Volume 13, No 2 International Journal of Radiation Research, April 2015 Different aspects of cytochalasin B Blocked cytome (CBMN cyt) assay as a comprehensive measurement tool for radiobiological studies, biological dosimetry and genome instability

M. Salimi1* and H. Mozdarani2

1Department of Medical Genetics, Medical Biotechnology Institute, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran 2Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

ABSTRACT Review article ► It is now universally accepted that DNA is the main target for damages caused by physical and chemical genotoxicants. Although there are different methods to measure directly the induced DNA damages but due to fast repair processes in cellular environment, most of the damages would be repaired even before sampling, therefore processed DNA damages, i.e. damages le * Corresponding author: unrepaired aer acng repair machinery is preferable to measure in various Dr. Mahdieh Salimi, exposure scenarios. Various cytogenec end points are introduced and Fax: +98 21 44787399 implemented such as metaphase analysis, sister chromad exchanges, E-mail: [email protected] premature condensaon, translocaon assay and micronucleus assay. All of these methods were extensively used for various purposes but Revised: Aug. 2014 among them micronucleus (MN) assay was found more praccal because of Accepted: Sept. 2014 ease of scoring, potenal for automaon as well as being nearly as sensive as other procedures. These characteriscs made MN assay very popular for Int. J. Radiat. Res., April 2015; screening of the effects of various genotoxic agents in vitro and in vivo. In this 13(2): 101-126 review we try to summarize the main aspects of applicaon of this method in radiobiological studies and genome instability related diseases. DOI: 10.7508/ijrr.2015.02.001 Keywords: Cytome assay, micronucleus, genome instability, radiobiological studies, biological dosim.

chromosomal aberration formation. There are INTRODUCTION various cytogenetic methods such as metaphase analysis, sister chromatid exchanges, premature

Downloaded from ijrr.com at 15:25 +0330 on Friday November 13th 2020 [ DOI: 10.7508/ijrr.2015.02.001 ] Ionizing radiations as well as a vast number chromosome condensation and micronuclei of chemical agents are capable of inducing DNA assay for assessment of induced in damage directly or indirectly via formation of cells expressed as chromosomal abnormalities. free radicals and oxidative agents. The most Among these methods micronuclei assay got important lesions induced in DNA include base greater attention because of ease of the scoring damage (bd), single strand break (ssb), double and potential for automation. In vitro micronu- strand break (dsb), DNA-DNA cross link and clei assay was irst performed by Heddle (1) on DNA-protein cross link. All DNA damages mono-nuclear lymphocytes. Later in 1985 when undergo various DNA repair processes and Fenech introduced cytochalasin-B blocked unrepaired DNA damage lead to genome micronucleus assay, where presence of instability of cell and formation of chromosomal micronucleus is scored in bi-nucleate lympho- abnormalities. The origin of chromosomal cytes allowing observation of chromosomal aberrations is not fully understood but there are damage after the irst division of cells, made this evidences showing DNA dsb as major cause of method more popular than before (2). Salimi and Mozdarani / Different aspects of cytome assay for radiobiological studies

As mentioned earlier The -block nuclear division (18) . The wide-spread use of the micronucleus (CBMN) assay is the preferred CBMN assay has made it feasible to launch an method for measuring micronucleus (MN) in international collaborative project, the HUMN cultured human and/or mammalian cells project (19) , with the following objectives: because scoring is speciically restricted to determining the important methodological, once-divided Bi-nucleated (BN) cells, which are demographic and life-style variables that

