Biological consequences of irradiation by low doses of technetium 99m: ultrastructural studies, p53 protein expression and cytogenetic effects.

Few studies concerning the potential genetic effects of diagnostic radionuclides used in nuclear medicine have been reported. The aim of this study was to evaluate the biological and cytogenetic consequences of two technetium 99m-labelled radiopharmaceuticals. Ultrastructural modifications of pulmonary cells were first investigated after injection of 99mTc labelled microspheres in the rat. On the same irradiated cells, nuclear expression of p53 protein was assessed using immunohistochemistry. Despite very high previously calculated doses delivered to pulmonary cells, no morpholological cell damage and no significant increase of nuclear expression of the p53 were noted. There was no correlation between the calculated dose and the ultrastructural biological damage. Secondly, a specific in vitro curve, activity/number of unstable chromosomal aberrations, corresponding to physical characteristics of 99mTc, was established to verify the potentiality of 99mTc to induce such aberrations. In vivo, cytogenetic effects were assessed on blood samples of 5 patients with various arthrosic and periarthrosic diseases obtained after bone scintigraphy. Aberration frequencies of both in vitro and in vivo irradiated lymphocytes were determined using the classical Fluorescence Plus Giemsa technique. No cytogenetic effects appeared with the routinely 99mTc injected activities as predicted by the in vitro curve.     

In vivo exposure of human lymphocytes to technetium-99m in nuclear medicine patients does not induce detectable genetic effects.

Recent reports have demonstrated that exposure of nuclear medicine patients to thallium-201 does not result in a detectable increase in mutation at the hprt locus in human lymphocytes. In an effort to study further the potential genetic effects of medical exposures to low dose radiation, we have examined chromosome aberrations and mutations in peripheral blood lymphocytes from nuclear medicine patients exposed to clinical doses of technetium-99m. Our results show that there is no exposure-related increase in chromosomal damage; furthermore, the data do not confirm earlier reports of exposure-related increases in mutations induced by technetium-99m.     

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.     

The characterization and transmissibility of chromosome aberrations induced in peripheral blood lymphocytes by in vitro alpha-particle radiation

Peripheral blood lymphocytes were irradiated in vitro with (213)Bi alpha particles at doses of 0, 10, 20, 50, 100, 200 and 500 mGy. Chromosome analysis was performed on 47-h cultures using single-color fluorescence in situ hybridization (FISH) to paint chromosomes 1, 3 and 5. The whole genome was analyzed for unstable aberrations to derive aberration frequencies and determine cell stability. The dose response for dicentrics was 33.60 +/- 0.47 x 10(-2) per Gy. A more detailed analysis revealed that the majority of aberrations scored as dicentrics were part of complex/multiple aberrations, with the proportion of cells containing complexes increasing with dose. Cells containing aberrations involving painted chromosomes (FISH aberrations) were further classified according to cell stability and complexity. The majority of cells with FISH aberrations were unstable. The proportion of aberrant FISH cells with complex/multiple aberrations ranged from 56% at 10 mGy to 89% at 500 mGy. A linear dose response for genomic frequencies of translocations in stable cells fitted the data from 0 to 200 mGy with a dose response of 7.90 +/- 0.98 x 10(-2) per Gy, thus indicating that they are likely to be observed in peripheral blood lymphocytes from individuals with past or chronic exposure to high-LET radiation. Comparisons with the dose response for low-LET radiation suggest an RBE of 13.6 for dicentrics in all cells and 3.2 for translocations in stable cells. Since stochastic effects of radiation are attributable to genetic changes in viable cells, translocations in stable cells may be a better measure when considering the comparative risks of different qualities of radiation.     

Structural genomic damages in workers of plutonium production

The research objective is assessment of structural genomic damages in plutonium workers. The study group included the Mayak nuclear workers subject to chronic occupational exposure to incorporated 239Pu and/or external gamma-rays. The analysis was performed based on the culture of lymphocytes in peripheral blood. The yield of intra-chromosomal exchange aberrations of chromosomal type on stained slides was analyzed using in situ fluorescent hybridization, mBAND. Linear relationships were revealed between (a) the total yield of chromosomal type aberrations (e.g. intra- and inter-chromosomal ones) and an absorbed dose from external exposure of the red bone marrow to gamma-rays, an absorbed dose from internal exposure to a-radiation from incorporated 239Pu; and (b) the yield of intra-chromosomal exchange aberrations of chromosomal type and an absorbed dose from exposure of the red bone marrow to 239Pu and 239Pu body burden.     

Evaluation of technetium-99m decay on Escherichia coli inactivation: effects of physical or chemical agents.

