Cytogenetic Effects of γ-Radiation in Onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Seedlings

The effect of γ-radiation on the cytogenetic parameters of root meristem cells of onion seedlings was studied in laboratory experiments (Allium-test). An increase in the overall frequency of chromosomal aberrations and micronucleus frequencies in seedling cells at low γ-radiation doses (≤0.1 Gy) was detected for the first time. At a maximum absorbed dose of 13 Gy, chromosomal aberrations were detected in the majority of cells in the anaphase and telophase stages of the cell cycle, and the number of cells with multiple aberrations increased. The main contribution to the overall frequency of chromosomal aberrations, in addition to multiple aberrations, is made by the bridge-type aberrations, fragments, and lagging chromosomes. The data obtained allow using the cytogenetic indices of Allium cepa seedlings to assess the biological effects of lowdose γ-radiation.     

Frequency of chromosomal aberrations after exposure to γ-radiation of human chorionic villi

We investigated the chromosomal damage induced by in vitro exposure to γ-rays of uncultured first trimester chorionic villi. Frequency and types of chromosomal aberrations at increasing doses of radiation have been evaluated on cytotrophoblast spontaneous metaphases obtained after a short term incubation. Our results indicate a direct correlation between radiation dose and aberration frequency.     

Excess processing of oxidative damaged bases causes hypersensitivity to oxidative stress and low dose rate irradiation

Abstract

Ionizing radiations such as X-ray and γ-ray can directly or indirectly produce clustered or multiple damages in DNA. Previous studies have reported that overexpression of DNA glycosylases in Escherichia coli (E. coli) and human lymphoblast cells caused increased sensitivity to γ-ray and X-ray irradiation. However, the effects and the mechanisms of other radiation, such as low dose rate radiation, heavy-ion beams, or hydrogen peroxide (H₂O₂), are still poorly understood. In the present study, we constructed a stable HeLaS3 cell line overexpressing human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) protein. We determined the survival of HeLaS3 and HeLaS3/hOGG1 cells exposed to UV, heavy-ion beams, γ-rays, and H₂O₂. The results showed that HeLaS3 cells overexpressing hOGG1 were more sensitive to γ-rays, OH^?, and H₂O₂, but not to UV or heavy-ion beams, than control HeLaS3. We further determined the levels of 8-oxoG foci and of chromosomal double-strand breaks (DSBs) by detecting γ-H2AX foci formation in DNA. The results demonstrated that both γ-rays and H₂O₂ induced 8-oxoguanine (8-oxoG) foci formation in HeLaS3 cells. hOGG1-overexpressing cells had increased amounts of γ-H2AX foci and decreased amounts of 8-oxoG foci compared with HeLaS3 control cells. These results suggest that excess hOGG1 removes the oxidatively damaged 8-oxoG in DNA more efficiently and therefore generates more DSBs. Micronucleus formation also supported this conclusion. Low dose-rate γ-ray effects were also investigated. We first found that overexpression of hOGG1 also caused increased sensitivity to low dose rate γ-ray irradiation. The rate of micronucleus formation supported the notion that low dose rate irradiation increased genome instability.     

Cytogenetic adaptive response induced by pre-exposure in human lymphocytes and marrow cells of mice

The frequencies of chromosomal aberrations bith in human lymphocytes and in mouse marrow cells exposed to low-level radiation were higher than in their unexposed controls. However, the frequencies of chromosomal aberrations in two kinds of cells pre-exposed to low-level radiation induced by a subsequent high dose of X-rays or γ-rays were lower than those of the groups only exposed to high-level radiation. This implies that adaptive responses for cytogenetic indicators might be induced by pre-exposure to low-level radiation. The results also show the existence of possible variations between individual lymphocytes.     

Radioprotective effects of Dragon's blood and its extract against gamma irradiation in mouse bone marrow cells

PurposeThe radioprotective effects of Dragon's blood (DB) and its extracts (DBE) were investigated using the chromosomal aberrant test, micronucleus and oxidative stress assay for anti-clastogenic and anti-oxidative activity.Materials and methodsAdult BALB/C mice were exposed to the whole body irradiation with 4 Gy ⁶⁰Co γ-rays. DB and DBE were administered orally once a day from 5 days prior to irradiation treatment to 1 day after irradiation. The mice were sacrificed on 24 h after irradiation. The cells of bone marrow were measured by counting different types of chromosomal aberrations and the frequency of micronuclei. Oxidative stress response was carried out by analysis of serum from blood.ResultsDB and DBE significantly decreased the number of bone marrow cells with chromosome aberrations after irradiation with respect to irradiated alone group. The administration of DB and DBE also significantly reduced the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE). In addition, DB and DBE markedly increased the activity of antioxidant enzymes and the level of antioxidant molecular. Malondialdehyde (MDA) and nitric oxide (NO) levels in serum were significantly reduced by DB and DBE treatment.ConclusionsOur data suggested that DB and DBE have potential radioprotective properties in mouse bone marrow after ⁶⁰Co γ-ray exposure, which support their candidature as a potential radioprotective agent.     