the cells that can express MN (2,3) . In the CBMN inluence this index of DNA damage, establishing assay, once-divided cells are recognized by their standardized protocols for the assay, and BN appearance after blocking cytokinesis with ultimately to determine the capacity of this cytochalasin-B (Cyt-B), an inhibitor of microila- biomarker to predict cancer risk, as well as the ment ring assembly required for the completion risk of other degenerative diseases. The term of cytokinesis (2,3) . Restricting scoring of MN in CBMN Cyt is now used because the original BN cells prevents confounding effects caused by cytokinesis-block micronucleus (CBMN) assay suboptimal or altered cell division kinetics, was restricted to measuring micronucleus (MN) which is a major variable in micronucleus (MN) frequency in binucleated cells but has since assay protocols that do not distinguish between evolved into a comprehensive “cytome” system non-dividing cells that cannot express MN and for measuring DNA damage, cytostasis, and cyto- dividing cells that can (2,3) . Because of its reliabil- toxicity. The ‘‘cytome’’ concept implies that every ity and good reproducibility, the CBMN assay cell in the system studied is scored cytologically has become one of the standard cytogenetic for its viability status (necrosis, ), its tests and an established comprehensive method mitotic status (mononucleated, BN, multinucleat- for assessing cytostasis and chromosome ed) and its chromosomal damage or instability stability, chromosome breakage, DNA misrepair, status (presence of MN, NPBs, NBUDs and chromosome loss, non-disjunction, necrosis, number of probe signals among apoptosis or genetic toxicology and radiation nuclei or MN of BN cell if such molecular tools sensitivity testing in human and mammalian are used in combination with the assay). For cells (4-16 ) . The CBMN assay is a multi-endpoint these reasons, it is now appropriate to refer to assay that measures not only chromosome this technique as the cytokinesis block MN damage [i.e., micronuclei relecting chromosome cytome (CBMN Cyt) assay. The protocol for breaks; nucleoplasmic bridges (NPB) relecting which has been previously detailed by Fenech chromosome rearrangements; and nuclear buds (18) . In brief, DNA damage events are scored (NBUD) relecting gene ampliication] but also speciically in once-divided binucleated (BN) other cellular events (such as apoptosis and cells and include micronuclei (MN) which is a necrosis). It was reported that the base-line biomarker of chromosome breakage and/or frequencies of MN frequency varied greatly whole chromosome loss, nucleoplasmic bridges between populations also age and gender were (NPBs), The nuclear buds (NBuds) also the Cyto- the two most important demographic factors static effects are measured via the proportion of Downloaded from ijrr.com at 15:25 +0330 on Friday November 13th 2020 [ DOI: 10.7508/ijrr.2015.02.001 ] inluencing MN frequency. The scoring criteria mono-, bi- and multinucleated cells and cytotoxi- used were probably the most important method city via necrotic and/or apoptotic cell ratios (18) . variable inluencing the score obtained (17) . In nucleoplasmic bridges (NPBs), a biomarker of Compared with other cytogenetic assays, DNA misrepair and/or telomere end fusions, quantiication of micronuclei confers several typically, a dicentric chromosome and an advantages, including speed and ease of acentric chromosome fragment are formed that analysis, no requirement for metaphase cells result in the formation of an NPB and an MN, and reliable identiication of cells that have respectively. Misrepair of DNA strand breaks completed only 1 nuclear division. This prevents could also lead to the formation of dicentric ring confounding effects caused by differences in cell and concatenated ring chromo- division kinetics because expression of the somes which could also result in the formation of micronuclei is dependent on completion of NPBs (20) . As mentioned an alternative mecha- Int. J. Radiat. Res., Vol. 13 No. 2, April 2015 102 Salimi and Mozdarani / Different aspects of cytome assay for radiobiological studies

