The low dose and low dose rate cytogenetic effects induced by gamma-radiation in human blood lymphocytes in vitro. II. the results of cytogenetic study

The yield of chromosome aberrations induced by gamma-radiation of 60Co in human blood lymphocytes in vitro at low doses (30 divided by 600 mGy) and low dose rates (0.70, 5.05, 59.2 mGy/min) was investigated. It was found that the observed level of chromosomal aberrations induced by gamma-irradiation was unaffected by the value of the dose rate when using constant dose rate and obtaining different doses by altering the exposure time. However, a relatively enhanced level of chromatid aberrations was found at 5.05 and 59.2 mGy/min dose rates in the dose range less than 250 mGy. We have found that the observed level of the sum of chromosomal aberrations induced by gamma-irradiation at doses less than 250 mGy and a dose rate of 59.2 mGy/min was essentially larger compared with the level extrapolated from high doses (above 300 mGy) using a linear-quadratic dose curve. This complied with our previous finding in 1976, 1977 when the enhanced level of dicentrics was only found at a high dose rate approximately 500 mGy/min. Such a non-linear cytogenetic effect does not manifest itself statistically significantly at dose rates of 0.70 and 5.05 mGy/min for the sum of chromosomal aberrations and does not manifest itself at all for dicentrics at all the examined dose rates.     

The low dose and low dose rate cytogenetic effects induced by gamma-radiation in human blood lymphocytes in vitro. I. the results of physical and dosimetric study

Physical and dosimetric characteristics of the gamma-radiation field which is formed in the room containing the 60Co radiation source were investigated on condition of an unclosed breech mechanism and the presence of the lead layer on the beam pathway. The inverse square law was approximately found for the dependence of the dose rate vs distance on the radiation source both for the unclosed breech mechanism and lead layer (4 and 9 cm wide) in the beam pathway. This finding indicated a non-significant contribution of the radiation scattered from the walls at the point of cytogenetic experiments. The Monte Carlo calculations showed that some changes in the efficient spectrum of gamma-radiation resulted in a decreased average energy of 60Co gamma-rays to 1.03, 1.17 and 1.07 MeV for the unclosed breech mechanism, behind 4 and 9 cm lead layers, respectively.     

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.     

Cause of death and neoplasia in mice continuously exposed to very low dose rates of gamma rays

Four thousand 8-week-old SPF B6C3F1 mice (2000 of each sex) were divided into four groups, one nonirradiated (control) and three irradiated. The irradiated groups were exposed to (137)Cs gamma rays at dose rates of 21, 1.1 and 0.05 mGy day(-1) for approximately 400 days with total doses equivalent to 8000, 400 and 20 mGy, respectively. All mice were kept until natural death, and pathological examination was performed to determine the cause of death. Neoplasms accounted for >86.7% of all deaths. Compared to the nonirradiated controls, the frequency of myeloid leukemia in males, soft tissue neoplasms and malignant granulosa cell tumors in females, and hemangiosarcoma in both sexes exposed to 21 mGy day(-1) were significantly increased. The number of multiple primary neoplasms per mouse was significantly increased in mice irradiated at 21 mGy day(-1). Significant increases in body weights were observed from 32 to 60 weeks of age in males and females exposed to 1.1 mGy day(-1) and 21 mGy day(-1), respectively. Our results suggest that life shortening (Tanaka et al., Radiat. Res. 160, 376-379, 2003) in mice continuously exposed to low-dose-rate gamma rays is due to early death from a variety of neoplasms and not from increased incidence of specific neoplasms.     

The effects of low doses of low-level gamma-radiation and phenozan injection on structural characteristics of mice spleen DNA

It is well known that AKR mice with spontaneous leucosis are more sensitive to ionizing irradiation as compared to normal F1 (CBA x C57BL) mice. A study on changes of the structural characteristics of spleen DNA and level of protein p53 in the blood serum under the action of low-level gamma-irradiation in a dose of 1.2 cGy and injections of 10(-14) or 10(-4) mol/kg phenozan was performed. The changes in the structural characteristics of DNA (the adsorption on nitrocellulose filters and number of double-strand breaks) and p53 content were observed for each line of mice under gamma-irradiation and each phenozan concentration. Both factors showed long-time post-effects, and structural changes in AKR DNA were consistent with the life span of these mice. Phenozan in the above doses has abolished the induction of double-strand breaks in case of irradiation of F1 mice in a dose of 1.2 cGy and showed long-time post-irradiation effect. These facts suggest a radioprotection property of phenozan.     