Technetium-99m (99mTc) has been used in nuclear medicine and in biomedical research to label molecular and cellular structures employed as radiotracers. Here, we have evaluated, on a DNA repair proficient Escherichia coli strain, the 99mTc decay inactivation and the influence of the (i) pre-treatment with metal ion chelators or of the (ii) treatment with a free radical scavenger on the protection of the cells against the lethal effect of the 99mTc. As SnCl2 is frequently used as a reducing agent in the 99mTc-labeling process, we have also studied the capability of SnCl2 to alter the biological effects induced by the 99mTc decay. As we are exposed to either chemical or physical agents in the nature, we have decided to study a possible influence of the ultraviolet solar radiation in the biological phenomena induced by the 99mTc decay. Our data point out (i) a very important role of the Auger and/or conversion electrons in the cytotoxicity induced by the 99mTc decay; (ii) SnCl2, the metal ion chelators and the free radical scavenger protect the cells against the lethal effect of the 99mTc; and (iii) near-UV does not alter the lethal effect of the 99mTc decay.     

Seventeen-year follow-up study on chromosomal aberrations in five victims accidentally exposed to several Gy of <Superscript>60</Superscript>Co γ-rays

On 25 June 1990, a radiation accident occurred in a ⁶⁰Co source radiation unit in Shanghai, due to violations in operation regulations. This accident resulted in the exposure of seven individuals to acute high-dose and dose-rate whole-body external irradiation. Conventional chromosomal aberration analysis, G-banding automatic karyotype analysis and/or fluorescent in situ hybridization (FISH) painting methods were used to analyze chromosomal aberrations in peripheral blood lymphocytes from five of the victims 24 h to 17 years after accidental exposure to 1.9–5.1 Gy of ⁶⁰Co γ-rays. The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at constant levels 1 month after exposure. Three months after exposure, the frequency was reduced by 20–40% in three victims, while no reduction was seen in the other two victims. Twelve years after exposure, the number of dicentrics and rings decreased by more than 90%, and did not reveal a dose-dependent relationship. However, even at 12–17 years after exposure, stable chromosome aberrations, dominated by translocations, remained at a high level in a dose-dependent manner. The frequency of stable chromosomal aberrations detected by FISH showed a similar dose-dependent relationship as that detected by karyotype analysis of G-banding chromosomes. The G-banding analysis also suggested that the pattern of chromosome breakpoints is random. The FISH data showed a decreasing tendency with time for chromosome translocation frequency in the peripheral lymphocytes, and the rate of reduction varied among different individuals. It is likely that the higher dose the victim received, the lesser the translocation frequency decreased with time. The G-banding data also showed that the rate of reduction of translocations is different among individuals. From 5 to 17 years after accidental irradiation, a very small reduction (~10%) of translocation frequency was observed in victims C and D, while there was about a 35% reduction (the highest among the victims) for victim G who received the smallest dose (1.9 Gy). These observations can be used to validate the existence of chromosomal aberrations in peripheral blood lymphocytes as a biological dosimeter for radiation exposures.     

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.     

Complex cytogenetic characteristic of people suffered from Chernobyl accident

A cytogenetic study was performed on Chernobyl cleanup workers, on their children, on persons evacuated from contaminated aeria (adult and children), on so named "veterans of particular risk" irradiated due to the accidents on the nuclear plant, testing of nuclear weapons etc. and on control donors. The yield of stable (FISH analysis) and of unstable chromosome aberrations, micronuclei in both lymphocytes and erythrocytes, HPRT mutations was found to be increased in exposed groups as compared to control ones. In children of liquidators and in evacuated children we observed genomic instability and increased in vitro chromosomal radiosensitivity. Acceleration of age accumulation of translocations characterized the exposed population in comparison with control group. People with the highest level of routine chromosome aberrations had cardiovascular and digestive diseases more often likely than those with the lowest level. In frame of International Project ECP-6--"Biological dosimetry" the dose-responses for dicentrics and translocations were constructed in dose range 0-100 cGy of gamma-irradiation on the base of data of 8 laboratories. On cancer patients undergone whole-body gamma-irradiation (every day at the dose 11.5 cGy to a total of dose 57.5 cGy) we constructed the dose-responses for the dicentrics and translocations and compared them with the dose-responses for these aberrations after the in vitro irradiation of lymphocytes of the same patients. For the dicentrics the effectiveness of the in vivo irradiation was less than of the in vitro one. No differences were found for translocations.     

The value of cytogenetic monitoring versus film dosimetry in the hot zone of a nuclear power plant.

Cytogenetic analysis was carried out in 41 workers prior to and following regular maintenance work in a nuclear power plant. Although film dosimetry did not show the maximal annual permitted dose in any of the examined subjects, cytogenetic analysis carried out following the work detected dicentric chromosomes in peripheral blood lymphocytes of 20 workers. According to our findings smoking habits and previous exposure to ionizing radiation had no effect on the increased number of chromosomal aberrations.     

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.     

Karyotypes of human lymphocytes exposed to high-energy iron ions

Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.     

Evaluation of the cytotoxic and mutagenic potentiality of technetium-99m in Escherichi coli.