nism for dicentric chromosome and NPB budding occurs during S phase and the NBUDs formation is telomere end fusion caused by are characterized by having the same morpholo- telomere shortening, loss of telomere capping gy as an MN with the exception that they are proteins or defects in telomere cohesion (21) , but linked to the nucleus by a narrow or wide stalk in this case the NPB is not necessarily accompa- of nucleoplasmic material depending on the nied by an acentric chromosome fragment or an stage of the budding process. The duration of the MN. NPBs occur when of dicentric nuclear budding process and the extrusion of the chromosomes are pulled to opposite poles of the resulting MN from the cell remain largely cell at . Rarely is it possible to observe unknown (23) . Using methods that can detect dicentric anaphase bridges before the nuclear centromeric regions of chromosomes helps to membrane is formed, because cells proceed take full advantage of the CBMN Cyt assay and through anaphase and telophase rapidly, obtain deeper understanding of the genotoxic completing cytokinesis, which ultimately results mechanism. This is best achieved by distinguish- in breakage of the NPB when the daughter cells ing between MN originating from whole separate (22) . However, in the CBMN assay, BN chromosomes and those originating from cells with NPBs are allowed to accumulate acentric fragments as well as to determine because cytokinesis is inhibited and the nuclear whether malsegregation of chromosomes is membrane is eventually formed around the occurring between nuclei in a BN cell that may chromosomes allowing an anaphase bridge to be or may not contain MN (18) . The use of MN size as observed as an NPB. Therefore, some MN may a discriminant is not recommended for human also originate from broken anaphase bridges cells or other cell types in which the size of although whether this actually happens in chromosomes is heterogenous because a small cytokinesis blocked cells remains unclear. The MN may contain either a fragment of a large importance of scoring NPBs should not be chromosome or a whole small chromosome. underestimated because it provides direct Centromeric regions of chromosomes can be evidence of genome damage resulting from detected indirectly using antibodies to the misrepaired DNA breaks or telomere end kinetochore proteins that are assembled on fusions, which is otherwise not possible to centromeres but this approach does not deduce by scoring MN only, which could distinguish between unique chromosomes and originate from either acentric chromosome may not detect chromosome loss occurring due fragments or chromosome loss (23) . The nuclear to absence of kinetochores on defective buds (NBuds), a biomarker of elimination of centromeres (18) . The preferred method is to use ampliied DNA and/ or DNA repair complexes in situ hybridization (ISH) to identify centromer- has been observed in cultures grown under ic regions using DNA or peptide nucleic acid strong selective conditions that induce gene pancentromeric probes, which allows MN ampliication as well as under moderate folic containing whole chromosomes to be reliably acid deiciency (24-28) . In vitro experiments with identiied. Furthermore, use of centromeric Downloaded from ijrr.com at 15:25 +0330 on Friday November 13th 2020 [ DOI: 10.7508/ijrr.2015.02.001 ] mammalian cells showed that ampliied DNA is probes that are speciic for a unique chromo- selectively localized to speciic sites at the some can be used to detect non-disjunctional periphery of the nucleus and eliminated via events (i.e., unequal distribution of unique nuclear budding to form MN during S phase of homologous chromosome pairs in daughter . Ampliied DNA may be eliminated nuclei) in BN cells (29) . It is also possible to use a through recombination between homologous combination of pantelomeric pancentromeric regions within ampliied sequences forming mini probes to determine the mechanism of MN and -circles of acentric and a telomeric DNA (double NPB formation cells (30-32) . The signiicance of minutes) , which localize to distinct regions these developments and the concept of the within the nucleus, or through the excision of CBMN assay as a ‘‘cytome’’ assay of chromoso- ampliied sequences after segregation to distinct mal instability in different ields are explained in regions of the nucleus. The process of nuclear the following sections. 103 Int. J. Radiat. Res., Vol. 13 No. 2, April 2015 Salimi and Mozdarani / Different aspects of cytome assay for radiobiological studies

