The estimation of molecular and cytogenetic effects in mice exposed to chronic low dose gamma-radiation

Molecular and cytogenetic parameters were estimated in male CBA/lac mice exposed to chronic low dose-rate gamma-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.     

Effects of Continuous Low Dose-Rate Irradiation: Computer Simulations

Abstract. The effects of continuous low dose-rate irradiation are studied with a computer model that incorporates cell kinetics and the accumulation and repair of radiation damage. This theoretical approach independently explores the effects on survival curves of a phase block, inherited damage and proliferation by dying cells. the computer model is a Monte Carlo simulation which follows the evolution in time of the family trees of a growing cell population under continuous irradiation. the model uses as input the measured phase-specific survival curves for acute exposures and the cell kinetic parameters to generate survival curves for continous low dose-rate irradiations. Cell survival curves for Chinese hamster lung cells (V79) for dose rates ranging from 15 to 500 cGy/h have been generated using various model assumptions. the model shows that for these cells a G₂ block will maximize cell killing for an optimum dose rate near 75 cGy/h. the effect on survival curves of inherited damage, as well as that of the proliferation by dying cells, is shown to increase monotonically with decreasing dose rates, and to be quite large at low dose rates.     

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.     

Effects of continuous low dose-rate irradiation: computer simulations

The effects of continuous low dose-rate irradiation are studied with a computer model that incorporates cell kinetics and the accumulation and repair of radiation damage. This theoretical approach independently explores the effects on survival curves of a phase block, inherited damage and proliferation by dying cells. The computer model is a Monte Carlo simulation which follows the evolution in time of the family trees of a growing cell population under continuous irradiation. The model uses as input the measured phase-specific survival curves for acute exposures and the cell kinetic parameters to generate survival curves for continuous low dose-rate irradiations. Cell survival curves for Chinese hamster lung cells (V79) for dose rates ranging from 15 to 500 cGy/h have been generated using various model assumptions. The model shows that for these cells a G2 block will maximize cell killing for an optimum dose rate near 75 cGy/h. The effect on survival curves of inherited damage, as well as that of the proliferation by dying cells, is shown to increase monotonically with decreasing dose rates, and to be quite large at low dose rates.     

Factors contributing to chromosome damage in lymphocytes of cigarette smokers

Cigarette smoking is generally believed to be responsible for a substantial number of human health problems. However, the causal relationship between smoking, the induction of biological effects and the extent of health problems among smokers have not been fully documented. Using the recently developed lymphocyte micronucleus (MN) assay, we have evaluated the chromosome aberration frequencies in 67 cigarette smokers and 59 matched non-smoking control subjects. We found that the mean MN frequency (per 100 cells) in the smokers was slightly higher than that found in the non-smokers (0.71 +/- 0.23 and 0.58 +/- 0.05 respectively; p less than 0.08). Factors which contribute to the expression of chromosome aberrations were also investigated. A significant age-dependent increase in MN frequencies was observed in both groups (p less than 0.05). Linear regression analysis showed that the age-dependent effects among smokers (r = 0.54; p less than 0.02) was further enhanced by cigarette consumption (r = 0.62; p less than 0.005). Consumption of low potency 'one-a-day' type multivitamins had no effect on MN frequencies in either sex of non-smokers and in the 1 male smoker who took multivitamins but vitamin intake consistently reduced the MN frequencies among female smokers. Using a challenge assay, fidelity of DNA repair was evaluated. Lymphocytes from both smokers and non-smokers were irradiated with single doses of 0 or 100 cGy of X-rays or with double doses of 100 cGy of X-rays each separated by 15 or 60 min (100/15 or 100/60). Chromosome translocation frequencies were consistently higher after irradiation in lymphocytes from smokers than in those from non-smokers. Statistically significant differences were detected when the cells were irradiated with the double doses of 100 cGy X-rays each separated by 60 min (p less than 0.05). These data suggest that lymphocytes from smokers made more mistakes in the repair of DNA damage than cells from non-smokers. Our studies provide new insights into the genotoxic effects of cigarette smoke and new information which may be useful for understanding the mechanisms for induction of health problems from smoking.     

The increase in the variability of microsatellite-associated repeats in the genome of gamma-irradiated male mice is tissue specific

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to measure the level of polymorphism of microsatellite (MCS)-associated repeating sequences of spleen, lung, and brain DNA in the F1 progeny of male BALB/c mice exposed to acute gamma-radiation at doses of 50 cGy and 200 cGy 15 days before mating with unirradiated females. The variability of MCS-associated sequences in the genome of brain and lung cells was higher as compared to the spleen cells of the progeny of unirradiated males. In the progeny of irradiated males, a 20% increase in MCS polymorphism of spleen DNA was found as an increase in the frequency of "non-parent" bands in DNA-fingerprints as against to the progeny of unirradiated males. Significant changes in this parameter were revealed for brain tissue and not for lung tissue only in the progeny of males exposed to 200 cGy. The results suggest a tissue-specific character of transmission of radiation-induced alterations in the genome of germ cells of male parents to the somatic cells of the progeny.     

