Prolongation of life span associated with immunological modification by chronic low-dose-rate irradiation in MRL-lpr/lpr mice

Chronic low-dose-rate gamma irradiation at 0.35 or 1.2 mGy/h prolonged the life span of MRL-lpr/lpr mice carrying a deletion in the apoptosis-regulating Fas gene that markedly shortens life due to severe autoimmune disease. Immunological modifications as indicated by a significant increase of CD8(+) T cells and a significant decrease of CD3(+) CD45R/B220(+) as well as CD45R/B220(+) CD40(+) cells were found in parallel with amelioration of total-body lymphadenopathy, splenomegaly, proteinuria, and kidney and brain syndromes.     

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.     

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.     

A Study of the Regenerative Potential of Bone Marrow Cells of Donor Mice that Carry the <Emphasis Type="Italic">egfp</Emphasis> Gene in Irradiated Mice

A study of the regenerative potential of bone marrow cells of donor mice that express the enhanced green fluorescent protein was conducted in mice irradiated at a dose of 7 Gy. Expression of this protein allowed us to carry out monitoring of the presence of donor cells in recipient blood over the entire lifespan of the recipient. The lifespan of young recipients increased by 93% after transplantation; for old recipients it increased by 15%. Total acceptance of the bone marrow, spleen, thymus, and blood of the recipient with donor bone marrow cells was demonstrated over the entire life of the recipient. Only the donor colonies were detected with the studied irradiation dose and number of transplanted cells (11.7 ± 0.4) · 10⁶ on the spleen surface. The percentage of bone marrow and spleen cells that expressed the CD117 and CD34 stem cell markers in the recipient mice was above the control level for a long period of time after the irradiation. More than half of the cells with CD117, CD34, CD90.2, and CD45R/B220 phenotypes in the studied organs were donor cells. Further detailed study of the peculiarities of the engraftment of bone marrow cells, both without preliminary treatment of recipients and after the effects of extreme factors, will allow improvement of the methods of cell therapy.     

Recovery course in mouse spleen and bone marrow after continuous irradiation.

The paper deals with the recovery process of some parameters in the spleen and bone marrow till day 60 after continuous irradiation with a daily dose of 476.5 mGy (50 R), 957 mGy (100 R) and 4785 mGy (500 R) up to the total accumulated dose of 9570 mGy (1000 R). The recovery process in the spleen and bone marrow are relatively significant and completed till day 28 or 60 respectively after irradiation.     

Gene expression profiles in mouse liver after long-term low-dose-rate irradiation with gamma rays

Changes in gene expression profiles in mouse liver induced by long-term low-dose-rate γ irradiation were examined by microarray analysis. Three groups of male C57BL/6J mice were exposed to whole-body radiation at dose rates of 17-20 mGy/day, 0.86-1.0 mGy/day or 0.042-0.050 mGy/day for 401-485 days with cumulative doses of approximately 8 Gy, 0.4 Gy or 0.02 Gy, respectively. The gene expression levels in the livers of six animals from each exposure group were compared individually with that of pooled sham-irradiated animals. Some genes revealed a large variation in expression levels among individuals within each group, and the number of genes showing common changes in individuals from each group was limited: 20 and 11 genes showed more than 1.5-fold modulation with 17-20 mGy/day and 0.86-1.0 mGy/day, respectively. Three genes showed more than 1.5-fold modulation even at the lowest dose-rate of 0.04-0.05 mGy/day. Most of these genes were down-regulated. RT-PCR analysis confirmed the expression profiles of the majority of these genes. The results indicate that a few genes are modulated in response to very low-dose-rate irradiation. The functional analysis suggests that these genes may influence many processes, including obesity and tumorigenesis.     