Value of CBMN Cyt assay in radiobiological tumor cell killing with minimal injury to normal studies: tissue. A cautious balance between the total In radiobiological studies, CBMN Cyt assay dose of radiation delivered and the threshold was used as a sensitive tool to measure variety limit of the surrounding normal critical of phenomena such as Radiosensitivity, tissues is essential to optimize outcomes. Th e Radioadaptation, Radioprotection, bystander prevention or treatment of early and late radio- effect and radiation biodosimetry. There are therapy effects would improve quality of life and some evidences as follow: increase cancer curability by intensifying therapies. In this sense, the role of radio Radio sensitivity protective compounds is of utmost importance Radiosensitivity is the relative susceptible- in clinical radiotherapy (48) . ness of cells, tissues, organs or organisms to One way to prevent DNA damage and the dangerous effect of ionizing radiation. Many potential disease development is to avoid biological factors affect radiosensitivity. exposure to mutagenic agents. Alternatively, the Inherent characteristic is one of the administration of chemoprotective agents may important reasons of differences in radiation increase resistance to mutagens/ sensitivity (33) . The physical speciications of and/or inhibit disease progression (48,49) . ionizing radiation such as its type (particle or Antioxidants are substances that can bind photon), energy and dose rate could alter the free radicals and signiicantly reduce or prevent biological response of organ or tissue to oxidation of the substrate. Speciically, antioxi- ionizing radiation. People with higher dants protect against free radical damage by radiosensitivity, most likely will suffer from preventing free radicals from attacking lipids, deterministic and stochastic effects in protein amino acids, and the double bond of radiotherapy (34) . In variety of studies the cytoki- polyunsaturated fatty acids and DNA bases, nesis-block micronucleus assay (CBMN) was thereby preventing cellular damage (47) . used as a tool to measure radiosensitivity (35-37) . Bystander effect Radioadaptation One of the major paradigm shifts in the ield There is a large body of evidence that of radiation biology was the discovery of radiation has a stimulating effect on a number of non-targeted effects such as the bystander effect biological processes and can induce resistance, a in which cells in the vicinity of radiation- phenomenon generally termed as 'adaptive damaged cells behave as though they were also response'. CBMN assay was used as a cytogenet- irradiated. The suitability of the CBMN Cyt assay ic technique to show these phenomena in variety to detect the bystander effect of IR exposure has of studies (8, 38-41) . also been demonstrated in a number of studies using different models, ranging from single cell Radioprotection layer models with/without inhibitors of gap Downloaded from ijrr.com at 15:25 +0330 on Friday November 13th 2020 [ DOI: 10.7508/ijrr.2015.02.001 ] Cytokinesis block micronucleus assay was junctions (50) to 3-D skin models in which it was used in variety of studies to evaluate the radio shown that MN and apoptosis induction was protective effect of different substances by highest in the vicinity of the radiation track (100 assess cellular DNA damage (42- 46) . It is well –800 microns) and declined progressively with known that exposure to certain environmental distance with no observed effect beyond 1,100 substances can cause genetic mutations. Because microns (51) . However, the extent to which the mutations are often associated with the develop- bystander effect is detectable in whole blood in ment of cancer and teratogenesis, information lymphocytes using the CBMN assay has not been regarding the mutagenic potential of various conclusively demonstrated, although indirect agents, especially those used in medicine, is evidence based on measurement of clastogenic crucial (47) . For example effective radiotherapy to factor effects following irradiation in vivo treat cancer patients should include maximal suggests this to be probable (52,53) . Int. J. Radiat. Res., Vol. 13 No. 2, April 2015 104 Salimi and Mozdarani / Different aspects of cytome assay for radiobiological studies