The phenomenon of the single-strand DNA breaks level decrease in blood system cells in first generation chronically irradiated mice

For the first time has been shown experimentally that chronic low dose-rate gamma-radiation (0.04 mGy/hr) exposure leads to decrease of the single-strand DNA breaks level in spleen cells in 2.3 times (p < 0.001) and blood leukocytes in 6.1 times (p < 0.01), a decrease of the apoptotic cells frequency in 1.3 times (p < 0.05) and an increase in the spleen relative mass ratio B 1.2 times (p < 0.001) in CBA mice offspings (F1) from the chronically irradiated parents and exposed to chronic irradiation during the embryonic and postembryonic periods. A hypothesis about the more compact chromatin structure of blood system cells in the individuals of the first generation from chronically irradiated mice is proposed.     

Protective effect of curcumin on gamma-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes

The present work is aimed at evaluating the radioprotective effect of curcumin, a naturally occurring phenolic compound on gamma-radiation induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analysed by using cytokinesis blocked micronucleus assay and dicentric aberration (DC). The gamma-radiation at different doses (1, 2 and 4Gy) were found to significantly increase micronuclei (MN), DC frequencies and TBARS level whereas the levels of GSH and antioxidant enzymes were significantly decreased. The maximum damage to lymphocytes was observed at 4Gy irradiation. Curcumin pretreatment (1, 5 and 10microg/ml) significantly decreased the frequency of MN and DC. The levels of TBARS decreased and activities of SOD, CAT and GPx significantly increased along with GSH levels. At 1Gy irradiation all the concentrations of curcumin (1, 5 and 10microg/ml) significantly protected the lymphocytes from radiation damage. At 2Gy irradiation, 5 and 10microg/ml of curcumin showed significant radioprotection. Since the highest damage was observed at 4Gy irradiation both 1 and 5microg/ml of curcumin pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10microg/ml of curcumin significantly protected the cultured lymphocytes from radiation damage. Thus, pretreatment with curcumin gives protection to lymphocytes against gamma-radiation induced cellular damage.     

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.     

Dose-rate effect on transgenerational mutation frequencies in spermatogonial stem cells of the medaka fish

The estimation of transgenerational genetic risk of radiation exposure to non-human species is crucial for the protection of ecosystems. Here we determined the frequency of specific-locus mutations at the five pigmentation loci in medaka spermatogonial stem cells after gamma irradiation at 0.03 cGy/min and 95 cGy/min. At each total dose, the mutation frequency was significantly lower in the 0.03-cGy/min group than in the 95-cGy/min group, suggesting a dose-rate effect. The ratio of the induced mutation frequency at 0.03 cGy/min to that at 95 cGy/min was approximately 0.42 from 0 to 1.9 Gy and approximately 0.33 from 1.9 to 4.75 cGy. In the mouse, this ratio is estimated to be 0.33 (Russell and Kelly, Proc. Natl. Acad. Sci. USA 79, 542-544, 1982). It is thus possible that the magnitude of the dose-rate effect on transgenerational mutation frequencies is comparable between mouse and medaka spermatogonia, suggesting similar dose-rate effects among vertebrates.     

Specific-locus experiments show that female mice exposed near the time of birth to low-LET ionizing radiation exhibit both a low mutational response and a dose-rate effect

Female mice were exposed to 300 R of 73-93 R/min X-radiation either as fetuses at 18.5 d post conception (p.c.) or within 9 h after birth. Combining the similar results from these two groups yielded a specific-locus mutation frequency of 9.4 X 10(-8) mutation/locus/R, which is statistically significantly higher than the historical-control mutation frequency, but much lower than the rate obtained by irradiating mature and maturing oocytes in adults. Other females, exposed at 18.5 days p.c. to 300 R of 0.79 R/min gamma-radiation, yielded a mutation frequency that was statistically significantly lower than the frequency at high dose rates. The low-dose-rate group also had markedly higher fertility. It appears that the dose-rate effect for mutations induced near the time of birth may be more pronounced than that reported for mature and maturing oocytes of adults. A hypothesis sometimes advanced to explain low mutation frequencies recovered from cell populations that experience considerable radiation-induced cell killing is that there is selection against mutant cells. The reason for the relatively low mutational response following acute irradiation in our experiments is unknown; however, the finding of a dose-rate effect in these oocytes in the presence of only minor radiation-induced cell killing (as judged from fertility) makes it seem unlikely that selection was responsible for the low mutational response following acute exposure. Had selection been an important factor, the mutation frequency should have increased when oocyte killing was markedly reduced.     