Stem cell kinetics in spleen and bone marrow after single and fractionated irradiation of infant mice

Summary The number and type of stem cells in spleen and bone marrow of mice were determined after exposure to a single dose of 150 R on day 6, to a single dose of 500 R on day 6 or day 9 or to a fractionated dose of 150 R + 350 R on day 6 and 9. The stem cells were assayed on the basis of colony forming units (CFU) in spleen and of incorporation of iododeoxyuridine in spleen and bone marrow of lethally irradiated host mice. During the first month of life, the number of stem cells in non-irradiated mice increases markedly in bone marrow and slightly in spleen. Irradiation causes a long-lasting depression in stem cells, particularly in bone marrow and affecting preferentially erythropoietic precursor cells. Following a dose of only 150 R, the number of CFU in bone marrow is still below control levels 24 days later. An exposure to 500 R fractionated between day 6 and 9 has a markedly greater effect on stem cells in the spleen than the same dose given in a single application either at day 6 or 9.Supported by the Schutzkommission am Ministerium des Innern der BRD and contract B232-76-1BIOB of the Biology Division of the Commission of the European Community (Publikation No. 1727)     

Disorders in the immune responses of T- and B-cells in mice administered intravenous verotoxin 2

To obtain a better insight into the pathogenesis of verotoxin-producing Escherichia coli (VTEC)-associated diseases, we explored the effect of verotoxin 2 (VT2) on the immune response in mice. The distribution of lymphocyte phenotypes and the lymphocyte immune response were examined after intravenous administration of VT2 to mice. Among the peripheral lymphocytes and splenocytes of 4-week-old C57BL/6 mice, there was first of all a decrease in T-cells, which began 24 h after intravenous administration of VT2 (50 ng/kg, lethal dose). The CD4+ cell subpopulations of the peripheral blood and spleen were significantly decreased at 24 h, while the B220+ splenocyte subpopulation was markedly decreased at 45 h after VT2 administration. In the thymus, a decrease in CD4+CD8+ cells was predominantly observed near death. Interestingly, in E. coli lipopolysaccharide (LPS)-responder mouse strains (C57BL/6 and C3H/HeN) cotreated with LPS, the susceptibility to VT2 was enhanced, and the increase in B220+ cells induced by LPS alone was suppressed. Furthermore, splenocytes from C57BL/6 mice treated with VT2 (50 ng/kg) 6-24 h earlier reduced LPS-induced proliferative responses to 50-52% of that in control cells, indicating that the effect of VT2 on the immunoresponse seen in vivo may be negatively exerted on the proliferation of the cells. In addition, the number of splenocytes that produced anti-sheep red blood cell antibody was decreased in mice treated with VT2. These results suggest that VTEC infection may eliminate CD4+ and CD8+ T-cells and B-cells by affecting their survival and proliferative responses, leading to reduced antibody production.     

Low-dose total-body γ irradiation modulates immune response to acute proton radiation

Health risks due to exposure to low-dose/low-dose-rate radiation alone or when combined with acute irradiation are not yet clearly defined. This study quantified the effects of protracted exposure to low-dose/low-dose-rate γ rays with and without acute exposure to protons on the response of immune and other cell populations. C57BL/6 mice were irradiated with ??Co (0.05 Gy at 0.025 cGy/h); subsets were subsequently exposed to high-dose/high-dose-rate proton radiation (250 MeV; 2 or 3 Gy at 0.5 Gy/min). Analyses were performed at 4 and 17 days postexposure. Spleen and thymus masses relative to body mass were decreased on day 4 after proton irradiation with or without pre-exposure to γ rays; by day 17, however, the decrease was attenuated by the priming dose. Proton dose-dependent decreases, either with or without pre-exposure to γ rays, occurred in white blood cell, lymphocyte and granulocyte counts in blood but not in spleen. A similar pattern was found for lymphocyte subpopulations, including CD3+ T, CD19+ B, CD4+ T, CD8+ T and NK1.1+ natural killer (NK) cells. Spontaneous DNA synthesis by leukocytes after proton irradiation was high in blood on day 4 and high in spleen on day 17; priming with γ radiation attenuated the effect of 3 Gy in both body compartments. Some differences were also noted among groups in erythrocyte and thrombocyte characteristics. Analysis of splenocytes activated with anti-CD3/anti-CD28 antibodies showed changes in T-helper 1 (Th1) and Th2 cytokines. Overall, the data demonstrate that pre-exposure of an intact mammal to low-dose/low-dose-rate γ rays can attenuate the response to acute exposure to proton radiation with respect to at least some cell populations.     

