Article

Micronucleus Induction in Human Lymphocytes: Comparative Effects of X Rays, Alpha Particles, Beta Particles and Neutrons and Implications for Biological Dosimetry

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Abstract

The cytokinesis-block micronucleus assay in peripheral blood lymphocytes has the potential for being a simple and rapid method of biological dosimetry. This technique has been used to study the induction of micronuclei in the blood from 12 donors after exposure to a range of radiations with track-averaged LET values ranging from 0.26 to 44 keV microns -1. Data based on the average response of the 12 individuals for 250 kVp X rays were found to agree well with results published previously from other laboratories using similar techniques. Low dose-limiting RBE values relative to 250 kVp X rays for the radiations studied were found to be 0.50 for strontium/yttrium-90 beta particles, 6.9 for 20-23 keV microns -1 alpha particles and 17 for 24 keV neutrons. The pattern of the variation of individual radiosensitivity was found to be complex and dependent on dose, and the evaluation of individual radiosensitivity based on the response at one dose only can be misleading. It is concluded that, although the cytokinesis-block micronucleus assay in blood lymphocytes is a radiobiologically appropriate technique to use for biological dosimetry, its practical implementation may be limited by a need to perform individual pre-exposure calibrations.

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... Joniani et al., 2002), (Masunaga et al., 2001), (Belyakov et al., 1999) Mikronukleustest " bezeichnet (Heddle et al., 1983). Zugleich entstanden weitere Mikrokern-Nachweisverfahren, um den zytogenetischen Schaden von Substanzen, die das Knochenmark nicht erreichen, in vitro zu erfassen (Heddle et al., 1983) 1998), (Catena et al., 1997), (Livingston et al., 1997), (Catena et al., 1996), (Mill et al., 1996), (Gantenberg et al., 1991). Mittlerweile ist die Cytochalasin-B-Methode aber umstritten, da Cytochalasin-B möglicherweise selbst Mikrokerne induziert und den Ablauf der Apoptose stört (Abend et al., 2000), (Abend et al., 1995). ...
... Chromosomen enthalten. Sie ähneln in Form, Färbeverhalten und Struktur dem Hauptkern, sind aber kleiner (Mill et al., 1996). Ihre Entstehung beruht auf Schäden in der DNS, die zu Chromatin-Fehlverteilungen in der Anaphase führen (Garewal et al., 1993). ...
... Röntgenbestrahlung Für die Zelllinie L929 liegen Vergleichsdaten in der Literatur vor (Abend et al., 2000). Instabilität dient (Kennedy et al., 1996), (Talbot et al., 1989) Relativen Biologischen Wirksamkeit (RBW) ab (Green et al., 2001), (Mill et al., 1996) (Ono et al., 1990). Diese soll daher im folgenden Abschnitt näher erläutert werden. ...
Article
The concept of radioimmunotherapy is to selectively eliminate tumor cells due to specific targeting with appropriate nuclides. In this study we used the alpha-emitter Bi-213 coupled to an antibody that binds to a mutated adhesion molecule. This altered E-cadherin (d9-E-cad) is expressed by diffuse type gastric cancer cells but not by normal tissue. The aim was to demonstrate specific therapeutic efficiency of antibody targeting by quantification of alpha-particle induced cell death. Furthermore the effects of alpha-radioimmunotherapy were compared to those of X-ray irradiation. The radiotoxicity was quantified via the MAA assay. At various time points after incubation with radioimmunoconjugates or after X-ray irradiation, cells were scored for the percentage of micronucleus formation (M), apoptosis (A), and abnormal cells (A) to evaluate different modes of cell death. We tested murine fibroblasts transfected with either wildtype E-cad (L929 wt) or mutant d9-E-cad (L929 d9) for radioinduced alterations in nuclear morphology. The cells were incubated with Bi-213-immunoconjugates directed against d9-E-cad (Bi-213-6H8). The MAA peak frequency was 13+/-3 % in L929 d9 cells with L929 wt cells and unirradiated controls not exceeding 2+/-1 %. Furthermore, we investigated human gastric cancer cells (HSC-45M2) constitutively expressing d9-E-cad. MAA formation occurred in a dose and time dependent manner with a maximum of 12+/-1.3 % after 10 minutes incubation and 19+/-3.3 % after 90 minutes incubation with 160 kBq/ml Bi-213-6H8. Incubation with 640 kBq/ml led to 21+/-3.6 % (10 minutes incubation) and 33+/-5.7 % MAA cells (90 minutes incubation). A group of HSC cells had been preincubated with unlabeled anti-d9-E-cad-antibody prior to application of Bi-213-6H8 to examine competitive inhibition of radioimmunoconjugate binding. An unspecific antibody labeled with Bi-213 was applied to another group of HSC cells to evaluate unspecific cell damage. Preincubation with unlabeled 6H8-antibody before application of Bi-213-6H8 as well as incubation with unspecific Bi-213-immunoconjugates significantly reduced cell death at the low activity concentration (160 kBq/ml) compared to incubation with Bi-213-6H8. At 640 kBq/ml, no difference was observed, most likely due to crossfire irradiation. Additionally, L929 d9 cells and HSC cells were irradiated with different X-ray doses. X-ray irradiation with 0, 1, 2, and 4 Gy equally induced MAA cells in both HSC and L929 d9 cells with about 12 % after irradiation with 1 Gy, 20 % with 2 Gy, and 30 % with 4 Gy. Tumorspecific Bi-213-labeled antibodies selectively induce cell death in antigen-expressing tumor cells depending on activity concentration and incubation time, whereas incubation with the unspecific radioimmunoconjugate or incubation with Bi-213-6H8 following preincubation with the unlabeled antibody had less effect. Thus, radioimmunotherapy with Bi-213-labeled specific antibodies targeting d9-E-cadherin may at appropriate activity concentrations provide a new strategy in the treatment of diffuse type gastric cancer.
... The cytogenetic effect of ␤-radiation on human cells reported in the literature refers to the exposure of blood lymphocytes to 3 H [11,12,16] and 131 I [17][18][19]. The effects of 90 Sr have been investigated by Hall and Wells [20] and Mill et al. [21] using the micronucleus assay as a parameter for cytogenetic damage evaluation and by Vulpis and Scarpa [22] using induced chromosome aberration analysis, with the radiation source being in direct contact with the blood sample. ...
... Similar results were obtained by Hall and Wells [20] and by Mill et al. [21], who analyzed the induction of micronuclei in human lymphocytes by 90 Sr ␤-radiation (1.0-1.6 Gy/min) at the dose range of 0.3-3.0 Gy. ...
... Gy. Hall and Wells [20] and Mill et al. [21] observed that external irradiation with 90 Sr was less effective than X-rays (0.5-2.0 Gy/min) in micronucleus induction. ...
Article
Among various environmental genotoxins, ionizing radiation has received special attention because of its mutagenic, carcinogenic and teratogenic potential. In this context and considering the scarcity of literature data, the objective of the present study was to evaluate the effect of 90Sr beta-radiation on human cells. Blood cells from five healthy donors were irradiated in vitro with doses of 0.2-5.0Gy from a 90Sr source (0.2Gy/min) and processed for chromosome aberration analysis and for comet assay. The cytogenetic results showed that the most frequently found aberration types were acentric fragments, double minutes and dicentrics. The alpha and beta coefficients of the linear-quadratic model, that best fitted the data obtained, showed that 90Sr beta-radiation was less efficient in inducing chromosome aberrations than other types of low linear energy transfer (LET) radiation such as 3H beta-particles, 60Co gamma-rays, 137Cs and 192Ir and X-rays. Apparently, 90Sr beta-radiation in the dose range investigated had no effect on the modal chromosome number of irradiated cells or on cell cycle kinetics. Concerning the comet assay, there was an increase in DNA migration as a function of radiation dose as evaluated by an image analysis system (tail moment) or by visual classification (DNA damage). The dose-response relation adequately fitted the non-linear regression model. In contrast to the cytogenetic data, 90Sr beta-radiation induced more DNA damage than 60Co gamma-radiation when the material was analyzed immediately after exposures. A possible influence of selective death of cells damaged by radiation was suggested.
... The environmental contamination resulting from the Chornobyl Nuclear Power Plant disaster (April 26,1986) offers a unique opportunity to examine in vivo biological effects of chronic, low-dose exposure to ionizing radiation. The effects of ionizing radiation at the chromosomal and molecular levels have been well documented for acute laboratory exposures [1][2][3][4]. Radiation protection measures have been adopted based on the results of laboratory investigations regarding low-dose radiation exposures, which suggest a linear, no-threshold, cumulative response. However, most low-dose laboratory exposures to radiation [3,5,6] are orders of magnitude greater than recent environmental levels of the Chornobyl exclusion zone, one of the most radioactive environments in the world. ...
... Radiation protection measures have been adopted based on the results of laboratory investigations regarding low-dose radiation exposures, which suggest a linear, no-threshold, cumulative response. However, most low-dose laboratory exposures to radiation [3,5,6] are orders of magnitude greater than recent environmental levels of the Chornobyl exclusion zone, one of the most radioactive environments in the world. ...
... Although a slight overlap was found in the confidence intervals ( Fig. 1) between day 0 and day 10, contingency 2 analysis of MN and PCEs across the four time periods showed a highly significant reduction in MN between day 0 and day 10 ( ,p Ͻ 0.001). Removal of the day 0 class from the 2 ϭ 24.66 3 contingency table showed that the remaining sample periods were not significantly different ( , p ϭ 0.92). There-2 ϭ 0.167 2 fore, the difference across samples was due to a decrease in MN from day 0 to day 10. ...
Article
Previous studies have demonstrated no difference in micronucleus (MN) frequencies between wild rodents chronically exposed to the environmental radiation contamination of the Chornobyl (Ukraine) exclusion zone and those inhabiting reference populations. The aim of the present study was to test the hypothesis that a population of bank voles (Clethrionomys glareolus) has developed radioresistance as a result of 14 years of chronic, low-dose radiation exposure. Naive voles were placed in environmental enclosures in the Red Forest region of the exclusion zone for 30 d. Blood samples were obtained at regular intervals, and the MN assay was used to assess chromosomal damage. Additionally, radionuclide uptake was monitored throughout the study, and dose was documented for each individual as well as for their offspring. Total dose for the voles experimentally exposed in this environment averaged 1.09 Gy (36.20 mGy d−1) for the 30-d study period. Our results indicate that exposure to radiation levels well above regulatory statutes did not result in an increased MN frequency. Furthermore, our results do not support the hypothesis that voles chronically exposed to these radiation levels have developed a genetic basis for radioresistance that is unique from that present in naive populations. The use of C. glareolus as a sentinel species for environmental studies of radiation contamination and the question of whether the MN assay is an appropriate endpoint for studies of low-dose, chronic radiation exposure are also discussed.