Effects of exposure to low-dose-rate (60)co gamma rays on human tumor cells in vitro

Cells of three asynchronously growing human tumor cell lines, PC3 (human prostate carcinoma), T98G and A7 (human glioblastomas), which have been shown previously to demonstrate low-dose hyper-radiosensitivity to low acute single doses, were irradiated with (60)Co gamma rays at low dose rates (2 cGy-1 Gy h(-1)). Instead of a dose-rate sparing response, these cell lines demonstrated an inverse dose-rate effect on cell survival at dose rates below 1 Gy h(-1), whereby a decrease in dose rate resulted in an increase in cell killing per unit dose. A hyper-radiosensitivity-negative cell line, U373MG, did not demonstrate an inverse dose-rate effect. Analysis of the cell cycle indicated that this inverse dose-rate effect was not due to accumulation of cells in G(2)/M phase or to other cell cycle perturbations. T98G cells in reversible G(1)-phase arrest also showed an inverse dose-rate effect at dose rates below 30 cGy h(-1) but a sparing effect as the dose rate was reduced from 60 to 30 cGy h(-1). We conclude that this inverse dose-rate effect in continuous exposures reflects the hyper-radiosensitivity seen in the same cell lines in response to very small acute single doses.     

The micronucleus assay as a biological dosimeter in hospital workers exposed to low doses of ionizing radiation

The health risk associated with low levels of ionizing radiation is still a matter of debate. A number of factors, such as non-target effects, adaptive responses and low-dose hypersensitivity, affect the long-term outcome of low-dose exposures. Cytogenetic bio-dosimetry provides a measure of the absorbed dose, taking into account the individual radiation sensitivity. The aim of the present study is to evaluate the value of the micronucleus (MN) test as a bio-dosimeter in hospital workers exposed to low doses of ionizing radiation. Blood samples were obtained from 30 subjects selected among workers exposed to X- and gamma-radiation, and 30 controls matched for sex, age and smoking from the same hospital. Micronucleus frequencies were analyzed by use of the cytokinesis-block method. The MN frequency was compared among the groups considering the confounding factors and the length of employment. No increase in the number of bi-nucleated cells with MN (BNMN), but a significant increase in the number of mono-nucleated cells with micronuclei (MOMN) was observed in exposed subjects compared with the controls. The relationship between MN frequency and accumulated dose (mSv) was evaluated. The length of employment did not affect the extent of MN frequency, but an increase of BNMN and MOMN cells was observed based on the accumulated radiation dose. Our study shows the sensitivity of the MN test in the detection of cytogenetic effects of cumulative exposure levels, suggesting the potential usefulness of this assay in providing a biological index in medical surveillance programs.     

Relative biological effectiveness of fission neutrons for induction of micronucleus formation in mouse reticulocytes in vivo

Following whole-body irradiation of ICR mice with various doses of fission neutrons or X-rays, the frequency of micronuclei (MNs) in peripheral blood reticulocytes was measured at 12 h intervals beginning immediately after irradiation and ending at 72 h after irradiation. The resulting time-course curve of MN frequency had a clear peak 36 h after irradiation, irrespective of the type of radiation applied and the dose used. The MN frequency, averaged as the unweighted mean over the experimental time course, showed a linear increase with increasing dose of either fission neutrons or X-rays. The linear response to X-rays supports reported conclusion that induction of MN formation in reticulocytes is a dose-rate independent phenomenon. The relative biological effectiveness (RBE) of fission neutrons to X-rays for MN induction was estimated to be 1.9 +/- 0.3. This value is considerably lower than the RBE value of 4.6 +/- 0.5 reported for the same fission neutrons for induction of lymphocyte apoptosis in the thymus of ICR mice that represents dose-rate independent, one-track event. Based on these results, we propose that MNs increased in reticulocytes after irradiation mostly represent acentric fragments caused by single chromosome breaks, and that some confounding factor is operating in erythroblasts for the formation of aberrations from non-rejoining DNA double-strand breaks more severely after high-LET radiation than after low-LET radiation.     