Radiation-induced chromosomal aberrations in the bone marrow of mice exposed to various doses of gamma radiation

The occurrence of chromosomal aberrations was studied at 1–14 days post-exposure in female BALB/c mice exposed to various doses of gamma radiation. The frequency of abnormal cells, chromatid and chromosome breaks, dicentrics, centric rings, acentric fragments and total aberrations increased with exposure dose, and it was highest at 7 Gy. A peak was recorded on day 1 post-exposure with a gradual decline thereafter. The chromosomal aberration yield reached a nadir on day 14 post-irradiation, without restoration to the control level. The best fit for the present data was by a linear-quadratic relationship between dose of radiation and the frequency of chromosomal aberrations.     

Possible mechanisms for occurrence of chromosomal restructuring. Interchromosomal contacts during metaphase and their role in chromosomal restructuring

The influence of colchicine-hypotonic treatment on interchromosomal aberrations at metaphase was studied in bone marrow cells of BALB mice irradiated by X-rays within the dose range from 0.25 to 1.50 Gy. In was found that after 30 min treatment with 0.002% colchicine of cells dividing 10 h following irradiation, the frequency both of chromosomal exchanges and interchromosomal contacts decrease about 3.5 times, the amount of chromosomal breaks increasing. It is calculated from the data of this experiment that two breaks induced by irradiation, which were scored at the same K metaphase as independent ones, appeared to be associated with each other at high frequency through exchange in the absence of colchicine or hypotonic treatment. It is assumed that regions of interchromosomal contacts at native metaphase are the most radiation-sensitive zones of the genome preferentially involved in chromosomal aberrations of X-irradiated cells.     

Chromosomal aberrations and micronuclei in lymphocytes of breast cancer patients after an accident during radiotherapy with 8 MeV electrons

In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.     

Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation

The aim of the present study was to analyse the dose rate effect of gamma radiation at the level of mutations, chromosomal aberrations, and cell growth in TK6 cells with normal as well as reduced levels of hMTH1 protein. TK6 cells were exposed to gamma radiation at dose rates ranging from 1.4 to 30.0 mGy/h (chronic exposure) as well as 24 Gy/h (acute exposure). Cell growth, frequency of thymidine kinase mutants, and of chromosomal aberrations in painted chromosomes 2, 8, and 14 were analysed. A decline in cell growth and an increase in unstable-type chromosomal aberrations with increasing dose rate were observed in both cell lines. A dose rate effect was not seen on mutations or stable-type chromosomal aberrations in any of the two cell lines. Reduction in the hMTH1 protein does not influence the sensitivity of TK6 cells to gamma radiation. This result fits well with data of others generated with the same cell line.     

Evaluation of radiation-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro results of an IAEA-coordinated programme

The results of an IAEA coordinated programme on radiation induced chromosomal aberrations in human peripheral blood lymphocytes in vitro are presented. In a master experiment, a whole blood sample from one donor was irradiated with 200 R of X-rays. Different fixation times from 46 to 82 h were used. The progression of cells into mitosis was monitored by BrdUrd incorporation. 14 investigators took part in the scoring of chromosomal aberrations. The main conclusions of this study are: (1) The mean frequencies of aberrations changed with fixation time. (2) The number of cells scored as aberrant by different laboratories was very similar, but there was variability in the number of aberrations scored per aberrant cell. (3) The differences in the frequencies of aberrations between laboratories were minimal when the scoring was restricted to the first major peak of mitotic activity and sufficient cells were scored.

It is concluded that using controlled experimental conditions, human peripheral blood lymphocytes can effectively be used as a reliable biological dosimeter for absorbed radiation dose.     