... Cytogenetic studies of 3 H [6, 7, 8], 131 I [9, 10, 11], 89 Sr [12] and 153 Sm [13, 14] have been conducted on human peripheral blood lymphocytes. Genotoxic effects of 90 Sr/ 90 Y, a highly energetic pure beta particle source (E max =2.27 MeV, E avg =1.13 MeV) [15] were reported by Vulpis & Scarpa [16], Hall & Wells [17], Mill et al. [15] and Oliveira et al. [18] using the analysis of chromosome aberrations, micronuclei and comets in human blood samples. Within this context, Chinese hamster ovary cells (CHO) have been used as a suitable biological system for genotoxic and cytotoxic studies because of their inherently advantageous characteristics such as belonging to a genetically stable cell line and having the ability to form colonies, a relatively rapid growth rate with a cell cycle of 12–14 h and a karyotype of 22€2 chromosomes [19]. ...
... Cytogenetic studies of 3 H [6, 7, 8], 131 I [9, 10, 11], 89 Sr [12] and 153 Sm [13, 14] have been conducted on human peripheral blood lymphocytes. Genotoxic effects of 90 Sr/ 90 Y, a highly energetic pure beta particle source (E max =2.27 MeV, E avg =1.13 MeV) [15] were reported by Vulpis & Scarpa [16], Hall & Wells [17], Mill et al. [15] and Oliveira et al. [18] using the analysis of chromosome aberrations, micronuclei and comets in human blood samples. Within this context, Chinese hamster ovary cells (CHO) have been used as a suitable biological system for genotoxic and cytotoxic studies because of their inherently advantageous characteristics such as belonging to a genetically stable cell line and having the ability to form colonies, a relatively rapid growth rate with a cell cycle of 12–14 h and a karyotype of 22€2 chromosomes [19]. ...
Article
Full-text available
Among various types of ionizing radiation, the beta emitter radionuclides are involved in many sectors of human activity, such as nuclear medicine, nuclear industries and biomedicine, with a consequently increased risk of accidental, occupational or therapeutic exposure. Despite their recognized importance, there is little information about the effect of beta particles at the cellular level when compared to other types of ionizing radiation. Thus, the objective of the present study was to evaluate the genotoxic and cytotoxic effects of (90)Sr/(90)Y-a pure, highly energetic beta source-on Chinese hamster ovary (CHO) cells and to compare them with data obtained with (60)Co. CHO cells irradiated with different doses of (60)Co (0.34 Gy min(-1)) and (90)Sr/(90)Y (0.23 Gy min(-1)) were processed for analysis of clonogenic death, induction of micronuclei (MN) and interphase death. The survival curves obtained for both types of radiation were fitted by the exponential quadratic model and were found to be similar. Also, the cytogenetic results showed similar frequencies of radio-induced MN between gamma and beta radiations and the MN distribution pattern among cells did not follow the expected Poisson probability pattern. The relative variance values were significantly higher in cells irradiated with (90)Sr/(90)Y than with (60)Co in all exposure doses. The irradiated cells showed more necrotic cells 72 h and 96 h after exposure to beta than to gamma radiation. In general, the (90)Sr/(90)Y beta-radiation was more damaging than (60)Co gamma-rays. The data obtained also demonstrated the need to use several parameters for a better estimate of cellular sensitivity to the action of genotoxic agents, which would be important in terms of radiobiology, oncology and therapeutics.
... In that study [2], the radiation responses from eight individuals, aged 23 to 57 years, were measured. In a later study [4], these data, together with data from a range of other radiations, were used to establish an RBE M (limiting relative radiobiological effectiveness) versus LET (linear energy transfer) relationship for micronuclei induction in human lymphocytes. More recently, as part of a wider study [5] of the relationship between individual sensitivity to radiation and individual sensitivity to chemotherapeutic agents [6], we had the opportunity to obtain blood samples from three of the eight donors used in the original study by Hall and Wells [2] and were able to reassess the response of these same individuals to the same source of radiation. ...
... In part this is due to a single individual who consistently recorded a very high spontaneous rate of ∼0.13 micronuclei per bi-nucleated cell. Factors which can affect the spontaneous frequency [4]. Despite this there are clearly no large differences in the average responses to radiation of the 1987 and 1998 populations as shown in figure 1, although the point at ∼3 Gy from the 1987 study is rather high. ...
Article
Full-text available
In this short note we describe the results of a unique 11 year follow-up of the induction of micronuclei by radiation in three individuals. These individuals were all part of two larger studies carried out in 1987 and 1998 respectively, each having similar population characteristics. No significant differences in the average radiation response of these two populations were observed, nor were there any apparent differences in the 1987 and 1998 responses of two of the three individuals reassessed. Data from the third individual (and from a wider study reported elsewhere) do, however, provide some evidence for an age dependence. It is concluded that significant individual variations in the age-dependent responses to radiation may exist, and that while for some individuals there is no increase in radiosensitivity with age, for others there is. Such age dependences may be diluted by studying age-related responses in whole populations of limited size rather than by following individuals over a long period of time. The results reported here are from a limited data set and it is important that further studies are carried out to provide evidence for or against the existence of an age-dependent response to radiation in some individuals.
... In vitro. Mill et al. 1996 20, 23, cyclotron In vitro. Durante et al. 1992 177, microbeam source Figure 6. ...
Article
Purpose: Adverse outcome pathways (AOPs) provide a modular framework for describing sequences of biological key events (KEs) and key event relationships (KERs) across levels of biological organization. Empirical evidence across KERs can support construction of quantified AOPs (qAOPs). Using an example AOP of energy deposition from ionizing radiation onto DNA leading to lung cancer incidence, we investigate the feasibility of quantifying data from KERs supported by all types of stressors. The merits and challenges of this process in the context of AOP construction are discussed.Materials and methods: Empirical evidence across studies of dose-response from four KERs of the AOP were compiled independently for quantification. Three upstream KERs comprised of evidence from various radiation types in line with AOP guidelines. For these three KERs, a focused analysis of data from alpha-particle studies was undertaken to better characterize the process to the adverse outcome (AO) for a radon gas stressor. Numerical information was extracted from tables and graphs to plot and tabulate the response of KEs. To complement areas of the AOP quantification process, Monte Carlo (MC) simulations in TOPAS-nBio were performed to model exposure conditions relevant to the AO for an example bronchial compartment of the lung with secretory cell nuclei targets.Results: Quantification of AOP KERs highlighted the relevance of radiation types under the stressor-agnostic intent of AOP design, motivating a focus on specific types. For a given type, significant differences of KE response indicate meaningful data to derive linkages from the MIE to the AO is lacking and that better response-response focused studies are required. The MC study estimates the linear energy transfer (LET) of alpha-particles emitted by radon-222 and its progeny in the secretory cell nuclei of the example lung compartment to range from 94 - 5 + 5 - 192 - 18 + 15 keV/µm.Conclusion: Quantifying AOP components provides a means to assemble empirical evidence across different studies. This highlights challenges in the context of studies examining similar endpoints using different radiation types. Data linking KERs to a MIE of 'deposition of energy' is shown to be non-compatible with the stressor-agnostic principles of AOP design. Limiting data to that describing response-response relationships between adjacent KERs may better delineate studies relevant to the damage that drives a pathway to the next KE and still support an 'all hazards' approach. Such data remains limited and future investigations in the radiation field may consider this approach when designing experiments and reporting their results and outcomes.
... The effects of gamma radiation on people exposed to its action, in accidents or therapeutic uses, are difficult to estimate in an biological approach [1]. The estimates of physical dosimetry underscores interpersonal biological diversity [2]. Nucleic acids and proteins are affected by radiation, both directly or by free radical action due water radiolysis [3]. ...
Article
Full-text available
Gamma irradiation affects people in several situations, with few if any sensitive biological assay of its action. Nucleic acids and proteins are affected by radiation, but only the former was used in most dosimetric techniques. The irradiation of proteins promotes structural modifications attributed to free radicals from water radiolysis. Theoretically, antibodies induced by irradiated proteins could recognize these radical-related new epitopes, allowing their use as a probe. Human erythrocyte membrane proteins(HEMP), few and well defined molecules, are certainly exposed to radiation, being the ideal target. With this rationale, we study the production of antibodies in mice immunized with 60 Co irradiated HEMPs. Membranes from hypotonic lysis with differential centrifugation of A+ erythrocytes, were irradiated in a Gammacell 220 with 400, 800 and 1600Gy, and used as immunogen for Balb/c mice, after SDS-PAGE. Irradiated HEMP induced antibodies recognize only irradiated human erythrocytes in an intact cell indirect immunofluorescence assay(ICIIFA). When used in Western-blot against non-irradiated HEMPs, those sera recognize most proteins, suggesting a pool of abs directed both to native, as detected by Western Blot, or irradiated, as detected by ICIIFA, HEMPs. Those data confirmed our assumptions, allowing the use of those abs in the search for a method of biological dosimetry.
... Radiation protection measures have been adopted based on the results of laboratory investigations regarding low-dose radiation exposures, which suggest a linear, no-threshold, cumulative response. However, most low-dose laboratory exposures to radiation [3,5,6] are orders of magnitude greater than recent environmental levels of the Chornobyl exclusion zone, one of the most radioactive environments in the world. Several studies have challenged cumulative effects and linear no-threshold hypotheses and, instead, have reported hormesis from exposure to low doses of ionizing radiation789. ...
Article
Previous studies have demonstrated no difference in micronucleus (MN) frequencies between wild rodents chronically exposed to the environmental radiation contamination of the Chornobyl (Ukraine) exclusion zone and those inhabiting reference populations. The aim of the present study was to test the hypothesis that a population of bank voles (Clethrionomys glareolus) has developed radioresistance as a result of 14 years of chronic, low-dose radiation exposure. Naive voles were placed in environmental enclosures in the Red Forest region of the exclusion zone for 30 d. Blood samples were obtained at regular intervals, and the MN assay was used to assess chromosomal damage. Additionally, radionuclide uptake was monitored throughout the study, and dose was documented for each individual as well as for their offspring. Total dose for the voles experimentally exposed in this environment averaged 1.09 Gy (36.20 mGy d(-1)) for the 30-d study period. Our results indicate that exposure to radiation levels well above regulatory statutes did not result in an increased MN frequency. Furthermore, our results do not support the hypothesis that voles chronically exposed to these radiation levels have developed a genetic basis for radioresistance that is unique from that present in naive populations. The use of C. glareolus as a sentinel species for environmental studies of radiation contamination and the question of whether the MN assay is an appropriate endpoint for studies of low-dose, chronic radiation exposure are also discussed.
... The relationship between MN frequency and age of donors has been considered by several authors, with discordant results. Some reports have described a positive correlation between age and MN frequency in human lymphocytes (Fenech and Morley, 1986;Ghosh et al., 1990;Migliore et al., 1991;Ganguly, 1993;Mill et al., 1996;Thierens et al., 1996), while other cytogenetic studies on MN have not shown an age effect (Huber et al., 1989;Sinues et al., 1991). In the present study no relationship was found between the frequency of MN and donor age in both controls and patients. ...
