DNA damage events are scored specifically in once-divided binucleated cells. The assay has been applied to the biomonitoring of in vivo exposure to genotoxins, in vitro genotoxicity testing and in diverse research fields, such as nutrigenomics and pharmacogenomics. It has also been shown to be important as a predictor of normal tissue and tumor radiation sensitivity and cancer risk. This protocol also describes the current established methods for culturing lymphocytes, slide preparation, cellular and nuclear staining, scoring criteria, data recording, and analyses.
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This article has been updated Abstract The cytokinesis-block micronucleus cytome assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. The assay is being applied successfully for biomonitoring of in vivo genotoxin exposure, in vitro genotoxicity testing and in diverse research fields such as nutrigenomics and pharmacogenomics as well as a predictor of normal tissue and tumor radiation sensitivity and cancer risk. The procedure can take up to 5 days to complete.
Download PDF Introduction MNi originate from chromosome fragments or whole chromosomes that lag behind at anaphase during nuclear division Fig. In the CBMN assay, once-divided cells are recognized by their BN appearance after blocking cytokinesis with cytochalasin-B Cyt-B , an inhibitor of microfilament ring assembly required for the completion of cytokinesis 3 , 4 , 5 , 6 , 7.
Restricting scoring of MNi in BN cells prevents confounding effects caused by suboptimal or altered cell division kinetics, which is a major variable in micronucleus MN assay protocols that do not distinguish between non-dividing cells that cannot express MNi and dividing cells that can 6 , 8 , 9. Because of its reliability and good reproducibility, the CBMN assay has become one of the standard cytogenetic tests for genetic toxicology testing in human and mammalian cells.
MN originate from either lagging whole chromosomes or acentric chromosome fragments. NPBs originate from dicentric chromosomes that may be caused by misrepair of double strand DNA breaks or telomere end fusions. These events can only be observed in cells completing nuclear division, which are recognized by their BN appearance after cytokinesis blocking with Cyt-B.
Full size image Over the past 17 years, the CBMN assay has evolved into a comprehensive method for measuring chromosome breakage, DNA misrepair, chromosome loss, non-disjunction, necrosis, apoptosis and cytostasis Figs.
This method is now also used to measure NPBs, a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair, and to measure NBUDs, a biomarker of gene amplification 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , In addition, cytostatic effects are readily estimated from the ratio of mono-, bi- and multinucleated cells.
The ratios of mononucleated, BN, multinucleated, necrotic and apoptotic cells are used to determine mitotic division rate or NDI a measure of cytostasis and cell death cytotoxicity. For a wider selection of photomicrographs of different types of cells and biomarkers scored in the CBMN Cyt assay, refer to Fenech et al.
Full size image Figure 4: Centromeric probes to determine mechanism of MN formation and aneuploidy. The use of molecular techniques for identifying a an MN originating from a lagging acentric chromosome fragment, b a MN originating from a lagging whole chromosome and c non-disjunction of a specific chromosome leading to aneuploid daughter nuclei.
The yellow spots in the nuclei and MNi of the BN cells on the left of each panel show the centromeric or kinetochore pattern of staining when pancentromeric probes or kinetochore antibodies are used. The light blue spots in the nuclei and MNi of the BN cells on the right of each panel show the pattern of centromeric staining when a centromeric probe specific to the chromosomes involved in MN formation or non-disjunction events is used.
The example shown is for a hypothetical cell with only two pairs of chromosomes. Pancentromeric probes should be used only for distinguishing between MNi originating from chromosome breaks centromere negative and chromosome loss centromere positive. Chromosome-specific centromere probes should be used only to measure malsegregation owing to non-disjunction or chromosome loss involving unique chromosomes.
It is important to note that pancentromeric probes cannot be used to determine non-disjunction because of the difficulty in reliably counting all centromeres within nuclei.
Pancentromeric and telomeric probes can be used to distinguish I between MNi originating from whole chromosome loss a and MNi originating from acentric chromosome fragments b and II NPBs from dicentric chromosomes resulting from misrepair of DNA strand breaks b and dicentric chromosomes caused by telomere end fusions c.
The yellow dots represent probes that hybridize to the centromeric region of chromosomes. The light blue dots represent probes that hybridize with the telomeric sequences in chromosomes. Full size image We have proposed that NPBs between nuclei in BN cells should be scored in the CBMN assay because they provide a measure of chromosome rearrangement, which is otherwise not achievable in this assay if only MNi are scored 3 , 22 , 23 , 35 , 36 , 37 , 38 , NPBs occur when centromeres of dicentric chromosomes are pulled to opposite poles of the cell at anaphase.
Rarely is it possible to observe dicentric anaphase bridges before the nuclear membrane is formed, because cells proceed through anaphase and telophase rapidly, completing cytokinesis, which ultimately results in breakage of the NPB when the daughter cells separate However, in the CBMN assay, BN cells with NPBs are allowed to accumulate because cytokinesis is inhibited and the nuclear membrane is eventually formed around the chromosomes allowing an anaphase bridge to be observed as an NPB.
Typically, a dicentric chromosome and an acentric chromosome fragment are formed that result in the formation of an NPB and an MN, respectively Figs. Misrepair of DNA strand breaks could also lead to the formation of dicentric ring chromosomes and concatenated ring chromosomes which could also result in the formation of NPBs An alternative mechanism for dicentric chromosome and NPB formation is telomere end fusion caused by telomere shortening, loss of telomere capping proteins or defects in telomere cohesion 40 , but in this case the NPB is not necessarily accompanied by an acentric chromosome fragment or an MN.