DNA repair defect in AT cells and their hypersensitivity to low-dose-rate radiation

Ataxia telangiectasia (AT) and normal cells immortalized with the human telomerase gene were irradiated in non-proliferative conditions with high- (2 Gy/min) or low-dose-rate (0.3 mGy/min) radiation. While normal cells showed a higher resistance after irradiation at a low dose rate than a high dose rate, AT cells showed virtually the same survival after low- and high-dose-rate irradiation. Although the frequency of micronuclei induced by low-dose-rate radiation was greatly reduced in normal cells, it was not reduced significantly in AT cells. The number of gamma-H2AX foci increased in proportion to the dose in both AT and normal cells after high-dose-rate irradiation. Although few gamma-H2AX foci were observed after low-dose-rate irradiation in normal cells, significant and dose-dependent numbers of gamma-H2AX foci were observed in AT cells even after low-dose-rate irradiation, indicating that DNA damage was not completely repaired during low-dose-rate irradiation. Significant phosphorylation of ATM proteins was detected in normal cells after low-dose-rate irradiation, suggesting that the activation of ATM plays an important role in the repair of DNA damage during low-dose-rate irradiation. In conclusion, AT cells may not be able to repair some fraction of DNA damage and are severely affected by low-dose-rate radiation.     

The rate of progression of radiation-transformed mammary epithelial cells is enhanced after low-dose-rate neutron irradiation

Studies in this laboratory have shown enhancement of the mammary tumorigenic effects of neutron irradiation after low-dose-rate neutron exposures. To investigate possible reasons, a mammary cell system was used which allows quantitation of initiated mammary epithelial cells and examination of the progression of these radiation-altered cells toward the neoplastic phenotype. Female BALB/c mice were irradiated with fission-spectrum neutrons at dose rates of 1 rad/min or 1 rad/day. Twenty-four hours or 16 weeks after irradiation, mammary cells were obtained by enzymatic dissociation. Mammary outgrowths were derived by injection of 10(4) cells into gland-free fat pads of 3-week-old female BALB/c mice. The frequency of ductal dysplasias in outgrowths from cells irradiated at high or low dose rates was similar. Persistence of dysplasias differed markedly. Few of the dysplasias in outgrowths derived from cells irradiated at the high dose rate persisted, while a large fraction of the dysplasias in outgrowths derived from cells irradiated at low dose rate persisted. When cells remained in situ for 16 weeks prior to dissociation a higher frequency of persistent altered cells was also observed in outgrowths derived from cells irradiated at low neutron dose rates. These data suggest that low-dose-rate neutron exposures enhance the probability of progression of carcinogen-altered cells rather than increase the numbers of initiated cells.     

Influence of a sublethal irradiation on the immune response of the mouse to sheep erythrocytes]

In the CBA mice, the immunological response of the spleen cells (RFC and PFC direct and indirect) against the sheep erythrocytes is highly depressed by a 400 R dose of X rays. The recovery is not complete at the 30th day after irradiation. The response of the bone marrow cells either irradiated or unirradiated to the antigenic stimulation is very low.     

No lengthening of life span in mice continuously exposed to gamma rays at very low dose rates

Late effects of continuous exposure to ionizing radiation are potential hazards to workers in radiation facilities as well as to the general public. Recently, low-dose-rate and low-dose effects have become a serious concern. Using a total of 4000 mice, we studied the late biological effects of chronic exposure to low-dose-rate radiation as assayed by life span. Two thousand male and 2000 female 8-week-old specific-pathogen-free (SPF) B6C3F1 mice were randomly divided into four groups (one nonirradiated control and three irradiated). Irradiation was carried out for approximately 400 days using (137)Cs gamma rays at dose rates of 21 mGy day(-1), 1.1 mGy day(-1) and 0.05 mGy day(-1) with total doses equivalent to 8000 mGy, 400 mGy and 20 mGy, respectively. All mice were kept under SPF conditions until they died spontaneously. Statistical analyses showed that the life spans of mice of both sexes irradiated with 21 mGy day(-1) (P < 0.0001) and of females irradiated with 1.1 mGy day(-1) (P < 0.05) were significantly shorter than those of the control group. Our results show no evidence of lengthened life span in mice continuously exposed to very low dose rates of gamma rays.     

p53-Mediated gene activation in mice at high doses of chronic low-dose-rate γ radiation