Radiation biodosimetry a number of additional assays (61) have been Biological dosimetry or Biodosimetry, is worked out and validated, including the now mainly performed, in addition to physical well-established micronucleus (MN), transloca- dosimetry, with the aim of individual dose tion and premature chromosome condensation assessment. It is a tool used to assess the dose assays. More recently, molecular biomarker received by an individual when physical dosime- methods such as the gamma-H2AX assay have try is missing or after accidental exposures due been proposed (62) . Numerous studies, to large scale radiological events or terrorist performed both on animals and humans, have attacks (54) .The risk of developmental demonstrated a close relationship between abnormalities and cancer risk increases with dicentric chromosomes induced in peripheral ionizing radiation (IR) (55) . In the case of blood lymphocytes (PBLs) under in vitro and in accidental exposures to ionizing radiation or in vivo conditions. This allows dose estimation of the situation of a terrorist attack, it becomes an accidentally exposed person by comparing essential to measure the exposure of each the observed aberration yield of dicentrics to an individual and the resulting genotoxic effects of in vitro calibration curve. The power of that exposure, which depends on genetic and dicentrics for dose estimation is related to the constitutional susceptibility. To achieve this low and constant spontaneous dicentric rate in objective, it is important to develop a reliable the healthy population (about one dicentric per biological dosimeter that can be applied in a 1000 metaphases), and by the fact that minimally-invasive manner and that can dicentrics are speciically induced by ionizing estimate dose and genetic effects accurately radiation. After whole-body exposure with low across the spectrum of doses that are biological- linear energy transfer (LET) radiation, doses ly- and medically-relevant. Such a tool would be down to _0.1 Gy can be detected. However, in useful in classifying subjects who require urgent cases of exposure to low doses of radiation, a medical attention; i.e., those whose long-term disadvantage of the dicentric assay is the time risks of cancer might have been increased and needed for microscopic scoring analysis of a those who need to be reassured that their suficient number of metaphases. As MN can exposure is unlikely to pose a serious risk in the arise from exposure to various clastogenic short or long-term (56) . agents in the form of acentric chromosome At present, the dicentric chromosome assay fragments, as well as to aneugenic agents as (DCA) is the only recognized tool that provides whole chromosomes, they are not radiation sensitive and robust biological dose estimates speciic. As a consequence, the CBMN assay is and has been used extensively for accidental often used as a general toxicology test (6) . overexposures for many years (57-60) . In case of a However, because ionising radiation is a strong radiation accident, the irst information comes clastogenic agent, and thus a potent inducer of especially from physical dose reconstruction, MN, the CBMN assay has proven to be a very blood count data and from the clinical symptoms reliable, thoroughly validated and standardized Downloaded from ijrr.com at 15:25 +0330 on Friday November 13th 2020 [ DOI: 10.7508/ijrr.2015.02.001 ] that exposed persons might display. All these technique in the ield of radiation biology to sources of information may be combined with evaluate in vivo radiation exposure of the results of biological dose assessments to occupational, medical and accidentally exposed obtain a clearer evaluation of the case. Biological individuals and to assess individual in vitro dosimetry using cytogenetic methods is of radiosensitivity or cancer susceptibility (9,11, 12, particular importance because it takes into 14,60,62-73) . Radiation induced chromosome account inter-individual variation in susceptibil- aberrations such as MN observed in PBL are ity to radiation damage. As mentioned earlier, mainly the result of unrepaired or misrepaired for many years, the dicentric assay performed in double strand breaks by the non-homologous PBL was the only method available; and still end joining (NHEJ) repair pathway (70,74) . today, it is the gold standard for cytogenetic Biological dosimetry, based on the analysis of radiation dosimetry. However, in the past years, micronuclei (MN) in the cytokinesis-block 105 Int. J. Radiat. Res., Vol. 13 No. 2, April 2015 Salimi and Mozdarani / Different aspects of cytome assay for radiobiological studies

