|Year : 2017 | Volume
| Issue : 4 | Page : 180-185
Technical note on cytokinesis-arrested binucleated cell and micronucleus assay
Karthik Kanagaraj1, Venkateswarlu Raavi1, Shangamithra Visweswaran1, Tamizh Gnanasekaran Selvan2, Shanmugapriya Dhanashekaran1, Venkatachalam Perumal1
1 Department of Human Genetics, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India
2 PROCyTOX Commission for Atomic Energy and alternative Energies (CEA), Fontenay-aux-Roses and Paris-Saclay University, Fontenay-aux-Roses Cedex, France
|Date of Web Publication||8-Jan-2018|
Dr. Venkatachalam Perumal
Department of Human Genetics, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Conventionally, many biomarkers are being in use as a measure to genotoxicity in occupational exposure to chemicals, pesticides, radiation, and drug screening. Of which, the micronucleus assay is a preferred choice for many of those applications owing to its simplicity and rapidity. The assay methodology has evolved in cell preparations, staining, and scoring methods: from quantifying the DNA damage in mononucleated cells and binucleated cells; solid (Giemsa) and fluorescence staining (propidium iodide/DAPI); and manual and automated microscopy scoring and flow cytometry. Despite the advantages, preparation of cells with good morphology to interpret DNA damage from a different type of cells remains a challenge in particular for laboratory being the processes of developing the assay. Therefore, the aim of the present report was to explain the micronuclei (MN) assay and means to overcome the troubleshoot for reliable outcome measure using cytokinesis-arrested micronucleus (CBMN) assay from suspension and adherent cultures.
Keywords: DNA damage, genotoxicity, micronuclei
|How to cite this article:|
Kanagaraj K, Raavi V, Visweswaran S, Selvan TG, Dhanashekaran S, Perumal V. Technical note on cytokinesis-arrested binucleated cell and micronucleus assay. J Radiat Cancer Res 2017;8:180-5
|How to cite this URL:|
Kanagaraj K, Raavi V, Visweswaran S, Selvan TG, Dhanashekaran S, Perumal V. Technical note on cytokinesis-arrested binucleated cell and micronucleus assay. J Radiat Cancer Res [serial online] 2017 [cited 2018 Jul 22];8:180-5. Available from: http://www.journalrcr.org/text.asp?2017/8/4/180/222442
| Introduction|| |
Change in the biomolecules such as nucleic acid, protein, lipid, and carbohydrates, which can be measurable followed by exposure in live models (cells, tissues, and organisms), is known as a biomarker. Chromosome alterations are used to relate the exposure and are commonly known as cytogenetic markers of exposure. Of the various assays employed to derive a relationship between exposure and altered chromosome morphology, the micronuclei (MN) is quite popular and widely used owing to its simple assay methodology and rapid quantification of induced changes. Analysis of MN has been widely used as an in vitro genotoxicity; biomonitoring for humans followed by exposures to environmental or pharmaceutical agents; prediction of risk for developing cancer in individuals occupationally or accidentally exposed to carcinogens; chronic infection; to monitor repair ability of damaged DNA; infertility; and to estimate in vivo radiation exposure, i.e. accidental, occupational, and medical., That is the number of publications documented in the indexed databases are the evidences for the popularity of the assay.
Ever since the inception of the assay, it has undergone many modifications to meet the requirement: the assay methods, stains used to visualize the changes, cell analysis options, etc., The MN were initially known as Howell–Jolly bodies, as they were first identified in red cell precursors by William Howell and Justin Jolly. At that time, they were presumed as bits and pieces of nuclei of red blood cells circulating at organs with pathological features. Matter and Schmid coined the term MN based on its size and appearance. Time was considered as limiting step that has slowed the development of screening chemicals for mutagenicity; however, the MN assay was developed as a rapid method for a qualitative assessment of chromosomal breakage. Nevertheless, the assay developed by Matter and Schmid was not able to distinguish between those cells that did not divide and those divided. The assay developed by Fenech and Morley allows to choose cells between the phases of mitosis; addition of spindle poison, the cytochalasin-B (Cyto-B), inhibits cell division at cytokinesis in a cycling cell and results in the formation of cells with two nucleus. The entire methodology is known as cytokinesis-blocked micronucleus (CBMN) assay. Thus, in addition to measuring MN, the assay permits to identify other changes such as nucleoplasmic bridges (NPBs), nucleoplasmic bud, necrosis, or apoptotic cells and to derive nuclear division index (NDI) (cytome assay). It is also possible to delineate the origin (chromosome number) and composition (acentric chromosomes/whole chromosome) of MN with fluorescence in situ hybridization (FISH). Despite those developments, the assay methods remain a challenge at least in the hands of beginners indented to use the assay. Therefore, it was intended to summarize the available information and to share our experience to the community for the effective implementation of MN assay for routine application.