The time course of the changes in the expression of p53-mediated genes in vivo after high doses of chronic low-dose-rate γ radiation remains unclear. Here we analyzed peripheral blood cell counts and the expression of p53-mediated genes in the spleens of mice chronically irradiated at low dose rate (0.0167 Gy/h) for 1-40 days. Low-dose-rate irradiation induced p53-dependent chronic decreases in white blood cell (WBC) counts in p53 wild-type mice. Upregulation of p53-mediated genes by low-dose-rate radiation was confirmed in the whole spleen cells from the p53 wild-type mice, while suppressed gene expression was observed in the spleen cells of p53-deficient mice. The expression of p21 and Bax in radiosensitive cells such as T and B lymphocytes from low-dose-rate irradiated mice at 10, 20, and 40 days were increased, although that of Mdm2 in both the lymphocytes was decreased at 20 and 40 days. Moreover, spleen weights for low-dose-rate irradiated mice were decreased at 20 and 40 days. Thus downregulation of Mdm2 in both T and B lymphocytes by low-dose-rate radiation may cause higher p53 activation; further, higher p53 expression may determine the radiosensitivity and cause a reduction in the spleen weights in low-dose-rate irradiated mice. These results indicate that p53 may be chronically activated by low-dose-rate radiation.     

Effect of continuous irradiation with a very low dose of gamma rays on life span and the immune system in SJL mice prone to B-cell lymphoma

Ionizing radiation has been shown to have dose- and dose-rate-dependent carcinogenic effects on the hematopoietic and lymphoreticular systems. We report here that continuous exposure to a low dose of gamma rays influences the course of spontaneous B-cell lymphoma in SJL mice. We studied the biological effects of 10 cGy year(-1) gamma rays on the life span of 560 4-week-old SJL/J female mice and on various parameters of the cell-mediated immune response. Life span was slightly prolonged. The mean survival was 397 days for controls and 417 days for irradiated mice that died with lymphoma (P = 0.34). In lymph nodes and spleen, lower percentages of CD4+ and CD8+ T cells were observed in irradiated mice before 32 weeks. Interestingly, the percentages of CD49+ NK cells were increased in the spleens of irradiated mice at 28 weeks (0.61 +/- 0.08% compared to 0.43 +/- 0.12% in controls, P = 0.01) and at 32 weeks (0.62 +/- 0.24% compared to 0.33 +/- 0.09%, P = 0.02), while NK cell activity remained unchanged in exposed mice. These results provide further support for the absence of harmful effects of a continuous very low dose of radiation on life span and incidence of lymphoma in SJL mice.     

Chromosome aberration frequencies and chromosome instability in mice after long-term exposure to low-dose-rate gamma-irradiation

Chronological changes of chromosome aberration rates related to accumulated doses in chronically exposed humans and animals at a low-dose-rate have not been well studied. C3H female specific pathogen-free mice (8 weeks of age) were chronically irradiated. Chromosome aberration rate in mouse splenocytes after long-term exposure to low-dose-rate (LDR) gamma-rays was serially determined by conventional Giemsa method. Incidence of dicentrics and centric rings increased almost linearly up to 8000 mGy following irradiation for about 400 days at a LDR of 20 mGy/day. Clear dose-rate effects were observed in the chromosome aberration frequencies between dose rates of 20 mGy/day and 200 Gy/day. Furthermore, the frequencies of complex aberrations increased as accumulated doses increased in LDR irradiation. This trend was also observed for the incidences of micronuclei and trisomies of chromosomes 5, 13 and 18 in splenocytes, detected by micronucleus assay and metaphase fluorescence in situ hybridization (FISH) method, respectively. Incidences of 2-4 micronuclei and trisomy increased in mouse splenocytes after irradiation of 8000 mGy at a LDR of 20 mGy/day. These complex chromosome aberrations and numerical chromosome aberrations seem to be induced indirectly after radiation exposure and thus the results indicate that continuous gamma-ray irradiation for 400 days at LDR of 20 mGy/day induced chromosomal instability in mice. These results are important to evaluate the biological effects of long-term exposure to LDR radiation in humans.     

