The Estimation of Molecular and Cytogenetic Effects in Mice Exposed to Chronic Low Dose-Rate Gamma-Radiation

Molecular and cytogenetic parameters were estimated in male CBA/lac mice exposed to chronic low dose-rate -radiation (62 cGy/year) for 40, 80, 120, 210, and 365 days. After 40 days of exposure (6.7 cGy), spleen lymphocyte susceptibility to hydrogen peroxide was shown to increase. However, beginning from the day 120 of the treatment (20.4 cGy), the opposite effect was observed. An increase in number of the DNA–protein crosslinks was recorded in spleen lymphocytes only on day 40 of the experiment. The number of DNA breaks increased significantly beginning from day 120 of the experiment, as shown by the DNA-comet method. On the day 210 of irradiation, the frequency of abnormal sperm heads in the mice significantly increased. The number of normochromatic micronucleated erythrocytes of the peripheral blood remained unchanged.     

Changes in the number of DNA-protein cross links in spleen lymphocytes of mice exposed to low intensity low dose gamma irradiation

Levels of DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB) in spleen lymphocytes were studied in mice exposed to low-intensity gamma-radiation (1.7 mGy/day) for 1, 4, 10, 20, and 30 days. The spleen mass and count of lymphocytes isolated from this organ also has been investigated. The significant increase in the DPC level as compared to the control occurred on the 10-th and 30-th days of irradiation at doses of 1.7 and 5.1 cGy, accordingly. The number of spleen lymphocytes normalized to organ mass significantly decreased on the 4-th and 30-th days of the experiment. No increase was found in levels of alkali-labile sites and SSB. In contrast, the increase in the amount of duplex form DNA was recorded on the 4-th and 30-th days of the experiment. Our indicate that DPC formation after irradiation at low doses represents some form of cellular response to the damaging agent.     

The effect of chronic exposure to cadmium and gamma radiation at low doses on the genetic structures in mice

The DNA-protein cross-links (DPC) in mouse thymocytes and spleen lymphocytes, the number of abnormal sperm heads (ASH) and the number of micronuclei (MN) in normochromatic erythrocytes (NCE) of peripheral blood were studied in mice exposed to long-term low-intensity gamma radiation (0.072 cGy/days) and/or cadmium with drinking water (0.01 mg Cd2+/l) for 20, 40 and 80 days. The dependence of DPC level on the total dose (exposure time) of gamma radiation and/or cadmium is nonlinear. The maximal level of DPC in cells of lymphoid organs upon exposure to gamma radiation or cadmium was recorded on the 40-th day, and under combined exposure on the 20-th day of the experiment. The long-term exposure to cadmium or gamma radiation causes an increase in the ASH frequency. The increase in frequencies of MN in NCE and reciprocal translocations in spermatocytes was not found.     

Estimation of genetic effects of chronic exposure to low-dose rate gamma-radiation by cytogenetic methods and DNA-comet assay

The study of genetic effects in CBA/lac mice exposed for 1 year to constant low dose-rate gamma-radiation at a dose-rate 63 cGy/year has been carried out. We have shown the significant increase in the DNA breaks' level in spleen lymphocytes by comet-assay beginning from the total absorbed dose of 20 cGy. It is possible that the DNA breaks' level increase resulted from the structural rearrangement of chromatin or induction of lymphocyte proliferation. The results obtained by micronucleus test have proved that the mutagenic effect of chronic low dose-rate gamma-radiation depends on cell type and respectively on cell proliferation rate, cell differentiation, etc. So, by the end of experiment the significant increase in the frequency of PCE with micronuclei (MN) was observed. However, in contrast, the frequency of NCE with MN was not increased. No significant increase in the percent of lung cells with MN was registered.     

Effect of low-level irradiation on incidence rate and development of malignant neoplasms

Low-level irradiation (1.2-2.4 cGy, dose-rate 0.6 cGy/day) leads to a significant acceleration of the development of spontaneous leukosis in AKR mice: decrease in the average and the maximum life-span of animals leukosis-carriers (by 20 and 120 days, respectively) and an increase in the leukosis incidence rate (%). The introduction of antioxidant phenozan (beta-(4-hydroxy-3,5-ditertbutylphenyl) propionic acid) results in a considerable antileukosis effect: the average life-span increases by more than 40 days and the leukosis incidence rate decreases by 6%.     