| Materials and Methods|| |
The reagents used were cell culture medium (e.g., RPMI-1640), growth factors (Fetal Bovine Serum, FBS), broad-spectrum antibiotics (penicillin, streptomycin, and gentamicin), mitogens (phytohemagglutinin-M, PHA-M), inhibitor of spindle assembly to prevent cytokinesis (Cyto-B), stains and dyes (Giemsa, propidium iodide [PI]/ 4',6-diamidino-2-phenylindole (DAPI), and fluorochrome-conjugated DNA probes for centromere, telomere, or whole chromosomes), methanol and glacial acetic acid, triton X-100, and trypsin-ethylenediaminetetraacetic acid.
The salts used were potassium chloride, potassium dihydrogen orthophosphate, and sodium bicarbonate.
Tissue culture flasks (T25 and T75), centrifuge tubes (15 and 50 ml), microfuge tubes, microtips (10, 200, and 1000 μl), and P60 Petri plates were used.
Centrifuge, water-jacketed CO2 incubator, hemocytometer, microscopes (bright field, phase contrast, and fluorescence), laminar airflow, micropipettes, vortex, and weighing balance were used.
The assay can be performed in any tissue sample and cell lines/cell strains. Here, we present the methodology to prepare binucleated (BN) cells from blood lymphocytes and cell lines (Human Adult Dermal Fibroblast).
CBMN assay for blood lymphocytes: The method described by Fenech and Morley has been modified and described; it consists of four steps, namely (i) culture initiation, (ii) harvest, (iii) casting, and (iv) staining.
- Culture initiation: Briefly, to 1 ml of blood, 9 ml of complete culture media (80% RPMI-1640 and 20% FBS) was added, stimulated with PHA-M (20 μg/ml) and incubated at 37°C in 5% CO2 in water-jacketed incubator. At 44th h of incubation, Cyto-B (6 μg/ml) was added aseptically and incubated further for another 28 h
- Harvesting the culture: At the end of the 72nd h of incubation, the cultures were transferred into the 15 ml centrifuge tubes and centrifuged at 800 rpm for 8 min at 37°C. The supernatant was discarded and the cell pellet was disturbed by adding 10 ml of 0.075 M prechilled hypotonic solution (potassium chloride). The sample was again centrifuged at 600 rpm for 6 min and supernatant was discarded. To the pellet, 10 ml of Carnoy's fixative (methanol: acetic acid, 5:1) was added. After the third wash with Carnoy's fixative, the cell pellet was resuspended in desired volume of fixative (500–1000 μl)
- Casting: The glass slides were washed and rinsed in the distilled water, then kept in ice-cold water before casting. The cell suspension was gently mixed with pasture pipette; few drops of the suspension were dropped onto precooled slides from a height of about 5 cm. The excess liquid was wiped with the tissue and slides were dried on a hotplate at a temperature of 25°C–30°C
- Staining: The slides were stained with either solid stain (Giemsa) or fluorescence dyes (PI/DAPI). For Giemsa staining, the dried slides were immersed in the Giemsa stain (5%) for 15–20 min and were rinsed in distilled water and air-dried. For long-term storage, it is recommended to cover slides with cover glass using DPX. The slides are observed under bright-field microscope (×40) for cells at different phases in cell cycle. In alternate they can be stained with PI/DAPI (1 μg/ml), the slides can be observed with fluorescence microscope.
Cytokinesis-blocked micronucleus assay for cell lines: The methodology to prepare BN cells from cell lines is easy when compared to blood lymphocytes. Confluent cells in cultures were detached using trypsin; cells were resuspended in cell culture medium (~1 ml) and the cells were counted using hemocytometer. Approximately 2–3 × 103 cells were seeded into P60 dishes with suitable media and incubated at 37°C in a 5% CO2 incubator. While initiating the culture or at 24th h, Cyto-B (3 μg/ml) is supplemented to the medium to get BN cells. After 48 h, the cells were washed with phosphate-buffered saline (PBS), fixed with ice-cold fixative (methanol: acetic acid, 3:1), and air-dried. Then, the cells were stained with PI/DAPI and analyzed using a fluorescence microscope (×40) with an appropriate filter. Based on the scoring criteria as described, one thousand BN cells need to be analyzed to derive MN frequency.