Effects of a perfluorochemical emulsion on the response of BA1112 rat rhabdomyosarcomas to continuous low-dose-rate irradiation

The effects of the combination of a perfluorochemical emulsion (Fluosol DA, 20%) and carbogen (95% O2, 5% CO2) on the response of BA1112 rat rhabdomyosarcomas to continuous low-dose-rate irradiation were examined. Tumors were irradiated locally in unrestrained, unanesthetized rats at a dose rate of 0.98 Gy/h, using a specially designed 241Am irradiator system. Cell survival was measured using a colony formation assay. The tumor cell survival curves were fitted to linear relationships of the form ln S = - alpha D, where alpha for air-breathing rats was 0.104 +/- 0.005 Gy-1, as compared to 0.137 +/- 0.009 Gy-1 for rats treated with Fluosol plus carbogen. The increase in the slope of the survival curve produced by the treatment with Fluosol and carbogen was highly significant with a P value of 0.0015. The radiosensitization factor for the combination of Fluosol/carbogen plus continuous low-dose-rate irradiation was 1.32 +/- 0.11. Slightly less radiosensitization was observed with continuous low-dose-rate irradiation than in previous experiments using acute high-dose-rate irradiation. The diminished sensitization with Fluosol/carbogen during continuous low-dose-rate irradiation probably reflects the intrinsically lower oxygen enhancement ratio (OER) of low-dose/low-dose-rate irradiation, reoxygenation of the tumors during the prolonged treatment times used for continuous low-dose-rate irradiation, and the decrease in the levels of circulating perfluorochemicals during the 30-h irradiations. More importantly, the significant level of radiosensitization observed in the experiments with continuous low-dose-rate irradiation suggests that hypoxic cells persist in BA1112 tumors during continuous low-dose-rate irradiations and that the response of these tumors to continuous low-dose-rate irradiation can be improved by adjunctive treatments which oxygenate these radioresistant hypoxic tumor cells.     

Fractionated, low-dose-rate ionizing radiation exposure and chronic ulcerative dermatitis in normal and Trp53 heterozygous C57BL/6 mice

The influence of low-dose-rate chronic radiation exposure and adaptive responses on non-cancer diseases is largely unknown. We examined the effect of low-dose/low-dose-rate fractionated or single exposures on spontaneous chronic ulcerative dermatitis in Trp53 normal or heterozygous female C57BL/6 mice. From 6 weeks of age, mice were exposed 5 days/week to single daily doses (0.33 mGy, 0.7 mGy/h) totaling 48, 97 or 146 mGy over 30, 60 or 90 weeks, and other Trp53+/- mice were exposed to a single dose of 10 mGy (0.5 mGy/min) at 20 weeks of age. The 90-week exposure produced an adaptive response, decreasing both disease frequency and severity in Trp53+/+ mice and extending the life span of older animals euthanized due to severe disease. The 30- or 60-week exposures had no significant protective or detrimental effect. In contrast, the chronic, fractionated exposure for 30 or 60 weeks significantly increased the frequency and severity of the disease in older Trp53+/- mice, significantly decreasing the life span of the animals required to be euthanized for disease. Similarly, the single 10-mGy exposure also increased disease frequency in older animals. However, the chronic, fractionated exposure for 90 weeks prevented these detrimental effects, with disease frequency and severity not different from unexposed controls. We conclude that very low-dose fractionated exposures can induce a protective adaptive response in both Trp53 normal and heterozygous mice, but that a lower threshold level of exposure, similar in both cases, must first be passed. In mice with reduced Trp53 functionality, doses below the threshold can produce detrimental effects.     

Effect of continuous irradiation on the thrombopoietic system in mice

In this paper the changes of the megakaryocyte number of the spleen and those of thrombocyte number of the peripheral blood were studied during continuous irradiation with a dose rate of 9.57 to 957 mGy/day. Beginning with a dose rate of 95.7 mGy/day the thrombocyte number of the blood and the megakaryocyte number of the spleen of irradiated animals decreased significantly. Whereas the thrombocyte number remained permanently decreased, the cell number of the megakaryocyte type is increased temporarily to a clearly higher level as compared with the controls on the 100th day of irradiation approximately. This is especially true for the middle dose rate. During this time of irradiation nucleolar hyperchromatosis as well as pycnosis were observed in many megakaryocytes.