Modulation of natural killer activity in mice by prolonged administration of various doses of dietary retinoids

Groups of BALB/C mice received a diet supplement of 0 (group C), 200 (group A 200), 500 (group A 500), and 1,000 (group A 1,000) IU retinol palmitate (RP)/mouse/day in drinking water for 450 days. At progressive time intervals, mice from each group were tested for natural killer (NK) activity and for the percentage of large granular lymphocytes (LGL) in the spleen. In groups A 200, A 500 and A 1,000, a dose-dependent increase in NK activity was evident 50 days after the beginning of RP supplementation and was accompanied by a parallel increase of LGL number in the spleen. In group A 1,000, the increase of spontaneous or Poly I:C-induced cytotoxicity persisted until day 160. By contrast, inhibition of Poly I:C-induced NK cytotoxicity was found in this group at day 450.     

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.     

A comparison of lung metastases and natural killer cell activity in daily fractions and weekly fractions of radiation therapy on murine B16a melanoma

C57BL/6J male mice were inoculated with 5 X 10(5) B16a melanoma cells. Seven days post-inoculation, when the tumor had grown to 8.0-10.0 mm in diameter, 120 tumor-bearing mice were randomly divided into three groups: (1) sham-irradiated controls, (2) mice receiving 200 cGy five times a week for 6 weeks, and (3) mice receiving 800 cGy once a week for 4 weeks. Thirty mice in each group were sacrificed 47 days postinoculation. Ten mice in each group were observed for the survival time data. The primary tumor was significantly smaller and the number of lung metastases were significantly fewer in mice treated with 800 cGy once a week compared to mice treated with 200 cGy five times a week. When natural killer (NK) cell activity was assessed against YAC-1 tumor targets, it was found to be significantly higher in mice treated with a single large weekly dose of irradiation. These results show that B16a melanoma responds more favorably to a single large dose of irradiation administered once a week compared to the smaller conventional fraction administered five times a week. This beneficial effect correlates with an increase in NK activity, indicating that there may be a causal relationship.     

Life shortening in mice exposed to fission neutrons and gamma rays. VIII. Exposures to continuous gamma radiation

Data are presented on the mean aftersurvival of male B6CF1 mice exposed for 22 h per day, 5 days per week, to 60Co gamma radiation at dose rates of 1.36 to 12.64 x 10(-3) cGy/min for 23 weeks or 1.36 to 6.32 x 10(-3) cGy/min for 59 weeks. For deaths from all causes, linear dose-response curves were obtained with slopes (days of life lost/cGy) of 0.158 +/- 0.016 and 0.077 +/- 0.002 for 23- and 59-week exposures, respectively. These values were not significantly altered when the analysis was restricted to those mice dying with tumors (92% of the total) or to those presumably dying from tumors (82% of the total). Analysis of mortality rates showed that about 90% of the radiation-specific excess mortality was tumor related. The 59-week exposure series induced only a small increase in the number of days of life lost/cGy/weekly fraction over that induced by 23 weeks of irradiation, 4.53 +/- 0.15 compared to 3.64 +/- 0.36 days lost/cGy/weekly fraction. This lower than expected value for 59 weeks of exposure may signal the approach to the final linear, additive, injury term postulated from earlier studies at this laboratory with low-dose-rate, daily, duration-of-life 60Co gamma irradiation.     

Thymus, peripheral lymphoid tissues and immunological responsiveness of the pituitary dward mouse (author's transl)]

Snell's pituitary dwarf mice (dw) were used for studies on the relationship between hypophysis and lymphoid organs. The age-dependent changes of thymus or spleen weights of dwarf mice were compared with those of normal littermates. The suppression of growth of the thymus or spleen in dwarf mice was recognized at 5th day of age. Although involution of the thymus varied among animals, a strong positive correlation was demonstrated between relative thymus weight and body weight in 30 approximately 40 days old dwarf mice. Lymphoid organs of dwarf mice were reconstituted by injection of growth hormone and or thyroxin. Relative thymus weight significantly increased in dwarf mice when the treatment with growth hormone started at 7 days of age, but the same treatment at 3 months of age did not show any effect on the increment of relative thymus weight. On the other hand, the antibody-forming capacitiy against sheep erythrocytes of dwarf mice was significantly increased even when the treatment with growth hormone was started at 3 months of age. A marked increase in the number of lymphoid cells in dwarf mice was observed by treatment with thyroxin, even if treatment was started either at 7 days or 3 months of age. Similar changes were also obtained in the antibody-forming capacity.     