In vivo cytogenetic studies on mice exposed to ethylene dibromide

The pesticide, ethylene dibromide (EDB), was evaluated with in vivo cytogenetic assays to determine its genotoxicity. CD1 male mice were exposed to EDB through intraperitoneal injections. Bone marrow cells isolated from femora were analyzed for sister-chromatid exchange (SCE), chromosome aberration and micronucleus formation. The results showed that only certain concentrations of EDB tested caused a slight but significant increase in SCEs and chromosome aberrations. However, these increases were not dose-related. No increase in the polychromatic erythrocytes with micronuclei was observed following EDB exposure. Also, EDB did not cause cell-cycle delay in comparison with controls. Thus, it appears that EDB is not an effective genotoxic agent in vivo in mice.     

AP-PCR assay of DNA alterations in the progeny of male mice exposed to low-level gamma-radiation

By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-doses of gamma-radiation was investigated. Male BALB/c mice exposed to 10-50 cGy were mated with unirradiated females 15 days after irradiation. DNA was isolated from biopsies taken from tail tips of 2-month-old progeny. Preliminary AP-PCRs were carried out with 17 primers representing core sequences of micro- and/or minisatellites or their flanking oligonucleotides. Best quantitatively reproduced AP-PCR fingerprints of genomic DNA were obtained with one of these primers, a 20-mer oligonucleotide flanking the micro-satellite locus Atplb2 on mouse chromosome 11. Comparative analysis of individual fingerprints of AP-PCR products obtained on DNA templates from the progeny of irradiated and intact males revealed an increased variability of micro-satellite-associated sequences and an increased frequency of "non-parental bands" in DNA-fingerprints from the progeny of males chronically exposed to gamma-radiation 15 days before mating (at the postmeiotic stage of spermatogenesis). The results show that increased micro-satellite instability can be initiated by irradiation of the male parent to subsequently arise or be transmitted to the soma of the F1 generations.     

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.     

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.     

The effect of low dose rate on metabolomic response to radiation in mice

Metabolomics has been shown to have utility in assessing responses to exposure by ionizing radiation (IR) in easily accessible biofluids such as urine. Most studies to date from our laboratory and others have employed γ-irradiation at relatively high dose rates (HDR), but many environmental exposure scenarios will probably be at relatively low dose rates (LDR). There are well-documented differences in the biologic responses to LDR compared to HDR, so an important question is to assess LDR effects at the metabolomics level. Our study took advantage of a modern mass spectrometry approach in exploring the effects of dose rate on the urinary excretion levels of metabolites 2 days after IR in mice. A wide variety of statistical tools were employed to further focus on metabolites, which showed responses to LDR IR exposure (0.00309 Gy/min) distinguishable from those of HDR. From a total of 709 detected spectral features, more than 100 were determined to be statistically significant when comparing urine from mice irradiated with 1.1 or 4.45 Gy to that of sham-irradiated mice 2 days post-exposure. The results of this study show that LDR and HDR exposures perturb many of the same pathways such as TCA cycle and fatty acid metabolism, which also have been implicated in our previous IR studies. However, it is important to note that dose rate did affect the levels of particular metabolites. Differences in urinary excretion levels of such metabolites could potentially be used to assess an individual’s exposure in a radiobiological event and thus would have utility for both triage and injury assessment.     

The characteristics of the realization of cytogenetic damage in mammalian and plant cells exposed to low doses of radiation]

A complicated character of the cytogenetic injury dependence upon radiation dose was revealed after low-level gamma irradiation of Vicia faba seedlings and Chinese hamster fibroblasts. The dependence was linear with low-level secondary exposure to 70 GeV protons. The authors discuss a threshold nature of induction of the cytogenetic damage repair responsible for a high outcome of damages under the effect of low-level gamma radiation.     

Membrane structure alterations in rat blood lymphocytes under external low dose rate gamma-radiation exposure

The influence of a 0.72 cGy/day dose rate of gamma-radiation on plasma membranes of peripheral blood lymphocytes of rats exposed to the doses of 1.5, 15, 30, 60 and 100 cGy was studied. Parameters characterizing the viscosity and the polarity of lipid bilayer and also an external membrane surface properties were examined using fluorescent probes pyrene and 1-anilinonaphthalene-8-sulfonate (ANS). Was shown the membrane structural parameters alterations after animal exposure to the doses of 1.5, 15, 60 and 100 cGy, being of a nonmonotonous nature as the dose accumulated. After exposure to the doses lower then than 30 cGy spectral changes were revealed not in each particular experiment that was probably caused by the individual peculiarities of radiation response development. After exposure to the doses higher than 30 cGy the changes were of reproducible character. After a 1.5 cGy dose a slight lipid bilayer polarity decrease and ANS binding parameter multidirectional changes were observed. After exposure to 15, 60 and to 100 cGy was shown polarity elevation and repartition of polar groups within the bilayer, the increase of viscosity of more polar membrane regions and also ANS fluorescence reduction mostly at the expense of quantum yield decrease. After the exposure of 60 cGy was observed a viscosity decrease in hydrophobic regions along with viscosity increase in more polar regions and after a 100 cGy dose accumulation an essential surface charge shift was found. Revealed alterations indicate the reorganization of external membrane surface and of intensification of oxidative processes in lipid bilayer.     