Cytogenetic control over the safety of merthiolate

The capacity of merthiolate (also known as thimerosal), a mercury organic compound used as preservative in vaccines, to induce chromosomal aberrations was studied in vivo in experiments on mice. The metaphasic method of the registration of anomalies was used. In a dose of 3.3 micrograms/kg body weight merthiolate did not produce a damaging effect on the chromosomes of marrow, spleen and embryonal liver cells. Cyclophosphamide (in a dose of 25-50 micrograms/kg) used as positive control induced a significant rise in the frequency of chromosomal transformations.     

Dose and time dependence of chromosomal aberration yields of bone marrow cells in male Chinese hamsters after a single i.p. injection of aflatoxin B1

The frequency of chromosomal aberrations in bone marrow cells, after a single i.p. aflatoxin B1 (AFB1) dose, was examined in male Chinese hamsters (Cricetulus griseus). There was a significant increase in aberrant cells within 5 days of administration of a dose of 0.1 micrograms-5 mg AFB1/kg, and on the 36th day. After a single dose of 5 mg AFB1/kg the enhanced frequency of aberrant cells was monitored up to day 104 with no sign of a decrease to control level. The results indicate that the minimum mutagenic effect of an AFB1 dose in this system is 0.1 micrograms/kg. Attention is drawn to the long-term presence of chromosomal aberrations even after a single i.p. exposure to AFB1.     

Search for clastogenic factors in the plasma of locally irradiated individuals

In studies reported in the 1960s and in several investigations since, plasma from irradiated individuals was shown to induce chromosomal aberrations when transferred into normal blood cultures. In the present study, the aim was to investigate the occurrence of these clastogenic factors (CF) using markers representing DNA damage produced in reporter lymphocytes that are treated with plasma from locally exposed individuals. Blood plasma was obtained from clinical patients with benign conditions before and after they had received radiation to small treatment volumes. Three patient groups were studied: (I) marginal resected basal cell carcinoma, (II) painful osteoarthritis of the knee, and (III) painful tendinitis of the elbow or the heel. Patients in each treatment group obtained the same fractionated treatment regimen, ranging from a total dose of 40 Gy (8 × 5 Gy, 2 factions/week) to a very small volume (1-3.5 cm3) in group I to a total dose of 6 Gy (6 × 1 Gy, 2 fractions/week) for groups II and III (treatment volumes 800-1150 cm3 and 80-160 cm3, respectively). The presence of CF in the plasma was investigated through cytogenetic (chromosomal aberrations, micronuclei) assays and kinetics of early DNA damage (γ-H2AX foci) in reporter cells. With the experimental settings applied, local radiation exposure had no apparent effect on the induction of CF in patient plasma; no deviations in chromosomal aberrations or micronucleus or focus induction were observed in reporter cells treated with postexposure plasma with respect to pre-exposure samples when the mean values of the groups were compared. However, there was a large interindividual variation in the plasma-induced DNA-damaging effects. Steroid treatment of patients was demonstrated to be the most influential factor affecting the occurrence of plasma factors; plasma from patients treated with steroids led to significant reductions of γ-H2AX foci and reduced numbers of chromatid aberrations in reporter cells. In addition to the locally exposed patients, newly obtained plasma samples from three radiological accident victims exposed in 1994 were examined. In contrast to the patient data, a significant increase in chromosomal aberrations was induced with plasma from two accident victims.     

Radiation induced chromosomal instability in human T-lymphocytes

Chromosomal instability in proliferating mammalian cells is characterized by a persistent increase of chromosomal aberrations and rearrangements occurring de novo during successive cell generations. Recent results from many laboratories using a variety of cells and cytogenetic end points show that this phenotype can be induced by low as well as high LET irradiation. A typical feature of chromosomal instability in primary human G₀-lymphocytes exposed to γ-irradiation at both high dose rate (45 Gy h⁻¹) and low dose rate (0.024 Gy h⁻¹) is the appearance of novel aberrations in the clonal progeny of the irradiated cell, many generations after the exposure. The same phenotype was observed in lymphocytes that were allowed to recover for 5 days in G₀ after the radiation exposure, as well as in hprt-mutant T cell clones. These results demonstrate that neither the acute genotoxic stress caused by high dose rate as compared to low dose rate irradiation, nor a hypothesized conflict between mitogen induced growth stimulation and growth arrest due to radiation damage, seem to be critical conditions for the development chromosomal instability in these cells. In contrast to observations in other cells, no evidence of a persistent decrease of cloning ability was observed in the progeny of radiation-exposed human lymphocytes, and no alteration was observed in their sensitivity to a second radiation exposure. Furthermore, the frequency of CA-repeat length variation at three loci was not increased in the progeny of X-irradiated T cells as compared to non-irradiated cells, which indicates that microsatellite instability is not part of the chromosomal instability phenotype in human T-lymphocytes.     