micronucleus (CBMN) assay can be used as an thoroughly validated and standardized tech- alternative method for scoring dicentric chromo- nique proposed high-throughput alternative to somes in the ield of radiation protection. The the DCA evaluate in vivo radiation exposure of cytokinesis block micronucleus cytome (CBMN occupational, medical and accidentally exposed Cyt) assay in peripheral blood lymphocytes is individuals. Compared to the gold standard, the one of the few available techniques with the re- dicentric assay, the CBMN assay has the quired characteristics of sensitivity, speciicity, important advantage of allowing economical, transportability, and reproducibility to be an easy and quick analysis (62) . Similar to the DCA, effective biological dosimeter of ionizing radia- the CBMN assay measures damage to the tion exposure and of genetic susceptibility to the chromosomes (e.g. clastogenic damage); harmful effects of ionizing radiation (72,73,75) . however, a slightly different culturing procedure The CBMN Cyt assay has also been applied and scoring technique is applied. A key successfully to identify radioprotective agents advantage of the CBMN Cyt assay over and to determine the impact of nutritional status metaphase analysis is the ease with which on susceptibility to the genome-damaging effects micronuclei can be scored and the high percent- of ionizing radiation (IR) (8,10-12, 72,73, 76- 79) . Many age (up to 60%) of scoreable binucleated cells. studies have shown that the number of radiation This makes it feasible to score thousands of cells -induced MN is strongly correlated with dose visually with unequivocal identiication of MN. and quality of radiation. Although the DCA is These characteristics are necessary to have sufi- very speciic to radiation damage and has a low, cient statistical power to detect small effects (56) . stable background, it is limited by the extensive The main disadvantage of the CBMN assay is time and expertise required to perform the related to the variable micronucleus (MN) scoring. Traditionally, 500–1000 metaphase background frequency in lymphocytes among spreads are analyzed for each sample to provide unexposed subjects due to a variety of factors sensitive (detection limit of 0.15–0.20 Gy) and including: age, gender, smoking and diet (82) by accurate biological dose estimates in situations which only in vivo exposures in excess of 0.2–0.3 where only a small number of individuals are Gy X-rays can be detected. Also in the large-scale involved. However, this assay is extremely labor Fenech study (83) , investigating variables intensive and time consuming, requiring several inluencing baseline MN frequencies, an increase days for analysis of a single sample. In the case of 0.31 MN/1000 BN cells/year was measured in of a large-scale radiological event, where a male population. In females, the spontaneous potentially thousands of people may be MN yields are higher compared to males and the concerned about radiation exposure, biological increase of MN with age is more prominent: an dosimetry using the conventional DCA is not increase of 0.58 MN/1000 BN cells/year was feasible. Lloyd et al. (80) have suggested that the observed by the Ghent group (60) , this in good conventional DCA may be modiied in such a agreement with the increase of 0.52 MN/1000 situation, by decreasing the number of BN cells/year reported by Fenech (83) . Investiga- Downloaded from ijrr.com at 15:25 +0330 on Friday November 13th 2020 [ DOI: 10.7508/ijrr.2015.02.001 ] metaphase spreads analyzed, yet maintaining tion of the MN content using a pan-centromeric sensitivity to detect clinically relevant doses. In a luorescence in situ hybridization (FISH) probe mass casualty event, it is generally agreed that showed that the age increase of baseline MN only those individuals receiving a whole-body frequencies can be attributed almost completely equivalent dose of ~1.5 Gy would require any to centromere-positive MN, relecting an medical intervention (81) . In order to achieve that increased chromosome loss with age (60) . By level of sensitivity, scoring only 50 metaphase using an X-chromosome-speciic centromeric spreads (or 30 dicentrics) would be required (80) . probe, it was shown that the X-chromosome is However, using such an approach would still almost always involved in this spontaneously only allow a modest increase in daily throughput occurring chromosome loss (84) . This inding may using highly trained biological dosimetrists . The explain also the gender difference observed in CBMN assay has become, in the last years, a spontaneous MN frequencies. However, at Int. J. Radiat. Res., Vol. 13 No. 2, April 2015 106 Salimi and Mozdarani / Different aspects of cytome assay for radiobiological studies