Article
Full-text available
To evaluate the genetic damage induced by tuberculosis infection and also by a combination of therapeutic exposure to anti-tuberculosis (anti-TB) drugs (isoniazid + rifampicin + pyrazinamide + ethambutol or streptomycin) we have considered chromosome aberrations (CA) and micronuclei (MN) in binucleate peripheral blood lymphocytes from 36 patients diagnosed with pulmonary tuberculosis prior to receiving anti-TB drugs in the first phase and in the second phase after 6 months of therapy with anti-TB drugs. The same cytogenetic study has also been performed on 36 healthy individual controls. It was shown that the frequencies of both CA and micronucleated binucleate cells increased significantly after therapy with anti-TB drugs as compared with TB patients and controls. In addition, we observed that both cytogenetic markers (CA and MN) in patients before treatment with anti-TB drugs were significantly (P: < 0.05) higher than controls. No relationship was found between the frequency of MN and donor age. Thus, apart from drug effects, infection with tuberculosis is associated with increased CA and MN.
... Linear component of the dose response relationship for dicentric or micronucleus induction in human lymphocyte dependence on linear energy transfer (LET). Data: NRPB: [1] [2] [3], GSF: [4], Kyoto: [5], MPL: [6] [7] [8] and BC: [9]. ...
Article
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Controversy exists regarding the biological effectiveness of low energy x-rays used for mammography breast screening. Recent radiobiology studies have provided compelling evidence that these low energy x-rays may be 4.42 +/- 2.02 times more effective in causing mutational damage than higher energy x-rays. These data include a study involving in vitro irradiation of a human cell line using a mammography x-ray source and a high energy source which matches the spectrum of radiation observed in survivors from the Hiroshima atomic bomb. Current radiation risk estimates rely heavily on data from the atomic bomb survivors, and a direct comparison between the diagnostic energies used in the UK breast screening programme and those used for risk estimates can now be made. Evidence highlighting the increase in relative biological effectiveness (RBE) of mammography x-rays to a range of x-ray energies implies that the risks of radiation-induced breast cancers for mammography x-rays are potentially underestimated by a factor of four. A pooled analysis of three measurements gives a maximal RBE (for malignant transformation of human cells in vitro) of 4.02 +/- 0.72 for 29 kVp (peak accelerating voltage) x-rays compared to high energy electrons and higher energy x-rays. For the majority of women in the UK NHS breast screening programme, it is shown that the benefit safely exceeds the risk of possible cancer induction even when this higher biological effectiveness factor is applied. The risk/benefit analysis, however, implies the need for caution for women screened under the age of 50, and particularly for those with a family history (and therefore a likely genetic susceptibility) of breast cancer. In vitro radiobiological data are generally acquired at high doses, and there are different extrapolation mechanisms to the low doses seen clinically. Recent low dose in vitro data have indicated a potential suppressive effect at very low dose rates and doses. Whilst mammography is a low dose exposure, it is not a low dose rate examination, and protraction of dose should not be confused with fractionation. Although there is potential for a suppressive effect at low doses, recent epidemiological data, and several international radiation risk assessments, continue to promote the linear no-threshold (LNT) model. Finally, recent studies have shown that magnetic resonance imaging (MRI) is more sensitive than mammography in detecting invasive breast cancer in women with a genetic sensitivity. Since an increase in the risk associated with mammographic screening would blur the justification of exposure for this high risk subgroup, the use of other (non-ionising) screening modalities is preferable.
... Ionizing radiation (IR) consists of alpha, beta and neutron particles, as well as X and gamma rays. As a result of ionization, chemical reactions are initiated and they lead to major disorders of a number of cell molecules, including DNA [1][2][3]. Radiation-induced DNA damage initiates the signaling the transduction pathway, known as the DNA damage response (DDR), resulting in the activation of multiple cellular signaling molecules to determine the cell fate, including cell cycle arrest, apoptosis, senescence, autophagy and DNA repair [4,5]. ...
Article
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Ionizing radiation may be of both artificial and natural origin and causes cellular damage in living organisms. Radioactive isotopes have been used significantly in cancer therapy for many years. The formation of DNA double-strand breaks (DSBs) is the most dangerous effect of ionizing radiation on the cellular level. After irradiation, cells activate a DNA damage response, the molecular path that determines the fate of the cell. As an important element of this, homologous recombination repair is a crucial pathway for the error-free repair of DNA lesions. All components of DNA damage response are regulated by specific microRNAs. MicroRNAs are single-stranded short noncoding RNAs of 20–25 nt in length. They are directly involved in the regulation of gene expression by repressing translation or by cleaving target mRNA. In the present review, we analyze the biological mechanisms by which miRNAs regulate cell response to ionizing radiation-induced double-stranded breaks with an emphasis on DNA repair by homologous recombination, and its main component, the RAD51 recombinase. On the other hand, we discuss the ability of DNA damage response proteins to launch particular miRNA expression and modulate the course of this process. A full understanding of cell response processes to radiation-induced DNA damage will allow us to develop new and more effective methods of ionizing radiation therapy for cancers, and may help to develop methods for preventing the harmful effects of ionizing radiation on healthy organisms.
... ,1, [11][12][13][14][15][16][17][18] , , , ...
... Beta particles with a maximum energy of 2.2 MeV are emitted from the 90 Sr daughter product ( 90 Y). The source has a trackaveraged LET of 0.26 keV m Ϫ1 (16). 90 Sr/ 90 Y dosimetry. ...
Article
Full-text available
Considerable controversy currently exists regarding the biological effectiveness of 29 kVp X rays which are used for mammography screening. This issue must be resolved to enable proper evaluation of radiation risks from breast screening. Here a definitive assessment of the biological effectiveness of 29 kVp X rays compared to the quality of radiation to which the atomic bomb survivors were exposed is presented for the first time. The standard radiation sources used were (a) an atomic bomb simulation spectrum and (b) 2.2 MeV electrons from a strontium-90/yttrium-90 (90Sr/90Y) radioactive source. The biological end point used was neoplastic transformation in vitro in CGL1 (HeLa x human fibroblast hybrid) cells. No significant difference was observed for the biological effectiveness of the two high-energy sources for neoplastic transformation. A limiting relative biological effectiveness (RBE(M)) of 4.42 +/- 2.02 was observed for neoplastic transformation by 29 kVp X rays compared to these two sources. This compares with values of 4.67 +/- 3.93 calculated from previously published data and 3.58 +/- 1.77 when the reference radiation was 200 and 220 kVp X rays. This suggests that the risks associated with mammography screening may be approximately five times higher than previously assumed and that the risk-benefit relationship of mammography exposures may need to be re-examined.
... However, a difference in LET is less likely to explain the different bystander response of BT-474 to EBRT vs 90 Y irradiation. The LET of electrons arising from 137 Cs and 90 Y irradiation are ~1keV/m and 0.26keV/m respectively, compared to 60-100keV/m in the case of alpha emitters [47,167,168]. Furthermore, Anzenburg et al found that neither irradiation with X-rays or alpha particles elicited a bystander response in the DU145 cells themselves. ...
... The values for the a and b coefficients obtained here for electrons and gamma rays are comparable to those reported earlier (Prosser et al. 1988;Thierens et al. 1991). The differences in the in vitro dose-response coefficient values obtained from different laboratories for micronuclei induction in PBLs after low LET irradiation (Prosser et al. 1988;Sreedevi and Bharathi 1989;Littlefield et al. 1989;Thierens et al. 1991;Mill et al. 1996) suggest a certain degree of variation in different laboratories. The frequencies of micronuclei after irradiation with electrons as reported here are comparable with those reported by Prosser et al. (1988) and Fenech and Morley (1986), but for doses [2 Gy, our yields are lower than those observed by Kormos and Koteles (1988) and Sreedevi and Rao (1994). ...
Article
The effect of electrons and gamma irradiation on the induction of micronuclei in cytokinesis-blocked human peripheral blood lymphocytes was investigated to understand the relative biological effectiveness (RBE) of electrons compared with gamma rays. Blood samples were irradiated with an 8 MeV pulsed electron beam, at a mean instantaneous dose rate of 2.6 9 10 5 Gy s-1. Gamma irradiation was carried out at a dose rate of 1.98 Gy min-1 using 60 Co gamma source. A dose-dependent increase in micronuclei yield was observed. The dose-response relationships for induction of micronuclei fitted well to a linear-quadratic relationship and the coefficients a and b of the dose-response curve were estimated by fitting the data using error-weighted minimum v 2 method. The RBE of 8 MeV electrons were found to be near unity as compared with gamma rays.
... [6] In vitro MN test with CBMN of human peripheral blood lymphocytes has been used extensively to study chromosomal damage induced by ionizing radiation or chemicals. [7] Because radiation-induced MN showed a radiation dose and quality dependence, MN can be used as a biological dosimeter for radiation protection purposes [8] and MN assay has been recommended by ...
Article
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The cytokinesis-block micronucleus assay in peripheral blood lymphocytes is an established technique for biodosimetry. The aim of this project was to generate a X-ray induced micronuclei (MN) curve for peripheral blood lymphocytes taken from five healthy donors. The blood samples were irradiated with X-rays of 122 KeV at a dose rate of 0.652 Gy/min to doses of 0.5, 1, 2, 3, and 4 Gy. The blood samples were then cultured for 72 h at 37°C and processed following the International Atomic Energy Agency standard procedure with slight modifications. The result showed that the yields of MN frequencies were increased with the increase of radiation dose. Reconstruction of the relationship of MN with dose was fitted to a linear-quadratic model using Chromosome Aberration Calculation Software version 2.0. Due to their advantages, mainly, the dependence on radiation dose and dose rate, despite their limitation, these curves will be useful as alternative method for in vitro dose reconstruction and can support the preparedness for public or occupational radiation overexposure and protection. The results reported here also give us confidence to apply the obtained calibration curve of MN for future biological dosimetry requirements in Indonesia.
... The dose-response relationship for the induction of micronuclei in cultured cells following exposure to high dose rates and for a range of different radiation types can be described as linear or linear-quadratic (Heddle et al. 1991;Mill et al. 1996). However, when the dose rate is low, induced micronuclei do not follow linear kinetics. ...
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Purpose: This review evaluates the role of dose rate on cell and molecular responses. It focuses on the influence of dose rate on key events in critical pathways in the development of cancer. This approach is similar to that used by the U.S. EPA and others to evaluate risk from chemicals. It provides a mechanistic method to account for the influence of the dose rate from low-LET radiation, especially in the low-dose region on cancer risk assessment. Molecular, cellular, and tissues changes are observed in many key events and change as a function of dose rate. The magnitude and direction of change can be used to help establish an appropriate dose rate effectiveness factor (DREF). Conclusions: Extensive data on key events suggest that exposure to low dose-rates are less effective in producing changes than high dose rates. Most of these data at the molecular and cellular level support a large (2-30) DREF. In addition, some evidence suggests that doses delivered at a low dose rate decrease damage to levels below that observed in the controls. However, there are some data human and mechanistic data that support a dose-rate effectiveness factor of 1. In summary, a review of the available molecular, cellular and tissue data indicates that not only is dose rate an important variable in understanding radiation risk but it also supports the selection of a DREF greater than one as currently recommended by ICRP ( 2007 ) and BEIR VII (NRC/NAS 2006 ).