Effects of low‐level 50 Hz magnetic fields on the level of host defense and on spleen colony formation

The results of 3 sets of experiments on the effects of 22 μT sinusoidal 50 Hz magnetic fields (MF), applied for 1 h on 5 successive days (1 h/5 days), on the level of host defense and on spleen colony formation are reported. The first set of experiments shows the effects on the number of colony‐forming units (CFUs) on the spleen and on the cellularity of the thymus in mice. The MF exposures resulted in an increase in CFUs which was statistically significant with respect to the controls, but not with respect to the shams. Statistically significant changes in the thymic weight and thymic index with respect to both the controls and the shams were measured 1 h after the last MF exposure. In the second set of experiments, the mice were given a sublethal dose of X‐rays (6 Gy), which was followed by exposure 2 h later to the MF. The MF exposure was repeated at the same time of day for 5 days. The number of colonies per spleen showed a consistent, statistically significant increase with MF exposure and the number of CFUs per femur was decreased. In the third set of experiments, bone marrow was taken from mice which had been exposed to 22 μT fields and injected into mice which had been exposed to a lethal dose of X‐rays (9 Gy). The number of CFUs per femur in the recipient mice was shown to be reduced by a statistically significant amount at 1 and 4 days after injection. Bioelectromagnetics 20:57–63, 1999. © 1999 Wiley‐Liss, Inc.     

Influence of chronic exposure to low doses of γ-radiation and <Superscript>90</Superscript>Sr on the level of DNA breaks and cell sensitivity to hydrogen peroxide in the mouse spleen

Using comet assay, a statistically significant increase (p < 0.05) in the level of DNA breaks in spleen cells was revealed in male CBA/lac mice exposed to γ-radiation (1.7 mGy/day) or ⁹⁰Sr (150–250 Bq/day) for 210 days. The level of DNA breaks also increased under combined exposure to both γ-radiation and ⁹⁰Sr (p < 0.05), but to a lesser degree than under exposure to each of these factors alone. Upon additional in vitro treatment of spleen cells with hydrogen peroxide, the relative increase in the level of DNA breaks was smaller in cells of irradiated mice than in the control. The ratio of the level of DNA breaks after hydrogen peroxide treatment to that before this treatment in control mice was 4.2 ± 0.9, compared to 1.4 ± 0.6 in γ-irradiated mice, 1.9 ± 0.8 in ⁹⁰Sr-irradiated mice, and 2.3 ± 0.8 in mice exposed to both γ- and ⁹⁰Sr-irradiation.     

Effect of small doses of ionizing radiation on the level of catecholamines and corticosteroids in mouse adrenals

Effects of 137Cs gamma-radiation (0.06-0.54 cGy, 0.06 cGy/day) on the levels of catecholamines and corticosteroids in mouse adrenals were investigated. There were observed increase of these parameters after mice irradiation during 1-2 days and their decrease after mice irradiation during 9 days.     

Response of a brachytherapy model using 125I in a murine tumor system

The effects of low-dose-rate irradiation (brachytherapy) were investigated in vivo using a murine mammary adenocarcinoma (MTG-B) growing in the flank of C3H mice. For local tumor irradiations, a noninvasive cap was devised to cover the tumor and house three 125I seeds (average apparent activity 5.2 mCi each) located at 120 degree intervals around the circumference of the hemispherical cap (13 mm i.d.). Mice were secured during treatment in a tube allowing limited mobility while restricting access to the seeds. Tumors were exposed to a series of dose rates ranging from 14-40 cGy/h, and the total dose over the treatment interval (48 or 72 h) ranged from 830 to 2378 cGy. A total of nine experiments were conducted using the caps over a 10-week interval. In each experiment three groups (irradiated tumors, sham controls, and untreated controls) were analyzed, each containing 8-15 mice (N = 34, untreated control; N = 46, sham control; N = 91, brachytherapy irradiation). The brachytherapy results are compared to the effects of external beam irradiation in the same tumor system. A linear relationship was observed between the total radiation dose and doubling volume growth delay (GDDV) or treatment volume growth delay (GDTV) for the brachytherapy and external beam irradiation. The slopes of the dose-response curves are steeper for the acute dose (517 cGy/min) external beam irradiation (0.0072 day/cGy, GDDV; 0.00695 day/cGy, GDTV) than for the brachytherapy (0.0050 day/cGy, GDDV; 0.0057 day/cGy, GDTV) using both GDTV and GDDV end points. Comparison of the tumor volume regrowth slopes indicates that the tumor bed effect is larger for external beam irradiation than for brachytherapy, suggesting that the tumor bed effect may be dose-rate dependent.     

Spontaneous leukosis as a model for an investigation of low and very low physical and physico-chemical effects on oncological process

Kinetic investigation of spontaneous leukosis in AKR mice have been carried in connection with a number of indices: changes in the mass of principal organs of the immune systems (thymus, spleen, lymphatic nodes), liver, alterations of haematological data (the sum of leukocytes, the percentage composition of blood cells, the quantity of undifferentiated cells), changes of physico-chemical conditions in cells (NMR-investigation). The dynamics of some of these indices and also life-spans of animals with leukosis after irradiation with doses 1.2-2.4 cGy (dose-rate 0.6 cGy/day) have been investigated. The enhancement of the frequency of leukosis and shortening the average and maximum life-spans of irradiated mice has been found.     