Reliable results of the assay depend on the follow of well-defined criteria to include/exclude cells and DNA damage as MN formation.
Criteria for scoring binucleated cells
- The cells should have two distinct nuclei within the same cytoplasmic boundary
- The two nuclei should be approximately equal in size, staining pattern, and intensity
- The size of NPB should not wider than one-fourth of the largest nuclear diameter
- The two daughter nuclei in a BN cell may touch, but it should not overlap each other
- The cytoplasmic boundary of a BN cell should be intact and clearly distinguishable from the cytoplasmic boundary of adjacent cells.
Criteria for scoring micronuclei
- Shape of MN should be regular (round or oval)
- MN should be nonrefractile and clearly differentiated from artifact such as stained debris
- MN are not connected or associated with the primary nuclei
- MN may touch but not overlap with main nuclei and should be present within the cytoplasmic boundary of BN cells
- Staining intensity of MN should be equivalent to that of main nuclei
- The diameter of MN in cells generally varies between 1/16th and 1/3rd of the mean diameter of the main nuclei.
In general, the scoring is performed by scanning the slides systematically using consistent criteria to include/exclude a cell for analysis: It can be performed manually or using automated system with suitable software to identify the MN.
Manual scoring: Followed by staining, the slides are scanned at low magnification (×10) for the index and staining quality. Upon satisfaction, the slides are focused at higher magnification (×40) and analyzed sequentially, to record details such as X and Y coordinates of cells, number of MN in the BN cells, and cells with one, two, three, four, etc., nucleus for verification.
Automated scoring: To improve the speed of scoring to handle huge sample size during large-scale accidents, automated imaging tools were developed. While automated scoring increased the speed of analysis of samples, it resulted in compromise of the sensitivity and specificity. This is because the system identifies the BN cells with the following criteria: (i) two equal-sized nuclei, (ii) close to each other, (iii) daughter nuclei shape and size, and (iv) distance between the two daughter nuclei.
Micronuclei content analysis by fluorescence in situ hybridization
It is an established fact that MN can be originated either from broken chromosome of whole chromosome which failed to get incorporated into the daughter nuclei at the time of cell division, that is, the content depends on the nature of DNA-damaging agent. FISH with pan-centromeric probe alone or in combination of pan-centromere and telomere probes can help delineate the origin of DNA content. In brief, prepared slides with BN cells as described earlier are treated with a series of ethanol concentrations (80%, 90%, and 100%) for dehydration and air-dried. A diluted probe (1 μl of probe in 15 μl of hybridization buffer) was added on to the slides and sealed under glass coverslips, placed in a moistened hybridizing chamber (HyBrite TM, Vysis) which was programmed for 73°C for 5 min denaturation and 37°C for 16–18 h of hybridization. The slides were then washed with ×0.4 SSC/0.3% IGEPAL at 72°C ± 1°C for 15–20 s and ×2 SSC/0.1% IGEPAL at room temperature for 5–10 s. Coverslips were applied with antifade mountant containing DAPI (Vysis Inc., Downers Grove, USA) and stored at −20°C for an hour to enhance the probe signal. Those were analyzed using a fluorescence microscope (×100) with Fluorescein isothiocyanate (FITC) (excitation–emission: 490–525-Green) and DAPI (excitation–emission: 358–461-Blue) filters and scored as described earlier.
| Discussion And Interpretation|| |
MN formation: It is a known fact that MN can be originated either from broken chromosome or from whole chromosome, which failed to get incorporated into the daughter nuclei at the time of cell division. [Figure 1] shows the illustration of MN formation. To visualize the damage as MN, the cells need to be divided twice (two-cell division) upon the induction of damage. It is because, that during the first division, though the damage has occurred, the resultant MN will not express; only when the cell attempt to divide second time, it expresses as separate entity from that of the daughter nuclei. Moreover, the MN is considered as an unstable aberration as the cell attempt to divide; they are eliminated as the daughter nuclei have either partial or complete aneuploidy or genomic instability. Therefore, for a reliable measure of genotoxicity using the MN assay, BN cells should be considered.