... Their results, as reported in literature, showed a relatively high dispersion concerning α and β parameters for both type of (c) RJP64(Nos. 5-6), ID 702-1 (2019) v.2.1r20190102 *2019.7.8#012f5511 radiation [6,[9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. It is also worth mentioning that the actual measurement conditions are significantly more accurate which imposes a periodical re-examination of this model. ...
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To estimate at which extent the exposure to gamma and X-rays influences the Micronuclei (MN) frequency, a non-specific chromosomal aberration, the same amount of blood cells were exposed, in identical conditions, (extended uncertainties less than 0.8%), to gamma and X rays at gradually increased doses up to 3 Gy. The final results showed that, within this range, in both cases the MN frequency increases with the dose by following a second order polynomial (the linear-quadratic model), which coefficients are, within experimental uncertainties, almost coincident.
... Moreover, this pattern is highlighted because the dose-response curves for VHDR resemble those observed after exposure of PBMC to radiation of high linear energy transfer (LET) that is known to induce a high level of difficult to repair, complex DNA damage (Goodhead 2006). For MN in PBMC, upward linear-quadratic dose-response relationships are common for low LET radiation like photons (Depuydt et al. 2017), while linear or saturating dose-response relationships are observed for alpha particles (Johannes et al. 2010;Mill et al. 1996). For gene expression, the opposite is observed: the dose-response is downward linear quadratic for alpha particles and linear for photons . ...
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Many experimental studies are carried out to compare biological effectiveness of high dose rate (HDR) with that of low dose rate (LDR). The rational for this is the uncertainty regarding the value of the dose rate effectiveness factor (DREF) used in radiological protection. While a LDR is defined as 0.1 mGy/min or lower, anything above that is seen as HDR. In cell and animal experiments, a dose rate around 1 Gy/min is usually used as representative for HDR. However, atomic bomb survivors, the reference cohort for radiological protection, were exposed to tens of Gy/min. The important question is whether gamma radiation delivered at very high dose rate (VHDR—several Gy/min) is more effective in inducing DNA damage than that delivered at HDR. The aim of this investigation was to compare the biological effectiveness of gamma radiation delivered at VHDR (8.25 Gy/min) with that of HDR (0.38 Gy/min or 0.79 Gy/min). Experiments were carried out with human peripheral mononuclear cells (PBMC) and the human osteosarcoma cell line U2OS. Endpoints related to DNA damage response were analysed. The results show that in PBMC, VHDR is more effective than HDR in inducing gene expression and micronuclei. In U2OS cells, the repair of 53BP1 foci was delayed after VHDR indicating a higher level of damage complexity, but no VHDR effect was observed at the level of micronuclei and clonogenic cell survival. We suggest that the DREF value may be underestimated when the biological effectiveness of HDR and LDR is compared.
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A relative biological effectiveness (RBE) not much larger than unity is usually assumed for soft x-rays (up to approximately 50 keV) that are applied in diagnostic radiology such as mammography, in conventional radiotherapy and in novel radiotherapy approaches such as x-ray phototherapy. On the other hand, there have been recent claims of an RBE of more than 3 for mammography and respective conventional x-rays. Detailed data on the RBE of soft x-rays, however, are scarce. The aim of the present study was to determine the effect of low-energy x-rays on chromosomal damage in vitro, in terms of micronucleus induction. Experiments were performed with 25 kV x-rays and a 200 kV x-ray reference source. The studies were carried out on primary human epidermal keratinocytes (HEKn), human fibroblasts (HFIB) and NIH/3T3 mouse fibroblasts. Micronucleus (MN) induction was assayed after in vitro irradiation with doses ranging from 1 to 5.2 Gy. Compared to the effect of 200 kV x-rays, 25 kV x-rays resulted in moderately increased chromosomal damage in all cell lines studied. This increase was observed for the percentage of binucleated (BN) cells with micronuclei as well as for the number of micronuclei per BN cell. Moreover, the increased number of micronuclei per micronucleated BN cell in human keratinocytes and 3T3 mouse fibroblasts suggests that soft x-rays induce a different quality of damage. For all cell lines studied the analysis of micronucleus induction by 25 kV soft x-rays compared to 200 kV x-rays resulted in an RBE value of about 1.3. This indicates a somewhat enhanced potential of soft x-rays for induction of genetic effects.
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Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years+/-11.2, in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv+/-49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls (10.7 per thousand +/-5.2 versus 6.6 per thousand +/-3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C-MN) in exposed subjects than in controls (8.5 per thousand +/-4.9 versus 2.2 per thousand +/-1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C-MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.
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The purpose of this study was to evaluate the degree of cytological radiation damage to peripheral blood lymphocytes induced by 153Sm-EDTMP applied for palliation of metastatic bone pain. Blood samples from 16 patients (46-82 years old), 10 without previous radiotherapy and 6 with previous radiotherapy, were collected before and one hour after the administration of a mean activity of 41.7+/-5.8 MBq/kg of 153Sm-EDTMP. Then the lymphocytes were cultured for cytokinesis block micronucleus (MN) assay. The number of MNper binucleated cells (BC) in patients without previous radiotherapy before the treatment was of 0.030 (+/- 0.016) and after one hour 0.035 (+/- 0.013), although we could find inter individual differences. The basal MN/BC of the patients with no previous radiotherapy was similar to the controls. The increment in the percentage of BC with MN was similar in patients with and without previous radiotherapy. The observed mean of MN/BC is equivalent to a dose range of 0.05 to 0.10 Gy of 153Sm-EDTMP in vitro. The relatively low frequency of lymphocyte with micronuclei after the exposure to 153Sm-EDTMP supported the contention that radiation damage in lymphocytes of patients with painful bone metastases is minimal.
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An assay for biological dosimetry based on the induction of apoptosis in human T-lymphocytes is described. Radiation-induced apoptosis was assessed by flow cytometric identification of cells displaying apoptosis-associated DNA condensation. CD4 and CD8 T-lymphocytes were analysed. They were recognized on the basis of their cell-surface antigens. Four parameters were measured for both cell types: cell size, granularity, antigen immunofluorescence and DNA content. Apoptosis was quantified as the fraction of CD4-, or CD8-positive cells with a characteristic reduction of cell size and DNA content. At doses below 1 Gy, levels of radiation-induced apoptosis increased for up to 5 days after irradiation. Optimal dose discrimination was observed 4 days after irradiation, at which time the dose-response curves were linear, with a slope of 8% +/- 0.5% per 0.1 Gy. In controlled, dose-response experiments the lowest dose level at which the radiation-induced apoptosis frequency was still significantly above control was 0.05 Gy. After 5 days post-irradiation incubation, intra- and interdonor variations were measured and found to be similar; thus, apoptotic levels depend more on the dose than on the donor. The results demonstrate the potential of this assay as a biological dosimeter.
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The frequencies of gamma-ray-induced micronuclei (MN) in cytokinesis-blocked (CB) lymphocytes at several doses were measured in three donors of seven species (human, cattle, goat, pig, rabbit, chicken, fish). Measurements performed after irradiation showed a dose-related increases in MN frequency in each of the donors of human, cattle, goat, pig and rabbit. The relative sensitivity of cattle, goat, pig and rabbit in peripheral blood lymphocytes (PBLs) compared with human PBLs was estimated by best fitting linear-quadratic model based on the radiation-induced MN data over the range from 0 cGy to 400 cGy. In the case of MN frequency with 0.2, the relative sensitivities of cattle, goat, pig and rabbit PBLs were 0.86, 0.98, 0.41 and 0.39, respectively. These data indicate that the induction of MN in CB cells following irradiation is similar in human, cattle and goat PBLs, while PBLs from pig and rabbit were much less sensitive to the MN induction effects of gamma-radiation than those from human. The micronucleus counts failed to show any evidence of radiation damage in the cells from chicken and fish. Measurements performed after irradiation showed a dose-related decrease in the formation of binucleated cells. We concluded that the use of CB cell from fish and chicken for detecting the results of radiation exposure was highly questionable. Our in vitro radiobiological study confirmed that the cytogenetic response obtained in blood from selected breeds of mammalian species can be utilized for application in environmental studies.
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To study the relative biological effectiveness-linear energy transfer (RBE-LET) relationship for micronuclei (MN) and cell inactivation, in Chinese hamster cells irradiated with low-energy protons (0.88 and 5.04 MeV, at the cell entrance surface). Chromosome loss was also investigated by means of antikinetochore CREST staining. Cl-1 cells were exposed to different doses of X-rays, gamma-rays, 7.7 keV/microm and 27.6 keV/microm protons. The induction of MN, the distribution of MN per cell and the frequency of CREST-positive MN were evaluated in cytokinesis-blocked binucleated cells (BN cells) in the dose range 0.125-3 Gy. In parallel, cell survival experiments were carried out in samples irradiated with 0.5 to 4 Gy. MN yield and the frequency of BN cells carrying multiple MN (> or =2) were significantly higher after exposure to 27.6 keV/microm protons, compared with the other radiation types. In contrast, MN induction and MN distribution per BN cell were similar among 7.7 keV/microm protons, X- and gamma-rays up to 1 Gy. Cell survival experiments gave RBE values very close to those obtained with the MN assay. Both X-rays and 27.6 keV/microm protons yielded a significant proportion of CREST-positive MN at the highest doses investigated (0.75-3 Gy). Good correlations between MN induction and cell inactivation were observed for both low- and high-LET radiation, indicating that the MN assay can be a useful tool to predict cell sensitivity to densely ionizing radiation with implications for tumour therapy with protons.
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This study set out to analyze biomarkers for genotoxic events, e.g., oxidative DNA damage, chromosomal damage and hprt mutations, among flight personnel, who are known to be occupationally exposed to ionizing radiation of cosmic origin. Twenty-three flight engineers were recruited while ground personnel served as a matched control group. Cumulative radiation doses during flight were calculated on the basis of subjects' flight records assuming an exposure rate of 6 microSv per hour of flight. Oxidative DNA damage in peripheral lymphocytes from flight engineers appeared significantly increased in comparison with controls and was associated with cumulative exposure to cosmic radiation. Frequencies of peripheral lymphocyte chromosome aberrations, micronuclei and hprt mutations appeared also to be increased in flight engineers, but not significantly. It was also observed that DNA damage was higher in flight engineers with a relatively shorter flight history in comparison with flight engineers with higher cumulative exposures to radiation, suggesting adaptation to DNA damage caused by ionizing radiation. DNA repair activities measured as unscheduled DNA synthesis were clearly increased in the higher-exposed subgroup of flight engineers, and appeared significantly correlated with cumulative radiation dose, as well as inversely with oxidative DNA damage. The implications for cancer risk assessment in relation to exposure to cosmic radiation are discussed.
Article
The effect of dose rate on the induction of micronuclei (MN) in human peripheral blood lymphocytes was investigated over a range of dose rates from 0.125 Gy h(-1) to 178.2 Gy h(-1). The response of MN induction was fitted with a linear quadratic model and the alpha and beta coefficients were estimated. It was found that beta values decrease with decreasing dose rate as in the case of chromosomal aberration. At the dose rate of 0.125 Gy h(-1), pure linear response of MN induction was observed. An attempt was made to simulate the calibration curve for the purpose of biological dosimetry at different dose rates and exposure times. The yields when simulated with the exposure time or the dose rate are in agreement with experimental results.