Effects of albendazole on Gnathostoma spinigerum in mice.

Mice were infected with 5 advanced third-stage larvae of Gnathostoma spinigerum and, beginning on the 28th day postinfection, were treated orally with albendazole. In the first experiment, infected mice each received albendazole once a day (30, 60, or 90 mg/kg/day) for 21 consecutive days. In the second experiment, they received albendazole twice a day (30 and 30, 60 and 60, or 90 and 90 mg/kg/day) for the same length of time. All mice were killed 28 days after cessation of treatment and the carcasses were examined for parasites. With both regimens, the administration of albendazole significantly reduced the number of larvae. However, a complete larvicidal effect was obtained only with albendazole at the dosage of 90 mg/kg twice daily.     

Pre- and post-implantation losses of embryos in aging female IVCS mice

Mated female mice of the IVCS strain, aged 90 (control group), 180, 210, 240, 270, 300, 330, 360 and 420 days old, were studied for pre- and post-implantation loss of embryos. The percentage of pre-implantation loss in mice aged 90 to 210 days was 1.7/11.8 (14.4%) to 2.7/11.7 (23.1%). In mice aged 240 to 300 days it increased significantly as compared to the controls (46.5-90.2% versus 14.4%). It reached 100% in 300-days-old mice. The post-implantation loss of embryos and/or fetuses in mice aged 90 to 240 days was 1.0/10.2 (9.8%) to 2.5/9.0 (27.8%). In mice aged 270 to 300 days it increased significantly compared to the controls (100% versus 9.8%). The decrease in reproductive activity appeared first in a decrease in litter size, followed by a decrease in the number of blastocyst and implantation sites, and finally by anovulation during the process of aging in IVCS mice.     

Expression and potential role of inducible nitric oxide synthase in the central nervous system of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. We examined the pathogenic roles of nitric oxide (NO) and inducible NO synthase (iNOS) in TMEV-induced demyelinating disease (TMEV-IDD). The presence of iNOS was confirmed in the spinal cords of TMEV-infected mice using immunohistochemical staining with anti-iNOS antibody on day 0 (control) and days 15, 30, 60, and 120. Aminoguanidine (AG), a specific inhibitor of iNOS, was injected intraperitoneally (ip) on 1, 3, 5, 8, 10, and 12 days post-TMEV inoculation as induction phase or 15, 17, 19, 22, 24, and 26 days as effector phase. Control animals in each experiment received phosphate-buffered saline (PBS) ip at similar time intervals. Few iNOS-positive cells were observed in the spinal cords of naive SJL/J mice. In the early phase (day 15) of TMEV-IDD, an increase of iNOS-positive cells was detected in the leptomeninges and perivascular space of the spinal cords. The number of iNOS-positive cells was increased and reached its peak on day 60, when histology of the animals showed peak infiltration with inflammatory cells. The clinical course of TMEV-IDD on each day postintracerebral infection was significantly reduced in mice treated with AG in the effector phase, and there was no significant difference between mice treated with AG in induction phase versus those administered PBS. Thus, NO production via iNOS appears to be a pathogenic factor in the effector phase of TMEV-IDD.     

Role of radioadaptation on radiation-induced thymic lymphoma in mice

Thymic lymphoma (TL) was observed in different stages of development in 46% of male mice (23/50) following exposure to an acute challenge dose of 2 Gy ⁶⁰Co γ-rays. With an adapting dose of 1 cGy 24 h prior to the challenge dose of 2 Gy, similar growth of TL was seen in 42.5% of mice (17/40). TL was not found in unirradiated control mice (0/50) or in the group treated with 1 cGy (0/50). Multiple adapting doses for 5 or 10 consecutive days induced TL in 8/50 and 9/50 mice, respectively (17% in average). When multiple adapting doses were followed by the challenge dose, the yield of TL was much lower, 16% (8/50) and 30% (15/30), respectively. By 15, 30, 60, 90, and 120 days after exposure with 3 Gy of ⁶⁰Co γ-rays, TL developed in 30, 70, 70, 80 and 85% of the female mice, respectively. When mice were conditioned with an adapting dose of 1 cGy 24 h prior to the challenge dose, TL was not found 15 days post-irradiation, while about a 25% reduction in the occurrence of TL was noticed at all other intervals. The results suggested that an adapting dose could play a role in bringing about a change in terms of delay and inhibition of the acute effects of radiation, i.e., the onset of TL in mice.