In vivo cytogenetic effects of cooked food mutagens

Using a variety of in vivo cytogenetic endpoints, we have investigated the effects of several compounds formed during the cooking of meat. C57Bl/6 mice were used to test for an increase in the frequency of sister-chromatid exchanges (SCEs), chromosomal aberrations, and micronucleated erythrocytes by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). MeIQx and DiMeIQx did not induce SCEs in mouse bone marrow cells. PhIP induced sister-chromatid exchanges, but not chromosomal aberrations in bone marrow. In peripheral blood lymphocytes, PhIP did induce aberrations at 100 mg/kg, the highest dose tested. PhIP induced a low but significantly increased frequency of micronuclei in normochromatic but not polychromatic erythrocytes in bone marrow and peripheral blood. However, dose responses were not observed. With the exception of the SCEs induced by PhIP, these results contrast with observations made in vitro, where these compounds were found to have significant genotoxicity in mammalian cells and a very high mutation frequency in prokaryotic systems.     

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.     

Evaluation of the mutagenic potential of the antichagasic drug Rochagan in healthy and chagasic rodents

Benznidazole (bz) is the active component of the antichagasic drug Rochagan. Tests were carried out to detect the induction of chromosomal aberrations and micronuclei in rodent bone marrow cells and peripheral blood cells, respectively. Rats were exposed to acute treatment with Rochagan by gavage at total doses of 150, 300, 1500, 2000 and 3000 mg bz/kg body weight and killed at different times. In the chronic treatments, healthy and chagasic Balb/c mice were treated with Rochagan by gavage at a dose of 100 mg bz/kg/day for 10 and 25 days. No significant increase in frequency of chromosomal aberrations in bone marrow cells or of micronuclei in peripheral blood cells was detected in the animals acutely or chronically exposed to Rochagan in vivo.     

Correlation of the sister chromatid exchange level with chromosome aberrations induced by chemical mutagens in vivo

The data on the dose dependencies of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations during exposure of mouse bone marrow cells in vivo to 5 alkylating substances are provided. The efficacy of SCE induction was found to be higher than that of chromosomal aberrations. It was established that SCE induced by chemical mutagens in vivo and in vitro are more sensitive and stable tests than chromosomal aberrations.     

DNA topoisomerase II inhibition and gene amplification in V79/B7 cells

Topoisomerase II inhibitors such as etoposide (VP16) are able to stabilize the enzyme—DNA complex by trapping the topoisomerase on DNA without affecting its strand-break activity. To test if this inhibition resulting in chromosomal breakage via double-strand breaks could underlie gene amplification, we performed VP16 treatments followed by selection for PALA resistance in V79/B7 Chinese hamster cells. We found that VP16 induced PALA-resistant cells very efficiently, and in a dose-dependent manner. On the other hand VP16 in combination with 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase involved in DNA repair, reduced the frequency of PALA-resistant cells. Cytogenetic analysis revealed a higher number of chromosomal aberrations in VP16-treated cells than in cells treated with VP16 plus 3AB. These results suggest a correlation between frequency of chromosomal aberrations and frequency of PALA-resistant cells, and are consistent with models which consider chromosomal breakage as an important step in initiating gene amplification.     

AT cells are not radiosensitive for simple chromosomal exchanges at low dose

Cells deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome) show increased yields of both simple and complex chromosomal aberrations after high doses (>0.5Gy) of ionizing radiation (X-rays or γ-rays), however less is known on how these cells respond at low dose. Previously we had shown that the increased chromosome aberrations in ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex exchanges. The linear dose-response term for simple exchanges was significantly higher in NBS cells compared to wild type cells, but not for AT cells. However, AT cells have a high background level of exchanges compared to wild type or NBS cells that confounds the understanding of low dose responses. To understand the sensitivity differences for high to low doses, chromosomal aberration analysis was first performed at low dose-rates (0.5Gy/d), and results provided further evidence for the lack of sensitivity for exchanges in AT cells below doses of 1Gy. Normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, showed increased numbers of exchanges at a dose of 1Gy and higher, but were similar to wild type cells at 0.5Gy or below. These results were confirmed using siRNA knockdown of ATM. The present study provides evidence that the increased radiation sensitivity of AT cells for chromosomal exchanges found at high dose does not occur at low dose.