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The following manuscript integrates various studies of cellular radiation response. This review gives a background to the original article in the accompanying edition of the journal Acta Oncological. Every attempt has been made to void needless repetition of the text but to maintain the autonomy of both works. In most cases, figures or tables have been reproduced only once. The final aim of the studies was to understand better, and where possible, even improve on the success of cancer treatment using radiation therapy. From the start it was clear that qualitative aspects of cellular response to ionizing radiation could not have been predicted. Cells are normally exposed to very low levels of ionizing radiation. They are certainly not able to recognise this radiation, in spite of their rich spectrum of receptors. Cells can and do respond to the damage caused by radiation as it passes through, but the response to ionizing radiation is more complex than simply repair. Cellular programs are activated and processes are induced which interfere with the most fundamental aspects of biological life. The following is a tour-de-force to elucidate the principles behind programmed cellular response to ionizing radiation damage and to examine potential applications in the clinic. Cellular response is initiated by the deposition of energy during passage of radiation through sensitive cellular targets and the production of macromolecular damage by ionization events and free radicals. At radiation doses employed for clinical purposes, or at doses of relevance for radiation protection, the cell responds in an active manner. Radiation damage is recognize by intra-cellular receptor molecules and signals are subsequently transduced throughout the cell which mobilize proteins involved in executing appropriate cellular responses. In many cell types, responses often involve an attempt to repair the damage. If the cells are replicating, transitory arrests also occur before or during critical phases of the cell cycle. However, probably because they are restricted to fewer growth controls than cells in solid tissues, lymphocytes preferentially respond to DNA damage by inducing a process of cellular suicide; apoptosis, which effectively eliminates this potential source of carcinogenic risk. Cellular radiation response is the result of active molecular signalling and is not simply a passive physico-chemical process. The decision whether or not a cell should respond to radiation-induced damage either by induction of rescue systems, e.g. mobilization of repair proteins, or induction of suicide mechanisms, e.g. programmed cell death, is an expression of intricate cellular biochemistry. A cell must recognize damage in its genetic material and then activate an appropriate response. Cell type is important, the response of a fibroblast to radiation damage is both quantitatively and qualitatively different from a lymphocyte, and cellular transformation may also be accompanied by changes in radiosensitivity. The programmed component of radiation response is highly significant in radiation oncology and creates unique opportunities for enhanced treatment success. The hallmark of an active cellular response is signal production; the encoding of some relevant piece of information in a form which can be handled by the cell in order to make a 'decision' at the molecular level (i.e. whether or not a repair mechanism should be activated). The cell cycle arrests induced either by radiation damage or staurosporine are closely related phenomena governed by a common kinase signalling pathway which recognizes DNA damage and regulates the G2/M transition. Such signal pathways are believed to influence many of the programmed responses to radiation damage. Current estimates suggest that approximately 5 per cent of cancer patients receiving radiotherapy display adverse reactions due to hypersensitivity. These differences are often due to genetic defects which compromise programmed cellular response and result in abnormal radiosensitivity. Assays of radiation response have yielded much valuable information about the radiobiology of tumours. New developments in cellular and molecular biology increase our understanding of the programmed component of radiation response, helping us to take better advantage of this phenomenon in the clinical setting.
Article
Scoring of micronuclei (Mni) in cytokinesis-blocked human blood peripheral lymphocytes after an accidental radiation overexposure appears an easier and faster alternative for biological dosimetry than dicentrics analysis. However, an increase of Mni rate could be difficult to interpret particularly at low doses, because of the known variability of individual dose response and the unknown background frequency. Moreover, in case of nuclear emergency, there are the added problems of large samples numbers for processing and so screening time. In this paper, we wish to propose some solutions using both methodological and statistical approaches. Firstly, we have tried to check the micronuclei assay in order to obtain a sufficient number of micronuclei in binucleated cells in the shortest time possible, even at higher exposition dose. Two techniques were compared using frequencies of binucleated cells and micronuclei in normal lymphocytes and after exposure to gamma-irradiation (60 Co) for doses up to 6 Gy. Secondly, we have supposed that, if the individual radiosensitivity was a critical problem for dose estimation, conversely, it would be not possible to build a reference curve combining many individuals. Thus, we have assessed the Mni response from 47 carefully selected healthy male blood donors for gamma-irradiation between 0 and 4 Gy. We show in this study that a realistic dose-effect relationship could be always fitted. In addition, the related coefficients could be compared with other published dose-effect relationships for gamma rays. However, the background incidence calculated from these 47 healthy individuals were found to be larger as expected so that this approach increases the detection limit for which an overexposure suspicion could be significantly detected to 0.32 Gy.
Article
To investigate the chromosomal damage caused by gamma-irradiation in T lymphocytes and natural killer (NK) cells and compare this with apoptosis induction in both lymphocyte subsets. Apoptosis induction by gamma-irradiation in T lymphocytes and NK cells was quantified using the annexin V flow cytometric assay. The cytokinesis-block micronucleus (MN) assay was used to evaluate the induced cytogenetic damage. For the MN assays on NK cells, gamma-irradiated peripheral blood mononuclear cells were cultured and stimulated with interleukin 15 (IL-15). Afterwards the NK cells (characterized by the CD3-/CD56+ phenotype) were separated with the FACSort flow cytometer and the number of MN in the sorted binuclear cells was scored. Doses of 1 and 2 Gy gamma-irradiation were applied. Higher numbers of MN in NK cells were found compared with the MN yield in T lymphocytes. In contrast, NK cells were less than T lymphocytes prone to apoptosis after gamma-irradiation. The results support the view that cytogenetic damage and apoptosis after gamma-irradiation are not necessarily correlated.
Article
Information on Japanese A-bomb survivors exposed to gamma radiation has been used to estimate cancer risks for the whole range of photon (x-rays) and electron energies which are commonly encountered by radiation workers in the work place or by patients and workers in diagnostic radiology. However, there is some uncertainty regarding the radiation effectiveness of various low-linear energy transfer (low-LET) radiations (x-rays, gamma radiation and electrons). In this paper we review information on the effectiveness of low-LET radiations on the basis of epidemiological and in vitro radiobiological studies. Data from various experimental studies for chromosome aberrations and cell transformation in human lymphocytes and from epidemiological studies of the Japanese A-bomb survivors, patients medically exposed to radiation for diagnostic and therapeutic procedures, and occupational exposures of nuclear workers are considered. On the basis of in vitro cellular radiobiology, there is considerable evidence that the relative biological effectiveness (RBE) of high-energy low-LET radiation (gamma radiation, electrons) is less than that of low-energy low-LET radiation (x-rays, betas). This is a factor of about 3 to 4 for 29 kVp x-rays (e.g. as in diagnostic radiation exposures of the female breast) and for tritium beta-rays (encountered in parts of the nuclear industry) relative to Co-60 gamma radiation and 2-5 MeV gamma-rays (as received by the Japanese A-bomb survivors). In epidemiological studies, although for thyroid and breast cancer there appears to be a small tendency for the excess relative risks to decrease as the radiation energy increases for low-LET radiations, it is not statistically feasible to draw any conclusion regarding an underlying dependence of cancer risk on LET for the nominally low-LET radiations.
Article
The effect of electrons and gamma irradiation on the induction of micronuclei in cytokinesis-blocked human peripheral blood lymphocytes was investigated to understand the relative biological effectiveness (RBE) of electrons compared with gamma rays. Blood samples were irradiated with an 8 MeV pulsed electron beam, at a mean instantaneous dose rate of 2.6 × 105 Gy s−1. Gamma irradiation was carried out at a dose rate of 1.98 Gy min−1 using 60Co gamma source. A dose-dependent increase in micronuclei yield was observed. The dose–response relationships for induction of micronuclei fitted well to a linear–quadratic relationship and the coefficients α and β of the dose–response curve were estimated by fitting the data using error-weighted minimum χ 2 method. The RBE of 8 MeV electrons were found to be near unity as compared with gamma rays.
Conference Paper
Sm-153-EDTMP is a radiopharmaceutical used in nuclear medicine for relief of metastatic bone pain with promising results, but there are few Studies about the effects of (SM)-S-153-EDTMP in human cells. This study was conducted for the evaluation of the cytogenetic effects of 153Sm-EDTMP in blood lymphocytes from patients with bone metastases (without previous radio or chemotherapy), using the chromosome aberration technique. The degree of cytological damage found in in vivo blood cells of patients was compared with those found in in vitro in an adjusted dose-response curve. Blood samples were collected before and 1 hr after the administration of Sm-153-EDTMP (about 42.31 MBq/kg). The frequency of structural chromosome aberration per cell observed in I hr samples (0.054 +/- 0.035 CA/cell) was higher than basal ones (0.031 +/- 0.026 CA/cell), although this difference was not statistically significant (p = 0.101). For in vitro assay, blood samples were exposed to different concentrations of Sm-153-EDTMP, during 1 hr (0.37-1.11 MBq/ml). An increase in the frequency of chromosome aberration per cell as a function of the radioactive concentration was found. The data were adjusted by linear regression model (Y = 3.52 +/- 2.24 x 10(-2) + 11.15 +/- 3.46 x 10(-2) X). The frequency of aberration/cell found in vivo was 0.054 and for the same activity in vitro was 0.098, this difference being statistically significant (p = 0.02). This result may be related to blood clearance, osteoblastic activity and individual variability. For a more accurate analysis, the study of more donors is necessary.
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The purpose of the study was to evaluate the usefulness of the cytokinesis-block micronucleus (MN) assay in assessment of radiosensitivity of lymphocytes in cancer patients. Lymphocytes from 15 cervical cancer patients, 21 head and neck cancer patients, seven lung cancer patients and 19 healthy donors were analysed using MN assay. The proportion of binucleate cells (BC) in cancer patients ranged from 22 to 56% and was significantly lower than in the control group (38-68%). MN frequency assessed five times over 6 months in four healthy donors showed that the interindividual variation was significantly higher than intraindividual. Before (0 Gy) and after irradiation (2 and 4 Gy) no statistical differences in the mean number of MN/BC were observed between healthy donors and cancer patient groups. Nevertheless, statistical cluster analysis allowed each group of donors to be divided into radioresistant and radiosensitive subgroups of patients. They showed significantly different dose-response. Separate comparison of the mean MN frequency within all examined radioresistant and radiosensitive subgroups, showed statistically significant differences only after a dose of 4 Gy. At this dose, the lung cancer patients and cervical cancer patients from radiosensitive subgroups presented significantly higher radiosensitivity than the healthy donors. However, healthy donors from radioresistant subgroup did not differ significantly from cancer patients. This work has shown a high variation in interindividual radiosensitivity of donors and suggests the possibility of identifying radiosensitive patients on the basis of MN assay performed on lymphocytes.