The formation of anaphase bridges and NPBs has been observed in models of rodent and human intestinal cancer in vivo and shown to correlate with telomere length, indicating that NPB formation may also be used as a surrogate measure of critically short telomeres Real-time in vitro studies showed that anaphase bridges may be broken in more than one region during late anaphase, leading to the formation of acentric chromosome fragments and eventually MNi 26 , Therefore, some MNi may also originate from broken anaphase bridges although whether this actually happens in cytokinesis-blocked cells remains unclear.
The importance of scoring NPBs should not be underestimated because it provides direct evidence of genome damage resulting from misrepaired DNA breaks or telomere end fusions, which is otherwise not possible to deduce by scoring MNi only, which could originate from either acentric chromosome fragments or chromosome loss. Over the past decade, another unique mechanism of MNi formation, known as nuclear budding, has emerged. This process has been observed in cultures grown under strong selective conditions that induce gene amplification as well as under moderate folic acid deficiency 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , Shimizu et al.
Amplified DNA may be eliminated through recombination between homologous regions within amplified sequences forming mini-circles of acentric and atelomeric DNA double minutes , which localize to distinct regions within the nucleus, or through the excision of amplified sequences after segregation to distinct regions of the nucleus. The process of nuclear budding occurs during S phase and the NBUDs are characterized by having the same morphology as an MN with the exception that they are linked to the nucleus by a narrow or wide stalk of nucleoplasmic material depending on the stage of the budding process Figs.
The duration of the nuclear budding process, and the extrusion of the resulting MN from the cell remain largely unknown. In this process, Rad 51 recombination protein complexes are detectable throughout the entire nucleus 3 h after irradiation and then concentrate into distinct foci before being extruded from the nucleus as NBUDs. In a series of studies on folic acid deficiency in long-term primary human lymphocyte cultures, we carefully quantified the interrelationship between MNi, NPBs and NBUDs in an attempt to validate the use of these biomarkers and to determine more comprehensively the impact of folic acid deficiency, within the physiological range, on various aspects of genomic stability 35 , 36 , 37 , For example, the DNA methylation inhibitor 5-azacytidine induces undercondensation of the heterochromatin regions of chromosomes 1, 9 and 16, and the specific loss of these chromosomes as MNi in human lymphocytes in vitro 42 , The immunodeficiency, centromeric region instability, facial anomalies syndrome, which is caused by mutation in the DNA methyl transferase DNMT3B gene, is characterized by despiralization of heterochromatin of chromosomes 1, 9 and 16 and loss of this chromatin into NBUDs and MNi These events are likely to be relevant to the aging process because Suzuki et al.
It has been conclusively shown that in vivo aging leads to an increased MN frequency in lymphocytes and that loss of the X chromosome in females and males and loss of the Y chromosome in males are among the primary mechanisms explaining this increase 6 , 45 , Although it is evident that MN expression could be increased as a result of hypomethylation of satellite DNA, it is also possible that increased genome damage may be caused by hypermethylation of CpG islands within or adjacent to the promoter regions of housekeeping genes involved in cell-cycle check points and DNA repair.
To take full advantage of the CBMN Cyt assay and obtain deeper understanding of the genotoxic mechanism, it is essential to distinguish between MNi originating from whole chromosomes and those originating from acentric fragments as well as to determine whether malsegregation of chromosomes is occurring between nuclei in a BN cell that may or may not contain MNi.
This is best achieved by using methods that can detect centromeric regions of chromosomes. The use of MN size as a discriminant is not recommended for human cells or other cell types in which the size of chromosomes is heterogenous because a small MN may contain either a fragment of a large chromosome or a whole small chromosome.
Centromeric regions of chromosomes can be detected indirectly using antibodies to the kinetochore proteins that are assembled on centromeres 52 , 53 but this approach does not distinguish between unique chromosomes and may not detect chromosome loss occurring due to absence of kinetochores on defective centromeres The preferred method is to use in situ hybridization ISH to identify centromeric regions using DNA or peptide nucleic acid pancentromeric probes, which allows MNi containing whole chromosomes to be reliably identified.
Furthermore, use of centromeric probes that are specific for a unique chromosome can be used to detect non-disjunctional events i. The types of results that can be expected with the various techniques are illustrated schematically in Figure 4. It is also possible to use a combination of pantelomeric and pancentromeric probes to determine the mechanism of MN and NPB formation as explained in Figure 5.
For example, an NPB originating from a telomere end fusion would be expected to have a telomere signal within the bridge, but an NPB originating from a DNA misrepair event is less likely to exhibit a telomere signal within the bridge because double strand breaks are expected to occur more frequently in the non-telomeric sequences because they are much more abundant.
Most chemical agents and different types of radiation have multiple effects at the molecular, cellular and chromosomal level, which may occur simultaneously and to varying extents depending on the dose.
Furthermore, determining nuclear division index NDI and proportion of cells undergoing necrosis and apoptosis provides important information on cytostatic and cytotoxic properties of the agent being examined that is relevant to the toxicity assessment. In human lymphocytes, the NDI also provides a measure of mitogen response, which is a useful biomarker of immune response in nutrition studies and may also be related to genotoxic exposure 3 , 23 , 36 , 37 , 38 ,
Cytokinesis-block micronucleus cytome assay
Cytokinesis-Block Micronucleus Cytome Assay in Lymphocytes