Gy), the incidence of chromosomal instability leveled off. There was an increase in the frequency and complexity of chromosomal instability per unit dose compared to cells unsubstituted with BrdU. The frequency of chromosomal instability appeared to saturate around approximately 30%, an effect which occurred at much lower doses in the presence of BrdU. Changing the gamma-ray dose rate by a factor of 190 (0.092 to 17.45 Gy min(-1)) produced no significant differences in the frequency of chromosomal instability. The enhancement of chromosomal instability promoted by the presence of the BrdU argues that DNA comprises at least one of the critical targets important for the induction of this end point of genomic instability.     

Paternally inherited effects of gamma radiation on mouse preimplantation development detected by the chimera assay

It has previously been shown that type B spermatogonia in male mice treated with 0.05 Gy of X rays undergo an alteration expressed by progeny embryos as a cellular proliferation disadvantage in a chimera assay. We wished to obtain information on the assay's detection limit to ionizing radiation and on the radiosensitive target in male germ cells. Male mice were briefly irradiated with 137Cs gamma rays at nominal absorbed doses of 0.0, 0.0015, 0.005, 0.010, or 0.05 Gy and then mated for the next 8 weeks to untreated females. Four-cell embryos from treated males (experimental embryos) were paired with FITC-labeled embryos from untreated males (control embryos) to form aggregation chimeras. The chimeras were cultured for 30-40 h and examined under phase-contrast and UV illumination for the number of unlabeled cells (from the experimental embryo) and total chimera cell number, which were then expressed as "proliferation ratios" (No. unlabeled cells/total chimera cell No.). Significant decreases in proliferation ratios were observed at postirradiation weeks 4, 6, and 7 for the 0.01-Gy dose group and at weeks 5-6 for the 0.05-Gy dose group. In addition, significantly lower ratios were observed with early and mid four-cell embryos, but not with late four-cell embryos. These results suggest that mouse male germ cells express a radiosensitive target(s) whose detection limit by the assay lies at an absorbed dose between 0.005 and 0.010 Gy for brief gamma irradiation and whose effect on embryonic cell proliferation might decay by the second cleavage.     

The adaptive response modifies latency for radiation-induced myeloid leukemia in CBA/H mice.

We have investigated the effect of the adaptive response on acute myeloid leukemia (AML) induced in CBA/Harwell mice by a chronic radiation exposure. Groups of mice irradiated with a total dose of 1. 0 Gy at two different chronic dose rates (0.5, 0.004 Gy/h) had similar frequencies of AML. Compared to control animals that did not develop AML, irradiation at either of these dose rates did not change the longevity of the mice that did not die of leukemia. The survival rates of irradiated mice that did develop leukemia in the two groups were not different from each other, indicating that the dose rates produced similar responses and therefore were both chronic exposures. We then tested the ability of a chronic 10-cGy (0. 5 Gy/h) exposure to ionizing radiation, mild hyperthermia (40.5 degrees C whole-body, 60 min) or treatment with interleukin-1 (1500 U i.p.) to induce an adaptive response and modify the frequency or latency of AML which resulted from a subsequent (24 h later) 1.0-Gy (0.5 Gy/h) chronic radiation exposure. The frequency of radiation-induced leukemia was not changed in mice given any of the three adapting treatments 24 h prior to the chronic 1.0-Gy dose that induced leukemia. However, the latent period for development of AML was significantly increased by both the prior low radiation dose and mild hyperthermia treatment. Injection of interleukin-1, in contrast, may have reduced the latent period. Similar to the single 1.0-Gy chronic exposure alone, none of the adapting treatments prior to that exposure influenced the survival of animals that did not develop AML. These results indicate that an earlier exposure to a small adapting dose of radiation or to a mild heat stress can influence secondary steps in radiation-induced carcinogenesis.     

Threshold-like dose of local beta irradiation repeated throughout the life span of mice for induction of skin and bone tumors

The backs of female ICR mice were irradiated with beta rays from 90Sr-90Y three times a week throughout life. Previously we observed 100% tumor incidence at five different dose levels ranging from 1.5 to 11.8 Gy per exposure, but no tumor on repeated irradiation with 1.35 Gy for 300 days (Radiat. Res. 115, 488, 1988). In the present study, delay of tumor development was again seen at a dose of 1.5 Gy per exposure, with further delay at 1.0 Gy. The final tumor incidence was 100% with these two doses. At 0.75 Gy per exposure, no tumor appeared within 790 days after the start of irradiation, but one osteosarcoma and one squamous cell carcinoma did finally appear. These findings indicate a threshold-like response of tumor induction in this repeated irradiation system and further suggest that the apparent threshold may be somewhat less than 0.75 Gy per exposure.