Article
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The purpose of this study was to evaluate the degree of cytological radiation damage to lymphocytes induced by beta particle (71% E max = 810 keV) and gamma rays (29% E max = 103 keV) of l53Sm (J&farc12; = 46.3 h). Samarium-153 ethylene diamine tetramethylene phosphonate (153Sm-EDTMP) has been successfully applied as a radiopharmaceutical for palliation of metastatic bone pain at dose of 37 MBq/kg (lmCi/kg) intravenously. Blood samples from four healthy donors and three patients with no previous radiotherapy were exposed to 370, 555 (equivalent to the activity administrated in vivo) and 1110 kBq/mL during one hour in vitro. Then the lymphocytes were cultured for cytokinesis block micronucleus assay that has received increased attention for biological monitoring of radiation exposure. The MN induction in binucleated cells (BNC) at 370 and 555 kBq/mL was not significantly increased and showed no difference between the groups. This result may be explained as a consequence of the sensibility of this technique. The radiation damage to peripheral blood lymphocytes (PBL) exposed to 1110 kBq/mL may be considered to be equivalent to that observed after an external irradiation with 60Co at doses of 0.38 Gy in healthy donors (MN/BNC = 0.053 ± 0.041) and 0.51 Gy in patients (MN/BNC = 0.069 ± 0.040). This study showed that the use of 153 Sm-EDTMP induced no significant increase in the micronucleation of PBL at radioactive concentration lower than 555 kBq/mL (37MBq/kg) and also that the radiosensitivity of the patients was higher at 1110 kBq/mL than that of the healthy donors.
Article
KEYWORDS Beta radiation; biodosimetry; chromosomal aberrations; micronuclei ABSTRACT The yield and distribution of unstable chromosome aberrations induced in human lymphocytes by tritiated water (HTO) has been measured. Tritiated water was mixed with heparinised blood in calculated amounts so as to give 0.1 Gy to 1.5 Gy, 30min and 2 hours. After culturing for 48 hrs, the dicentric yield was measured as a function of dose to the blood. Using a linear quadratic dose-effect relation to fit the experimental data, a significant linear contribution was found. The α , β values were found to be 8.25 + 0.4 x 10 -2 Gy -1 and 6.4 + 0.2 x10 -2 Gy -2 respectively. Micronuclei yield at low doses could be fitted to a linear equation Y= C + α D and indicates a á coefficient of 0.172 ±. 0.003. This value is found to be 2.18 and 2.8 times higher than those previously reported for X and gamma rays respectively. Hence β- rays are found to be more efficient in producing two lesions with single ionizing tracks at low doses.
Article
This study set out to analyze biomarkers for genotoxic events, e.g., oxidative DNA damage, chromosomal damage and hprt mutations, among flight personnel, who are known to be occupationally exposed to ionizing radiation of cosmic origin. Twenty-three flight engineers were recruited while ground personnel served as a matched control group. Cumulative radiation doses during flight were calculated on the basis of subjects' flight records assuming an exposure rate of 6 μSv per hour of flight. Oxidative DNA damage in peripheral lymphocytes from flight engineers appeared significantly increased in comparison with controls and was associated with cumulative exposure to cosmic radiation. Frequencies of peripheral lymphocyte chromosome aberrations, micronuclei and hprt mutations appeared also to be increased in flight engineers, but not significantly. It was also observed that DNA damage was higher in flight engineers with a relatively shorter flight history in comparison with flight engineers with higher cumulative exposures to radiation, suggesting adaptation to DNA damage caused by ionizing radiation. DNA repair activities measured as unscheduled DNA synthesis were clearly increased in the higher-exposed subgroup of flight engineers, and appeared significantly correlated with cumulative radiation dose, as well as inversely with oxidative DNA damage. The implications for cancer risk assessment in relation to exposure to cosmic radiation are discussed. Environ. Mol. Mutagen. 32:121–129, 1998 © Wiley-Liss, Inc.
Article
Objective: The uptake of FDG was measured before, during, and after fractionated radiation in order to evaluate the potential of FDG-PET imaging as an indicator of tumor response.Methods: The study was performed with nude rats bearing the human neuroblastoma BE(2)C tumor xenografts. Tumors were irradiated with 10 fractions of 2 Gy using a 320 kVp X-ray unit. Following a baseline FDG-PET scan, repeat scans were performed weekly until animal sacrifice. The rodents were given up to 10 FDG-PET scans, over a period of up to 75 days posttreatment.Results and Conclusions: Neither, the average and maximum activity/cc of FDG tumor uptake, nor the respective standardized uptake values (SUV), correlated with tumor response. Instead, the total FDG uptake (defined as the product of the average FDG activity/cc with the tumor volume) correlated better with tumor response.
Article
The purpose of this study was to analyse the cytogenetic effect of exposing human peripheral blood lymphocytes (PBL) to a mixed beam of alpha particles and X-rays. Whole blood collected from one donor was exposed to different doses of alpha particles ((241)Am), X-rays and a combination of both. All exposures were carried out at 37 °C. Three independent experiments were performed. Micronuclei (MN) in binucleated PBL were scored as the endpoint. Moreover, the size of MN was measured. The results show that exposure of PBL to a mixed beam of high and low linear energy transfer radiation led to significantly higher than expected frequencies of MN. The measurement of MN size did not reveal any differences between the effect of alpha particles and mixed beam. In conclusion, a combined exposure of PBL to alpha particles and X-rays leads to a synergistic effect as measured by the frequency of MN. From the analysis of MN distributions, we conclude that the increase was due to an impaired repair of X-ray-induced DNA damage.
Conference Paper
The in vitro cytokinesis-block micronucleus cytome (CBMN-cytome) assay is a modified CBMN assay based on the assessment of micronuclei (MNi) in nucleated cells that have completed only one nuclear division. Along with micronucleus measurement, the CBMN cytome assay allows to assess relevant biodosimetric markers like nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), apoptotic and necrotic cells. The present study is aimed at obtaining in vitro dose response data for the induction of MNi, NPBs, NBUDs, apoptotic and necrotic cells in human lymphocytes using 60Co-gamma rays. An attempt has been made to validate possibility of applying NPBs and NBUDs as new biodosimetry endpoints. The 60Co gamma chamber with dose rate 1.2 Gy was used for irradiation. The dose range was selected was 0 to 6 Gy. A total of 1000 binucleated cells per dose were evaluated for the frequency of MNi, NPBs, NBUDs, apoptotic and necrotic cells. The MN induction was found to be linear quadratic and the dose response relationship can be represented by Y (MNi) = (0.04 ± 0.016) + (0.041 ± 0.014) D + (0.038 ± 0.002) D2 (with R2=0.998, and p < 0.0001). The NPBs are narrow DNA- containing bridges linking the two daughter nuclei, and represent an indicator of dicentric chromosomes resulting from mis-repaired DNA breaks or telomere end fusions, which can be a useful tool for biodosimetry. The results show a linear quadratic increase in NPBs with the radiation dose, and the dose response relation can be written as Y (NPBs) = (0.002 ± 8.57×10-4) + (0.0026 ± 8.57×10-5) D + (0.00243 ± 5.4×10-4) D2 (with R2=0.99, and p = 0.0046). The study concludes that NPBs frequency against radiation dose can be used for biodosimetry application, where as NBUDs frequency does not vary significantly with the radiation dose.
Article
Some recent studies have renewed concerns regarding the carcinogenicity of 'hot particles'. These claim an enhanced induction of malignant transformation following non-uniform compared with uniform irradiation. Enhancement factors, however, vary widely between studies. Even fewer animal studies are available and these tOO show divergent results. Determination of possible 'hot particle' enhancement factors is critically dependent on both reliable dosimetry and biological protocols. Some of these experiments are flawed for one or both of these reasons. In this study internationally validated protocols for dosimetry and malignant transformation in vitro have been used. Uniform and non-uniform sources of 90Sr/90Y and 170Tm β particles and 250 kV(p) X rays were used to measure the induced transformation frequency in C3H 10T 1/2 mouse fibroblasts. 'Hot particle' exposures were found not to be significantly different in their transforming ability (expressed as transformation frequency per surviving cell) than uniform irradiation, although moderate enhancement factors are possible. There are, however, suggestions of moderate enhancements for transformation frequency per cell at risk.
Article
Dose Rate Effect of Pulsed Electron Beam on Micronucleus Frequency in Human Peripheral Blood Lymphocytes The micronucleus assay in human peripheral blood lymphocytes is a sensitive indicator of radiation damage and could serve as a biological dosimeter in evaluating suspected overexposure to ionising radiation. Micronucleus (MN) frequency as a measure of chromosomal damage has also extensively been employed to quantify the effects of radiation dose rate on biological systems. Here we studied the effects of 8 MeV pulsed electron beam emitted by Microtron electron accelerator on MN induction at dose rates between 35 Gy min ⁻¹ and 352.5 Gy min ⁻¹ . These dose rates were achieved by varying the pulse repetition rate (PRR). Fricke dosimeter was employed to measure the absorbed dose at different PRR and to ensure uniform dose distribution of the electron beam. To study the dose rate effect, blood samples were irradiated to an absorbed dose of (4.7±0.2) Gy at different rates and cytogenetic damage was quantified using the micronucleus assay. The obtained MN frequency showed no dose rate dependence within the studied dose rate range. Our earlier dose effect study using 8 MeV electrons revealed that the response of MN was linear-quadratic. Therefore, in the event of an accident, dose estimation can be made using linear-quadratic dose response parameters, without adding dose rate as a correction factor.
Article
The present study is aimed at obtaining in vitro dose-response data for the induction of micronucleus (MN) and nucleoplasmic bridges (NPBs) in human lymphocytes using 60Co-gamma rays and 8 MeV pulsed electron beam. An attempt was made to validate the possibility of applying NPBs as new biodosimetry endpoint in the dose range of 0–6 Gy. A total of 1000 binucleated cells (BNCs) per dose point were evaluated for the frequency of MN and NPBs. From the study, it is clear that the dose-response increase of MN and NPBs is linear quadratic in nature. The study provides linear and quadratic parameter for biodosimetry application. The relative biological effectiveness value of the 8 MeV electron beam was estimated using slope values and is found to be 1.18 ± 0.01 for MN/BNCs, 1.27 ± 0.02 for the fraction of BNCs with MN, 1.16 ± 0.13 for MN/(BNCs with MN) and 1.09±0.01 for NPBs.
Article
Micronuclei (MN) were assessed in dermal fibroblasts from C3H HeJ and C57 Bl6 mice (6-10 mo of age) irradiated in vivo as a potential method of biodosimetry. Radiation-induced MN [per 1,000 binucleated (BN) cells], assessed in fibroblasts obtained 1 d post-irradiation, increased in a dose-dependent manner in the range of 1 - 10 Gy per single dose. Analysis at 1 wk post irradiation showed some attenuation of MN response in C3H HeJ male mice, suggesting partial recovery of DNA damage. This was not observed in C57 Bl6 mice. Monomicronucleated cells predominated in unirradiated fibroblasts, whereas in irradiated fibroblasts multimicronucleated cells predominated at dose levels above about 5 Gy (more than 1,000 MN per 1,000 BN cells). Modeling of the data indicate that assaying total MN in dermal fibroblasts from samples of irradiated skin taken up to 1 wk after irradiation can provide biodosimetric information (with an accuracy of 1-2 Gy) for doses up to at least 6 Gy with lesser accuracy at higher doses. Percentage of multimicronucleated cells may be useful as a substitute of total MN at the higher doses, but total percentage of micronucleated cells is of limited value as a substitute.