Elevated sodium chloride concentrations enhance the bystander effects induced by low dose alpha-particle irradiation

Previous studies have shown that high NaCl can be genotoxic, either alone or combined with irradiation. However, little is known about the relationship between environmental NaCl at elevated conditions and radiation-induced bystander effects (RIBE). RIBE, which has been considered as non-targeted bystander responses, has been demonstrated to occur widely in various cell lines. In the present study, RIBE under the elevated NaCl culture condition was assessed in AG 1522 cells by both the induction of gamma-H2AX, a reliable marker of DNA double-strand break (DSB) for the early process (<1h post irradiation), and the generation of micronuclei (MN), a sensitive marker for relative long process of RIBE. Our results showed that in the absence of irradiation, NaCl at elevated concentration such as 8.0, 9.0 and 10.0g/L did not significantly increase the frequency of gamma-H2AX foci-positive cells and the number of foci per positive cell comparing with that NaCl at a normal concentration (6.8g/L). However, with 0.2cGy alpha-particle irradiation, the induced fraction of gamma-H2AX foci-positive cells and the number of induced gamma-H2AX foci per positive cell were significantly increased in both irradiated and adjacent non-irradiated regions. Similarly, the induction of MN by 0.2cGy alpha-particle irradiation also increased with the elevated NaCl concentrations. With N(G)-methyl-l-arginine, an inhibitor of nitric oxide synthase, the induced fraction of foci-positive cells was effectively inhibited both in 0.2cGy alpha-particle irradiated and adjacent non-irradiated regions under either normal or elevated NaCl conditions. These results suggested that the cultures with elevated NaCl medium magnified the damage effects induced by the low dose alpha-particle irradiation and nitric oxide generated by irradiation was also very important in this process.     

Quantitative characteristics of radiation injury of the spermatogenic epithelium and the rate of its recovery after exposure to fast neutrons and gamma-radiation

The paper submits the results of studies on the kinetics of spermatogenous epithelium cell number after exposure to fast neutrons (60-300 cGy) and gamma-radiation (200-600 cGy). It was shown that a relative decrease in the quantity of spermatocytes is determined by an exponential dose-response curve with D0 of 35 and 120 cGy for neutrons and gamma-radiation respectively. For spermatides and spermatozoa a single D0 value of 20 and 55 cGy was obtained for neutrons and gamma-radiation respectively. As the radiation dose increases the recovery process in the epithelium is substantially decelerated. The equation T1/2 = T1/2(0)e0.0009D well describes the dependence of the half-recovery period T1/2 upon the equivalent dose.     

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

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

An individual variability of the adaptive response to irradiation in human cells. Approach to its determination

On human blood lymphoxytes with micronuclei (MN) assay and cytokinetic cytochalasin block and analysis of chromosome aberrations the change of cell population composition, adaptive response (AR) and phenomenon of enhanced radiosensitivity after low dose (5 cGy) and challenge doses (1.0 Gy) have been studied. Irradiation have been carried out in G1 and G2 phases of cell cycle (24 h and 48 h after PHA stimulation). Fixation of cells have been conducted after 50 h (2 h after demecolcin adding) and 72 h (24 h after cytochalasin adding) chromosome and MN assay. Evaluation criteria were the frequency of binucleated cells with MN on 1000 binucleated cells and the frequency of cells with chromatid aberration on 100 metaphases. It was shown that cell population constitution change, AR occurring depended on the individual peculiarity. The evaluation of AR presence by the indexes of bimucleated cells with MN frequency and cells with chromatid aberrations don't coincide (coincidence is observed in 3 cases from 15). It is supposed that in G2 phase after irradiation in challenge dose the MN assay and metaphase analysis can register different cells (24 h and 2 h after mitotic block). The cell population constitution change can probably influence on the AR evaluation but in isn't the AR mechanism. The main mechanism of AR forming * the protection from the damages by different ways. AR depends on many factors, individual peculiarities observes by the use of definite evaluation criteria, in individuals with definite genetic constitution. Perhaps these considerations permit to discuss the problem of AR universality.     

Molecular-cellular characteristic of blood lymphocytes in Hodgkin lymphoma

The molecular-cellular parameters complex has been studied on the blood lymphocytes of malignant Hodgkin's lymphoma (HL) patients: the frequency of cells with micronuclei (MN) and chromosome aberrations; the level of DNA single and double strand breaks - OR and DR DNA (DNA comet assay), oxidative status--the content of reactive oxygen species (ROS) by using nonfluorescent dye that is oxygenated in the cells to fluorescent reagent and detection of fluorescence intensity after there. It was shown that the patients with LH had the increased level of DR and OR DNA, the increased frequency of cells with chromosome aberrations and the number of aberrations per cell was increased too. The concentration of ROS is increased too for the most individuals with intoxication. In the process of the chemical and radiation therapy the increase of OR DNA level, the frequency of the cell with MN has been registered. The ROS concentration correlates with the level of DNA-strand breaks. So the blood lymphocytes of HL patients before treatment differ from the lymphocytes of healthy donors. The damage of genome and the change of oxidative status have been observed that can be additive markers for the HL diagnosis, their sensitivity to the treatment and the characteristic of lymphocytes changes by this disease.