|Figure 1: Illustration on formation of micronuclei from damaged chromosomes|
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Binucleated cells in solid-stained preparations
Staining of cells with Giemsa and scoring of MN manually is a widely adopted choice for the assay. BN cells without MN and cells with different numbers of MN are shown in [Figure 2]a. Cells at different phases of the cycle followed by mitogen stimulation are given in [Figure 2]b. For reproducible results, it is recommended to follow stringent and consistent criteria to select a BN cell and MN. Advantages of Giemsa-stained preparation are that upon staining, the slides can be mounted in DPX and stored even at room temperature for longer duration and can be analyzed leisurely. However, the beginners need sufficient training to identify the MN correctly, as at times, artifacts of stain can result into erroneous results.
|Figure 2: (a) A micrograph of binucleated cells without and with different number of micronuclei and (b) different phases of cell cycle after mitogen stimulation|
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Metafer, an automated imaging system to capture and identify MN among the BN cells, is available commercially. Rapid screening and analyzing higher sample size are the merits of automated scoring. A panel of BN cells captured using automated image analyzer is shown in [Figure 3]a. Repeated scoring of BN cells to reconfirm the obtained MN frequency is the advantage of automated imaging system. IMSTAR Pathfinder™ Screentox Auto-MN, Compucyte iCyte® Laser Scanning cytometer and Imaging flow cytometry are other platforms that are available for MN scoring.
|Figure 3: (a) A micrograph of binucleated cells captured using automatic image analyzer and (b) binucleated in cell lines stained with propidium iodide and DAPI|
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Binucleated cells in fluorescence-stained preparations
Misclassification of artifacts as MN in solid-stained preparations can be minimized by staining the BN cells with nucleic acid-specific fluorescence dyes. Fluorescence dyes such as PI/DAPI can reduce difference in the radiation-induced MN yield when they were scored in automation when compared to that of Giemsa-stained preparations. This is because unlike that of Giemsa, the fluorescence dyes are nucleic acid specific and permit to identify MN unequivocally. [Figure 3]b shows the BN cells stained with PI/DAPI after manual and automated scanning, respectively.
Origin of micronuclei and content analysis
Population exposures to chemicals, biological agents, and physical agents such as radiation, either alone or in combination, are high in case of mishandling, technical failure, or accidents. In turn, biological effects of those agents are varied and dependent on the agent involved, concentration and stability or biological half-life of the agent, incident conditions (weather, terrain, and time), and environment. While the MN assay in uniform stained BN cells enables to quantify the extent of DNA damage, content (whole chromosome or fragment/hot spots on any chromosome) analysis is not possible. [Figure 4] shows the BN cells after centromere FISH followed by counterstained DAPI. MN originated from the whole chromosome contain centromere and/or both centromere and telomere signal; whereas, MN derived from acentric fragments shows only the telomeric signal or without any fluorescence signal. Similarly, MN derived by the fusion of more than one acentric fragment also can be identified, if the MN shows more than one telomere signal. By doing m-FISH (probes for each chromosome labeled with different fluorochromosomes), one can identify hot spots if any among the genome to form DNA damage. Thus, FISH-MN permits to identify the content as well as derive the mechanism of DNA-damaging agent. Despite those advantages, FISH-MN is not practiced for routine applications owing the higher cost and self-life of DNA probes.
|Figure 4: A micrograph of binucleated cells stained with centromere fluorescence in situ hybridization probes and counterstained DAPI|
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Frequency of micronuclei
The frequency of MN can be obtained by scoring around 1000 BN cells. The frequency of aberration is generally expressed as either MN frequency/cell or micronucleated cells/cells with the standard error.
MN frequency = number of MN in BN cells/total number of BN cells scored.
Micronucleated cell frequency = BN cells with MN/total number of BN cells scored.
Standard error: Square root of number of MN/total number of BN cells scored.
Nuclear division index
The NDI provides a measure of the proliferative status of the viable cell fraction. It was calculated by scoring around 1000 viable cells and then grouped into cells with 1, 2, 3, or 4 nuclei, using the formula.
NDI = M1 + 2 (M2) +3 (M3) +4 (M4)/N
where, M1–M4 = the number of cells with 1–4, respectively, nuclei and N = total number of viable cells scored (excluding necrotic and apoptotic cells). This can be employed to investigate the cell cycle kinetics followed by genotoxic exposure.
Troubleshoot and means to overcome
The methodology for the CBMN assay has been well established. Excellent reviews on methods and its applications on various domains has been reported. However, any laboratory intend to utilize the assay for specific application for the first time, it is quite cumbersome and challenging to get sufficient and scorable BN cells. [Table 1] summarizes the experience of troubleshoot from our laboratory.
|Table 1: Troubleshoots and means to overcome the issues while performing the micronuclei assay|
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Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]