Article
Thesis (Ph. D.)--Texas Tech University, 2000. Includes bibliographical references.
Article
The irradiation of cells in culture is an essential part of many radiation biology experiments. Since these experiments necessarily involve the irradiation of cell culture vessels and nutrient medium, the possibility of effects due to the interactions of irradiated material with growing cells needed to be investigated. In the present study the micronucleus frequency in Chinese hamster ovary (CHO) cells as a function of such parameters as type of radiation, type of cell substrate, changes in cell environment, and time course of the effect were characterized. Observations of the persistence of micronucleus formation in irradiated CHO cells reveal that the number of cells containing micronuclei reaches its maximum within nine hours after irradiation and remain elevated for at least five days. The influence of the cell environment on micronucleus formation in CHO cells was examined by plating cells in preirradiated nutrient medium or on preirradiated cell culture vessels. In all experiments, pre-irradiation of the cell substrate (the culture dish or culture dish filled with medium) led to a significantly higher micronucleus frequency than when cells were plated on un-irradiated substrate. The difference is most pronounced at the lowest doses examined. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be confounding factors, particularly in the experiments which are intended to examine the response of cells exposed to low doses of ionizing radiation.
Article
The micronucleus assay in human peripheral blood lymphocytes is a sensitive indicator of radiation damage and could serve as a biological dosimeter in evaluating suspected overexposure to ionising radiation. Micronucleus (MN) frequency as a measure of chromosomal damage has also extensively been employed to quantify the effects of radiation dose rate on biological systems. Here we studied the effects of 8 MeV pulsed electron beam emitted by Microtron electron accelerator on MN induction at dose rates between 35 Gy min-1 and 352.5 Gy min-1. These dose rates were achieved by varying the pulse repetition rate (PRR). Fricke dosimeter was employed to measure the absorbed dose at different PRR and to ensure uniform dose distribution of the electron beam. To study the dose rate effect, blood samples were irradiated to an absorbed dose of (4.7±0.2) Gy at different rates and cytogenetic damage was quantifi ed using the micronucleus assay. The obtained MN frequency showed no dose rate dependence within the studied dose rate range. Our earlier dose effect study using 8 MeV electrons revealed that the response of MN was linear-quadratic. Therefore, in the event of an accident, dose estimation can be made using linear-quadratic dose response parameters, without adding dose rate as a correction factor.
Article
Full-text available
Here we compared the effectiveness of neutrons (E = 5·5 MeV) versus 60Co γ-rays in producing micronuclei (MN) in human lymphocytes. To obtain dose-response data, blood samples of six donors were irradiated with doses ranging from 0·1 to 5 Gy for γ-rays and 0·1–3 Gy for neutrons. A linear dependence of MN yield with dose was found for fast neutrons while for γ-rays a nonlinear dependence existed. For both radiation qualities no significant interindividual differences were found. Derived relative biological effectiveness values decreased with increasing dose. The MN frequency distributions were overdispersed with respect to the Poisson distribution, with neutrons showing higher dispersion values than with γ-rays. To compare the repair kinetics of both radiation qualities split-dose experiments were performed. A dose of 4 Gy γ-rays (3 Gy neutrons) was delivered either as a single exposure or in two equal fractions separated by time intervals ranging from 30 min to 10h (30 min to 7h for neutrons). The data showed for γ-rays a significant decline (30% ± 10%) in MN yield with interfraction time due to repair of DNA damage. This repair is a continuous process starting almost immediately after the first of the two doses and lasting 3–5h. For fast neutrons no decline was observed indicating irreparable damage.
Article
Full-text available
This paper reviews the development of boron neutron capture therapy (BNCT) and describes the design and dosimetry of an intermediate energy neutron beam, developed at the Harwell Laboratory, principally for BNCT research. Boron neutron capture therapy is a technique for the treatment of gliomas (a fatal form of brain tumour). The technique involves preferentially attaching 10B atoms to tumour cells and irradiating them with thermal neutrons. The thermal neutron capture products of 10B are short range and highly damaging, so they kill the tumour cells, but healthy tissue is relatively undamaged. Early trials required extensive neurosurgery to exposure the tumour to the thermal neutrons used and were unsuccessful. It is thought that intermediate-energy neutrons will overcome many of the problems encountered in the early trials, because they have greater penetration prior to thermalization, so that surgery will not be required. An intermediate-energy neutron beam has been developed at the Harwell Laboratory for research into BNCT. Neutrons from the core of a high-flux nuclear reactor are filtered with a combination of iron, aluminium and sulphur. Dosimetry measurements have been made to determine the neutron and gamma-ray characteristics of this beam, and to monitor them throughout the four cycles used for BNCT research. The beam is of high intensity (approximately 2 x 10(7) neutrons cm-2 s-1, equivalent to a neutron kerma rate in water of 205 mGy h-1) and nearly monoenergetic (93% of the neutrons have energies approximately 24 keV, corresponding to 79% of the neutron kerma rate).
Article
The relative biological effectiveness (RBE) of selected low-LET radiation modalities (55 kVp X-rays, 250 kVp X-rays, 60Co γ-rays, and 11 MeV electrons) was investigated for survival of two cell lines (V79 and CHO). Detailed measurements were made in the low (0 to 3 Gy) dose range using an image cytometry device to accurately determine the number of cells assayed at each dose point. Data were also collected in the high dose range (0 to 10 Gy) using conventional counting and plating techniques. RBE values (± 1 SE) varied from 1·0 ± 0·07 (V79 cells) and 1·2 ± 0·05 (CHO cells) at high doses to 1·3 ± 0·07 (V79) and 1·4 ± 0·1 (CHO) at low doses for 55 kVp X-rays, from 1·1 ± 0·05 (V79) and 1·1 ± 0·04 (CHO) at high doses to 1·1 ± 0·06 (V79) and 1·2 ± 0·2 (CHO) at low doses for 250 kVp X-rays, and from 1·1 ± 0·08 (V79) and 1·0 ± 0·04 (CHO) at high doses to 1·0 ± 0·06 (V79) and 0·9 ± 0·1 (CHO) at low doses for 11 MeV electrons. Only the low and high dose RBEs for 55 kVp X-rays relative to 60Co γ-rays were significantly different.
Article
The cytokinesis-blocked micronucleus technique has the potential of being a simple method of biological dosimetry for the rapid screening of individuals following large scale radiation accidents and for the assessment of suspected radiation over exposures. This technique was used to study the induction of micronuclei in human lymphocytes following irradiation in vitro by strontium/yttrium-90 beta rays. The lymphocytes were obtained from eight individuals aged between 23 and 57 years. The wide variation in dose-response for this limited sample suggests that this technique requires prior routine measurements on unirradiated personnel if the sensitivity of the technique is to be maximised. Beta irradiation in vitro appears to be less effective at inducing micronuclei than X-rays.
Article
The DNA contents of 75 micronuclei found in mouse bone marrow 48 h after a 260 R dose of γ-rays were measured individually and compared to 71 diploid G1 nuclei. The coefficient of variation for the diploid cells was 6.3%. The DNA content of micronuclei varied from 0.5 to 11.1% of the diploid G1 nuclei with a mean of 3.5%. These results are in agreement with the expectations based upon the hypothesis that micronuclei arise from acentric chromosomal fragments produced by random breakage of the mouse genome.
Article
An in vivo method for screening drugs, food additives, and other chemicals that might cause chromosomal aberrations has been tested. It is reliable, easy, and very much more rapid than the traditional method.
Article
Mutation induction at the hprt locus has been studied in V79-4 Chinese hamster cells irradiated with mono-energetic protons and alpha-particles with LET of 20.3 and 23 keV microns-1. The mutation frequency increased linearly with the dose for all the four radiation qualities investigated, so that effectiveness for mutation induction could be expressed by the slope of the relevant curve. This effectiveness did not significantly change with the small change in LET of each kind of particle, while sizeable differences were found between particles. Protons were more effective in mutation induction than alpha-particles with the same LET by a factor of about 2. This finding is similar to, although slightly larger than, the factors 1.5-1.8 found for inactivation of the same cells in the same series of experiments.
Article
This paper reports the effects of changes in dose-rate and dose-fractionation on the micronucleus (MN) yield in human lymphocytes exposed to 250 kV X-rays. For the investigation of dose-rate effects whole blood samples of four healthy donors were irradiated with doses ranging from 1 to 4 Gy given at various dose-rates between 0.2 and 40 Gy/h. For the higher doses (3 and 4 Gy) a decline in the MN yield became apparent when the dose-rate was reduced below 1.6 Gy/h. This effect was enhanced systematically by a further lowering of the dose-rate. For lower doses (1 and 2 Gy) the reduction in the MN yield was less pronounced: only a small effect was observed for two donors when a dose of 2 Gy was administered at a dose-rate of 0.2 Gy/h. In the split-dose experiment a dose of 4 Gy was delivered either as a single exposure or in two fractions of 2 Gy, separated by time intervals ranging from 30 min to 10 h. A continuous decrease of the MN yield with increasing interfraction time is observed: after an initial fast decline a further slight reduction in the MN yield occurs. The observed dose-rate and split-dose effects on the MN yield can be attributed to repair of sublethal damage.
Article
A collaborative study between two laboratories has been carried out in order to investigate the reproducibility of the cytokinesis-block micronucleus assay in human lymphocytes. A modified protocol to obtain good quality slides from whole blood cultures has been developed. The spontaneous frequencies of micronuclei in binucleated lymphocytes from five male subjects were evaluated in each laboratory at two cytochalasin-B concentrations (3 and 6 μg/ml). Our data show that 6 μg/ml cytochalasin-B was more effective than 3 μg/ml to arrest cytokinesis in all the donors analysed. The mean frequency of micronuclei in binucleated cells was significantly lower at 6 μg/ml compared to 3 μg/ml cytochalasin-B in both laboratories. The results obtained indicate a very high concordance between both sets of data supporting the validity of the proposed protocol.
Article
The dose response of the number of micronuclei in cytokinesis-blocked lymphocytes after in-vitro irradiation of whole blood with x rays in the dose range 0-4 Gy was studied for a heterogeneous population of 10 donors. One thousand binucleated cells were systematically scored for micronuclei. A linear-quadratic dose dependency of the micronucleus yields was observed. The data were used to derive the accuracy of the dose assessment with the technique in case of a radiation accident. It was shown that for doses of 2 Gy and higher, the uncertainty of the dose was predominantly due to interindividual differences in the x-ray-induced micronucleus yields. At the 95% confidence level, doses lower than 0.3 Gy could not be unequivocally detected with the cytokinesis-blocked micronucleus assay due to the variability of the baseline micronucleus frequency within the donor population and the poor statistical accuracy when scoring 1,000 cells. The precision and sensitivity of the method in the low-dose range could be improved by the knowledge of individual pre-irradiation baseline values and an increase of the number of cells scored by automation of the technique.