Since technetium-99m (99mTc) was introduced in medical research it has become one of the most employed radionuclides in nuclear medicine. 99mTc is ideal for routine use on the labeling of different radiopharmaceuticals due to its favorable characteristics. However, some biological effects have been described. These effects may be related to internal conversion electron and/or Auger electron emissions from 99mTc decay that present high linear energy transfer and can generate reactive oxygen species (ROS) in the medium. We evaluated in Escherichia coli K12S and Salmonella typhimurium TA102, both proficient in DNA repair, contribution of those decay emissions on the cytotoxicity induced by 99mTc, both either by generating lesions on DNA or by inducing alterations at membrane. We also studied the genotoxic and/or mutagenic potentiality of 99mTc, in Salmonella typhimurium, using the Ames test. The results showed that: i/ 99mTc is cytotoxic to the Escherichia coli K12S strains; ii/ this effect is related to the electrons (Auger and internal conversion) emissions, and iii/ the 99mTc is not mutagenic and/or genotoxic, when measured by Ames test.     

FISH-based analysis of 10- and 25-kV soft X-ray–induced DNA damage in 184A1 human mammary epithelial cells

Over the past years, several in vitro studies have been performed on DNA damage induced by soft X-rays, especially in the energy range below 50 keV. Radiation effects originating from such low-energy photons are relevant in the context of medical diagnostics, for example, mammography, or of accidental exposure to scattered radiation. The present study was initiated to investigate the X-ray energy-dependent induction of stable and unstable chromosomal aberrations in the human mammary epithelial cell line 184A1. Three colour fluorescence in situ hybridisation was applied to identify chromosomal damage in chromosomes 1, 8 and 17, induced by 10-kV or 25-kV soft X-rays as well as by 200-kV X-rays as a reference quality. The overall results confirm the X-ray energy dependencies published for human lymphocytes showing increasing chromosomal aberration frequencies and higher aberration complexity with decreasing X-ray energy and increasing dose. Comparing the obtained dose dependencies, ratios of 0.84 ± 0.09 and 1.22 ± 0.18 were revealed for stable translocations induced by 25- and 10-kV X-rays, respectively, using 200-kV X-rays as reference. Moreover, the analysis of the minimum number of breaks required to form the visible chromosomal damage resulted in similar ratios of 0.93 ± 0.07 for 25-kV X-rays and 1.25 ± 0.10 for 10-kV X-rays relative to 200-kV X-rays. In addition, non-DNA-proportional contributions of chromosomes 8 and 17 to the whole DNA damage and deviations from the expected 1:1 ratio of translocations and dicentrics were observed for cell line 184A1.     

Adaptive response of human lymphocytes for the repair of radon-induced chromosomal damage

In human lymphocytes low doses of X-rays can decrease the number of chromatid deletions induced by subsequent high doses of sparsely ionizing X-rays. Because of the concern with the carcinogenic effects of low doses of -particles from radon in homes, experiments were carried out to see if low doses of X-rays could also decrease the yield of chromosomal aberrations induced by subsequent exposure to radon. Human peripheral blood lymphocytes were irradiated with low doses of X-rays (2 cGy) at 48 h of culture, exposed to radon at 72 h of culture, and analyzed for the presence of chromatid aberrations at subsequent intervals. The frequency of chromatid aberrations induced by radon alone increased with time after exposure, indicating exaggerated differences in the stage sensitivity of cell cycle stages to high-LET radiation. Furthermore, the numbers of aberrations per cell did not follow a Poisson distribution but were over dispersed, as might be expected since high-LET radiations have a high relative biological effectiveness compared with low-LET radiations. Nevertheless, lymphocytes exposed to 2 cGy of X-rays before radon exposure contained approximately one-half the number of chromatid deletions compared with lymphocytes treated with radon alone and analzed at the same time. Thus, the putative chromosomal repair mechanism induced by low doses of sparsely ionizing radiation is also effective in reducing chromosomal aberrations induced by radon, which hitherto had been thought to be relatively independent of repair processes.     

Biological indication and dosimetry of unstable chromosome aberration frequencies in human lymphocytes

Review is devoted to the problems of biological (cytogenetic) dosimetry and indication of degree of radiation lesions based on analysis of unstable chromosome aberrations in lymphocytes of human peripheral blood. Effects of radiation in low doses on human chromosomes and methodology of interpretation of the character of dose cytogenetic curves are discussed. Traditional cytogenetic analysis remains the basic one for monitoring in groups of people with accidental irradiation.     

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.     

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.     

Radiation damage in patients treated by total-body irradiation, bone marrow grafting, and cyclosporin

The bone marrow (BM) and peripheral blood (PB) from 63 patients were assessed for the presence of chromosomal aberrations after bone marrow transplantation (BMT) following total body irradiation (TBI) for leukemia. Forty-one patients showed no abnormalities in either BM or PB, and 22 had aberrations in either BM or PB or both. Only stable aberrations were found in the BM, but both stable and unstable abnormalities were present in the PB, the majority showing only unstable aberrations. Among the 25 patients who had a leukemic relapse, clonal chromosomal abnormalities were found in the BM of 12 out of the 16 cases for whom marrow was studied at the time of the relapse. A statistically significant negative correlation between leukemic relapse and graft versus host disease (GvHD) was found, but the relationships between chromosome damage and leukemic relapse, GvHD, and the pretransplant radiation dose and between the radiation dose and both leukemic relapse and GvHD were not significant.     

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.