clinically relevant doses of ionizing radiation internationally by the International Atomic (~1.5 Gy), the CBMN assay may be suficiently Energy Agency for IR biodosimetry for in vitro sensitive to discriminate between exposed genetic toxicology testing in the pharmaceutical individuals and those with background levels of industry, for environmental genetic toxicology in MN. In fact, this assay has proved to be effective molecular epidemiology studies, and in estimating unknown doses in several increasingly for studying the genotoxic effects of accidental overexposures with results in close suboptimal diet (75, 85-89) . Micronucleus induction agreement with the DCA (57, 59, 63) . In the last following IR exposure in humans has been years, several improvements have been reported for cell types and tissues other than achieved, with the ultimate goals ; of further lymphocytes such as erythrocytes, hair root increasing the sensitivity of the CBMN assay for cells, and buccal cells (90) ; however, these low -dose detection by combining the assay with methods, unlike the CBMN Cyt assay in a luorescence in situ hybridization centromere lymphocytes, have not yet been adequately staining technique; of increasing the speciicity validated for IR biodosimetry and will therefore of the test for radiation by scoring nucleoplasmic not be discussed in this review. It has been bridges in binucleated cells and ; of making the shown in several studies that up to 1 Gy of low- assay optimally suitable for rapid automated LET (linear energy transfer) IR exposure gives analysis of a large number of samples in case of a an almost 1:1 ratio for expression of acentric large-scale radiation accident. The development fragments in metaphases and micronuclei in of a combined automated MN-centromere binucleated cells; however, at higher doses the scoring procedure remains a challenge for the eficiency of conversion of acentric fragments to future, as it will allow systematic biomonitoring MN tends to diminish possibly due to the of radiation workers exposed to low-dose inclusion of more than one acentric fragment radiation (62) . Scoring of NPBs in cytokinesis- within a MN or mitotic slippage of cells with blocked BN cells acts as a biomarker of dicentric multiple aberrations, which would lead to failure chromosome formation. In the cytokinesis-block of nuclear division and formation of a 4N micronucleus cytome (CBMN cyt) assay (18) , it is mononuclear cell that would not be scored in the possible to score NPBs joining the two nuclei in a conventional CBMN assay . While the dose- BN cell. NPBs originate from dicentric response for acentric fragments between 0–4 Gy chromosomes, which are induced by misrepair tends to be linear-quadratic, that for MN tends to of chromosome breaks. Scoring NPBs in the be closer to linear at low dose exposure (~2 Gy) CBMN cyt assay for radiation biodosimetry is (91) . important because the NPB index has a lower Results from in vivo exposure studies on background frequency than MN frequency; cancer patients undergoing radiotherapy unlike MN background yields, the NPB frequency showed that the CBMN Cyt assay eficiently is not affected by gender; NPBs in BN cells detects the accumulated dose-related increase in provide a direct method of measuring asymmet- chromosome damage, and the observed effects Downloaded from ijrr.com at 15:25 +0330 on Friday November 13th 2020 [ DOI: 10.7508/ijrr.2015.02.001 ] rical chromosome rearrangement in cells after a were linearly correlated with the equivalent single-cell division and NPBs can be scored body dose (56) . To assess the suitability of the eficiently because the CBMN cyt assay allows a CBMN assay for biological dosimetry, MN yields large proportion of BN cells to be accumulated have been analyzed in PBLs of different groups and furthermore, MN/NPB ratio may provide a of patients treated with fractionated partial body ingerprint of speciic genotoxic exposure. NPBs radiotherapy, for e.g. cervical cancer, prostate in lymphocytes are increased in a dose-related cancer or Hodgkin’s disease. The doses manner following exposure to ionizing radiation estimated by MN analysis agree quite well with and correlate well with dicentric and ring averaged whole body doses calculated from the chromosome frequencies in metaphases of the radiation treatment plans (92, 93) . Studies per- same lymphocyte cultures (62) . formed in thyroid cancer patients undergoing The CBMN Cyt assay has been adopted radioiodine treatment further demonstrated that 107 Int. J. Radiat. Res., Vol. 13 No. 2, April 2015 Salimi and Mozdarani / Different aspects of cytome assay for radiobiological studies

the CBMN assay is sensitive enough to detect and in the study reporting the accident of a low average whole -body doses from internal hospital worker exposed to a 50-kV contact exposure scenarios (69,94) . As the above- radiotherapy X-ray device during maintenance mentioned patient studies have shown that the (63) , several cytogenetic end points were scored. CBMN assay is a reliable biomarker for radiation In both radiation accidents, blood was sampled exposure, the CBMN assay has been used at different time points, varying between 1 frequently to measure radiation exposure in month (97) and 6 months (63) , after the accident radiation accidents and has been app