Article
Human peripheral blood lymphocytes were irradiated for 24 h in vitro with plutonium-239 citrate. The dose response for micronucleus induction using the cytochalasin B blocking technique fitted well to a linear relationship. Comparison with published data from the same laboratory using X-irradiation, indicated an RBE of 3.6 for alpha particles at low doses.
Article
We employed cytochalasin B to block cytokinesis in human lymphocytes exposed to 220 kV X-radiation. When 3 micrograms/ml of cytochalasin B was used, most cells escaped the block whereas at 6 micrograms/ml greater than 90% of the mitogen-responsive cells became binucleate. Using the higher concentration of cytochalasin B, we observed a linear-quadratic (i.e. Y = gamma + alpha D + beta D2) dose dependency for X-ray-induced micronuclei (MN) in preparations from three donors. When dose-response parameters were compared with those for total acentrics scored in first division metaphases, we observed no significant differences in estimates of the background (gamma) or linear (alpha) coefficients, but a 2-fold reduction in the beta coefficient for MN. We interpret our data as providing evidence that radiation-induced micronuclei are derived from acentric fragments (AF); that virtually all AF are recovered as MN in binucleate interphase daughter cells at low radiation doses; and that for our data, the relative proportion of AF that will be observed as independent MN decreases by a constant factor of approximately one-half as radiation dose increases.
The occurrence of micronuclei proved to be a sensitive biological indicator of clastogenic effects of many chemical and physical agents. The possibility of using the micronucleus technique in radiation protection as an alternative to the traditional chromosomal analysis has recently been followed with increasing interest. This review outlines the main biological and practical aspects of the micronucleus assay and discusses its potential to serve as a rapid and reliable measure of radiation overexposures and hypersensitivities.
Article
The cytokinesis-block method of Fenech and Morley (1985) has been tested for the enumeration and characterization of micronuclei in exponentially growing Chinese hamster cells in culture. The consistent dose-response relations were obtained in cultures treated with mitomycin C, caffeine and colcemid. Comparison with the chromosome aberration frequencies indicated that approximately 30% of the acentric chromosomes are expressed as micronuclei in the mitomycin C and caffeine treated cells. The size distribution of the micronuclei suggested that the base-line frequency of micronuclei is mainly a reflection of mitotic dysfunctions rather than chromosome structural aberrations.
Article
The response of V79 Chinese hamster cells to a 24 keV neutron spectrum has been compared with data for the response of V79 cells to a range of higher neutron energies. The linear energy transfer (LET) distributions of the neutron spectra were calculated and the expected responses of the cells to the different spectra were calculated using published track-segment data on the response of V79 cells to charged particles with various LET values. The response of the cells to 24 keV neutrons was predicted satisfactorily by the LET distribution, in spite of the fact that the maximum range of the recoil protons is only 0.5 micron. The response was not correctly predicted by the microdosimetric parameter y*D evaluated in a 1 micron diameter sphere.
Article
The yield of radiation-induced micronuclei in human lymphocytes was assessed by two methods, i.e., by incorporating bromodeoxyuridine or by inhibiting cytokinesis by cytochalasin for identification of cells which have undergone one cell division. The cytochalasin block method was found to be more efficient with a capacity to detect between 60 and 90% of the induced fragments. Dose-response characteristics and the results of fractionation experiments indicate that the yield of micronuclei reflects both classes of acentric fragments, i.e., those associated and independent of exchange type of aberrations.
Article
An examination of the cytokinesis-blocked micronucleus technique confirmed its potential usefulness as a method of biological dosimetry for radiation accidents. Several advantages and disadvantages of the system are discussed. It has been demonstrated that under the conditions of these experiments, the blocking agent, cytochalasin B does not induce micronuclei or unstable chromosome aberrations. The induction of sister-chromatid exchanges proved just significant. Analysis of the dose response for 250 kVp X-rays indicates that although the Y = alpha D + beta D2 model fits the data, the relationship does not correspond to that for total aberration induction as might have been expected. The background frequency of micronuclei and the value of the alpha coefficient are higher than for total aberrations and the beta term is lower. This indicates that simple incorporation of acentric chromosome fragments into micronuclei may not wholly account for the phenomenon.
Article
The dose-effect relationship of the frequency of micronuclei in cytokinesis-blocked human lymphocytes after in vitro irradiation of whole blood in the range from 0.2 to 4 Gy was studied. The linear-quadratic response obtained offers a useful technique for dose assessments in cases of radiation injuries above approx. 0.1 Gy whole body dose. The distribution in size of micronuclei in cytochalasin B treated cells ranges to larger diameters than in mononuclear lymphoblasts.
Article
The cytokinesis-block micronucleus technique was developed to overcome the kinetic problems inherent in the use of human lymphocytes for micronucleus assays. Using this technique the number of spontaneous micronuclei in lymphocytes from 42 individuals aged between 20 and 85 years was studied and was found to increase at a rate of 4.3% per year. Comparison with the results obtained with the conventional micronucleus assay confirmed that the conventional method markedly underestimates this age effect. The sensitivity of the cytokinesis-block method was determined by studying the effect of low-dose (less than 50 rad) X-irradiation. The results indicated that the dose-response was linear and a single in vitro exposure to 5 rad of X-rays could be unequivocally detected. We concluded that the cytokinesis-block micronucleus method is more sensitive and precise than the conventional micronucleus method and classical metaphase analysis, and that it will be of value for detecting chromosome damage induced in vivo by genotoxic agents.
Article
The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.
Article
Acridine Orange fluorescent staining was applied to the micronucleus test in mice and rats. Micronuclei emitted bright green fluorescence and were easily distinguished from micronucleus-like inclusions or contaminants. In rat bone-marrow cells, micronuclei with green fluorescence could be easily distinguished from granules accidentally dispersed from broken mast cells, which showed bright red fluorescence. Therefore, it is recommended that the Acridine Orange staining method be used to provide more reliable data in the micronucleus test.
Article
An improved slide preparation technique is described for the automated detection of micronuclei in binucleate cytokinesis-blocked human lymphocytes. This automated system (Discovery Image Analyser, Becton-Dickinson Image Cytometry Systems, Leiden, The Netherlands) searches for the pattern of two touching nuclei in binucleate cells instead of the edges of the cytoplasm. For this purpose several ratios of the fixative mixture, methanol/acetic acid, were checked. After fixation with a ratio of 25:1, touching nuclei were obtained in almost 100% of the binucleate cells. A hydrolysis treatment with 5 N HCl before staining with Romanowsky-Giemsa resulted in binucleate cells with dark-stained nuclei and micronuclei and a vaguely stained cytoplasm. The high visual contrast between cytoplasm and nuclear material obtained by this staining procedure makes an accurate automated detection of micronuclei feasible. Additionally, a 64 h culture time resulted in an optimal yield of binucleate cells. The results of manual micronucleus scoring on slides prepared with the 'manual protocol' and with the 'automation protocol' indicate no significant differences between both sets of data supporting the validity of the automation protocol.
Article
A comparison between manual and computer-based automatic scoring of micronuclei (MN) was performed in order to optimize the preparation technique and to validate the image analysis procedure. For this purpose whole human blood of three donors was either irradiated (1 Gy X-rays) or treated with the chemical mutagen methyl methane sulphonate (25 mg/ml) and cultivated in the presence of cytochalasin B to obtain binucleated cells with a high yield of MN. An algorithm for MN detection has been developed for Giemsa (G)- and Feulgen-Congo-Red (FCR)-stained slides. This algorithm contains a sequence of grey operators and binary operators necessary to detect nuclei and MN, and to efficiently reject artefacts. The output is a data file with measurements of cells and intracellular inclusions. From these features, information can be extracted concerning the frequency of the various cell classes (based on nuclearity), the presence of MN and various shape parameters. A close analysis of the automatic scoring of G- and FCR-stained cells, revealed that 59-86% of all automatically classified binucleated cytokinesis-blocked (CB) cells were correctly classified. Although some MN were overlooked during automated scoring, the results show that, on average, similar MN frequencies are obtained with automated and manual scoring. The errors which occurred were mainly due to the misclassification of CB cells, the non-detection of extremely small MN and the aggregation of MN to the main nucleus. The possibility of scanning high numbers of cells overnight, to relocate CB cells with potential MN and the quantitative character of the results offers good prospects for future use in the in vitro MN test.
Article
To use the micronucleus (MN) assay as a biological dosimeter, it is essential that the dose response to different radiation qualities is known. In this paper we present dose-response curves for MN induction by X rays (14, 50 and 350 kVp) and 60Co gamma rays. The dose responses for 14 kVp compared to 50 kVp and for 350 kVp compared to 60Co were found to differ significantly, whereas no significant difference could be demonstrated between 50 kVp and 350 kVp. Calculated RBE values with respect to 60Co gamma rays increase strongly with decreasing dose. The high RBE value (2.7) obtained for 14 kVp X rays in the low-dose region is of importance for radiological protection in clinical practice. A good linear correlation was found between the biological damage expressed by the RBE values for MN induction and the calculated physical parameter LET100,T expressing the energy deposition pattern in the medium. This correlation points to the fact that secondary phenomena involved in MN formation (repair, coagulation) do not differ greatly for the X-ray qualities used in this work. It can be concluded that MN induction in human lymphocytes can be used as a sensitive biological dosimeter for radiation damage by X rays and gamma rays.
Article
To investigate the effectiveness of high LET fast neutrons compared with low LET 60Co gamma-rays to induce apoptosis in resting lymphocytes. Apoptosis induction was quantified by light microscopic analysis of irradiated lymphocyte samples from healthy donors after 24 h culture. For the dose-response analyses doses ranging from 0.05 to 5 Gy were applied at 1.5 Gy/min (gamma-rays) or 0.2 Gy/min (fast neutrons). To investigate the role of DNA repair in apoptosis induction a dose of 2 Gy was also given at low dose-rate (0.006 Gy/min). Dose-response curves obtained with both radiation qualities were characterized by an initial steep increase in the number of apoptotic cells below 1 Gy, with a flattening of the curves at higher doses towards 5 Gy. The calculated relative biological effectiveness (RBE) values for fast neutrons were close to unity. When a 2 Gy dose was administered at low rather than high dose-rate no decrease in apoptotic cell yield was observed. The dose-response data confirm the high radiosensitivity of lymphocytes and demonstrate that their response to undergo early interphase cell death by apoptosis is largely independent of LET. The observations that apoptosis induction is independent of LET and dose-rate may suggest that initial DNA damage, as opposed to DNA repair, dominate the induction of apoptosis in resting lymphocytes.
Radiological-protection-related neutron studies using the C3H 10T1/2 cell system: I. The RBE of 24 keV neutrons; II. An investiga-tion into the inverse dose-rate effect using 2.5 MeV neutrons
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