Comparative evaluation of the carcinogenic effects of chronic exposure to tritium oxide and external gamma-radiation

In experiments of on rats a study was made of late effects of chronic action of tritium oxide (3HOH) during 6 months (37 X 10(4) Bq/g/day) and external gamma-radiation (137Cs) which were delivered in comparable daily and cumulative doses. It was shown that 3HOH produced a more pronounced blastomogenic effect. The RBE coefficient of tritium oxide approximated 1, with a reference to the average life shortening, and 4.2 and 2.5, with a reference to the incidence of malignant tumors and leukoses, respectively.     

Genotoxic and cytotoxic effects in the bone marrow of rats exposed to a low dose of paraquat via the dermal route

The genotoxic effect of the herbicide paraquat was studied in rat bone-marrow by means of the micronucleus assay. Paraquat at dose levels of 6, 15 and 30 mg/kg body weight was given to rats in a single application via the dermal route. Marrow was collected at 24, 48 and 72 h after the application. The micronucleus assay was done as recommended by standard procedures. Paraquat gave rise to an increase in the number of micronuclei in a dose-dependent manner. The number of micronucleated polychromatic erythrocytes showed a maximum at 48 h and the toxicity was further prolonged, as there was no complete recovery at 72 h. These findings suggest a genotoxic effect of paraquat even after exposure via dermal application.     

Detecting the cytogenetic effects in workers occupationally exposed to vincristine with four genetic tests

To study the human genetic damage induced by vincristine (VCR), the cytogenetic effects in workers occupationally exposed to vincristine were studied with micronucleus (MN) test, comet assay, hypoxantinepho-guanine phosphoribosyl-transferase (hprt) gene mutation assay and T-cells receptor (TCR) gene mutation assay. Fresh peripheral blood samples were collected from the workers and controls. Fifteen workers from a plant producing antineoplastic drug (vincristine) and 15 controls were matched according to age, gender and smoking. The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in 15 workers were 17.80+/-1.88 per thousand and 13.67+/-1.56 per thousand, respectively, which were significantly higher than those (3.73+/-0.80 per thousand and 3.13+/-0.59 per thousand) in controls (P<0.01). It was found in the comet assay that the mean tail length (MTL) of 15 workers and 15 controls were 1.72+/-0.15 microm and 0.71+/-0.01 microm, respectively, there was significant difference between workers and controls for MTL (P<0.05), but the difference between the mean tail moment (MTM, 0.29+/-0.03) of 15 workers and MTM (0.17+/-0.05) of 15 controls was not significant (P>0.05). The results of hprt gene mutation assay showed that the average mutation frequency of hprt (Mf-hprt) in workers was 1.03+/-0.02 per thousand, which was significantly higher than that (0.87+/-0.01 per thousand) in controls (P<0.05). Meanwhile, the results of TCR gene mutation assay indicated that Mfs-TCR of workers and controls were 2.52+/-0.34 x 10(-4) and 1.51+/-0.11 x 10(-4), respectively, there was a significant difference between workers and controls (P<0.01). It is found in the results of our study that the genetic damage is detectable in 15 workers occupationally exposed to vincristine.     

Cytogenetic effects in children exposed to low doses of ionizing radiation

The obtained data indicate that frequencies of different types of cytogenetic anomalies in investigated children groups living in radionuclide contaminated territories and children irradiated in utero have complicated patterns. The frequency of chromosomal anomalies in the investigated groups of children exceeds the average population level. At the same time, no statistically significant differences in frequencies of various types aberrations between groups of children were revealed.     

Cytogenetic and molecular cytogenetic investigations in relapse of B-cell chronic lymphocytic leukemia

Сhromosomal abnormalities have been analyzed in bone marrow cells of 61 patients with relapse of B-cell chronic lymphocytic leukemia. The cytogenetic results have allowed the structural stratification of the obtained karyotypes into ten groups of clones: normal, normal/near tetraploid, abnormal/normal, abnormal/ near tetraploid/normal, evolution of clonal chromosome abnormalities; evolution of clonal chromosome abnormalities/normal, evolution of clonal chromosome abnormalities/near tetraploid/normal, independent clones, independent/normal clones; and independent/near tetraploid/normal clones. The identified structural rearrangements included translocations, deletions, insertions, and duplications; however, deletions with the involvement of bands 17p12, 13q12–q14, 11q14, and 11q23 dominated (63.8%). The application of i-FISH helped to show the presence of one to four abnormalities per karyotype. The identified cytogenetic and molecular cytogenetic rearrangements may signify a multilevel nature of the process underlying the development of resistant karyotypes. The results obtained under both methods have revealed the presence of a heterogenic cell population with possibly different levels of chemotherapy resistance.