|Year : 2016 | Volume
| Issue : 2 | Page : 202-206
Cytokinesis-blocked micronucleus assay as a biomarker for risk of lung cancer
Keerthana Ganesh1, G Tamizh Selvan1, Surabi Ganga1, Solomon F.D. Paul1, Anita Ramesh2, J Vijayalakshmi1
1 Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India
2 Department of Medical Oncology, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India
|Date of Web Publication||29-Sep-2016|
Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Lung cancer is the second most leading cause of cancer mortality throughout the world. It is an imperative requirement to find reliable markers which can be used effectively to assess the initial analysis and predict the risk of lung cancer. The cytokinesis-blocked micronucleus (CBMN) assay is one of the well-developed methods to measure the proficiency of genomic instability in peripheral blood lymphocytes of individuals with different types of cancer. In our present study, we used the CBMN assay to investigate chromosomal aberrations in lung cancer patients.
Materials and Methods: Peripheral blood was collected from lung cancer patients (n = 12) and healthy subjects (n = 10). The blood samples were cultured with RPMI-1640, fetal bovine serum, stimulated with phytohemagglutinin, and incubated at 37°C. The cells were blocked by adding cytochalasin-B at 44 th h and analyzed for micronuclei, nucleoplasmic bridges, and nuclear buds (NBUD) in binucleated cells.
Results: The frequency of micronuclei, nucleoplasmic bridges, and NBUD were found to be significantly (P < 0.001) higher in cases, indicating escalated genetic damage in cancer patients than controls. This study also revealed a correlation between the age, duration of smoking, number of cigarettes, and chromosomal aberration present in the study subjects.
Conclusion: The CBMN assay is a valuable tool that can be employed to screen lung cancer cases for its simplicity, rapidity, and sensitivity.
Keywords: Chromosome damage, lung cancer, micronuclei, peripheral blood lymphocytes
|How to cite this article:|
Ganesh K, Selvan G T, Ganga S, Paul SF, Ramesh A, Vijayalakshmi J. Cytokinesis-blocked micronucleus assay as a biomarker for risk of lung cancer. Indian J Health Sci Biomed Res 2016;9:202-6
|How to cite this URL:|
Ganesh K, Selvan G T, Ganga S, Paul SF, Ramesh A, Vijayalakshmi J. Cytokinesis-blocked micronucleus assay as a biomarker for risk of lung cancer. Indian J Health Sci Biomed Res [serial online] 2016 [cited 2020 Jul 6];9:202-6. Available from: http://www.ijournalhs.org/text.asp?2016/9/2/202/191275
| Introduction|| |
Lung cancer is the second leading cause of cancer, the first being breast cancer in the global population, making it essential to improve to predict early risk and diagnosis.  The generation of the genomic instability phenotype is one of the early events in carcinogenesis, which enables an unstable cell to evolve into a cancer cell by achieving a greater proliferative capacity.  It is well known that cancer results from an accumulation of multiple genetic changes that can be mediated through chromosomal changes and can be cytogenetically detectable.  The level of genetic damage in peripheral blood lymphocytes can be hypothesized to the amount of damage in the precursor cells that lead to the carcinogenic process in target tissues.  Both cohort and nested case-control studies have shown evidence that cytogenetic biomarkers are positively correlated with cancer risk that has been strongly validated showing that chromosome aberrations are a marker of cancer risk reflecting both the genotoxic effects of carcinogens and individual cancer susceptibility. 
Approximately, 85% and 47% of the lung cancers in men and women are caused by tobacco smoking. The conventional smoke from the cigarette mouthpiece contains about 10 10 particles/ml.  Cigarette smoke contains at least 81 known carcinogens such as benzo[a] pyren, Nicotine-derived nitrosamine ketone (NNK), 1,3-butadiene and the radioisotope polonium-210, and free radicals and it induces oxidative damage in humans. International Agency for Research on Cancer (IARC) has evaluated, and studies have been conducted to prove the carcinogenicity in either laboratory animals or human models.
About 10-15% of lung cancer occurs in cases who have never smoked. Exposure to radon gas, asbestos, crystalline silica, polycyclic aromatic hydrocarbons, and heavy metals (e.g. cadmium) are recognized as pulmonary carcinogens by the IARC.  Chronic obstructive pulmonary disease is at greatest risk factor for lung cancer, which causes inflammation of the thoracic region. Apart from the external factors, the combination of genetic factors has also shown a higher risk for lung cancer, which is independent of smoking status.  The cytokinesis-block micronucleus (CBMN) assay is a distinguished method to evaluate the risk factors of cancer by measuring the residual chromosome damage levels and micronucleus (MN) frequency. The MN formation is due to exposure to clastogens and aneugens caused by oxidative stress.  The CBMN assay makes a valuable tool for screening; the multiple end points simultaneously generated lead to a better understanding of the underlying mechanisms involved in carcinogenic process that could in turn substantially improve risk predictions.  MN formation and other associated biomarkers of DNA damage (nucleoplasmic bridge [NPB] and nuclear buds [NBUD]), cell death, and cytostasis have become more evident in any stress-induced environment, may be due to carcinogenic, mutagenic, or radiation induced. , In addition, the altered mitotic activity, cytostasis, and cell death by apoptosis and necrosis can be studied using the nuclear division index (NDI) and nuclear division cytotoxicity index (NDCI) as reported earlier.  The presence of MN due to tobacco smoke has been reported in many studies irrespective of the smoking status in both active and passive smokers.  About 63,000 lung cancer cases are reported in India each year.  In this study, the assay is being applied successfully for biomonitoring of in vivo smoking/nonsmoking individuals to study higher cancer risk.
| Materials and Methods|| |
This is a case-control study consisting of individuals diagnosed with lung cancer (n = 12) and controls (n = 10). Their mean age is 58.2 ± 10 years. The study was approved by the Institutional Ethics Committee of Sri Ramachandra University (Ref No: CSP/15/JAN/39/07). About 2-3 ml of blood samples were collected from the study subjects after obtaining informed consent.
Cytokinesis-block micronucleus assay
The CBMN assay protocol was adopted from Fenech (2007). Each culture contained 80% of RPMI-1640 media, 20% fetal bovine serum, and 1 ml peripheral blood. About 400 μl of phytohemagglutinin (20 μg/ml) was used to initiate the culture. The total incubation time for all cultures was 72 h at 37°C in the presence of 5% CO 2 . At 44 th h of culture, the cells were blocked at cytokinesis by adding cytochalasin B (6 μg/ml). The cell contents were transferred to 15 ml sterile centrifuge tubes, spun at 600 rpm/7 min. The supernatant was discarded, to the cell pellet 8 ml of hypotonic solution (0.075M potassium chloride) and fixed with 5:1 methanol: glacial acetic acid and kept at 4°C until use. The cell pellet was washed two to three times, dropped onto a prechilled clean microscopic glass slide, prewarmed, and stained with Giemsa stain (8%) for 4-7 min. For each sample, 1000 binucleated cells were scored using a light microscope (×40 magnification). The scoring criteria were followed based on the guidelines HUMN Project (Fenech et al., 2003). The number of micronuclei, nucleoplasmic bridges, and NBUD was recorded.
Nuclear division index and nuclear division cytotoxicity index
The NDI provides a measure of proliferative status of the viable cell fraction. Therefore, this is an indicator of cytostatic effects and helps to measure the mitogenic response of lymphocytes, which is considered to be a useful biomarker for proper function of cellular systems and it is calculated using the formula:
NDI = (M1 + 2M2 + 3M3 + 4M4 )/N.
Where M1-M4 represents the number of cells presenting with 1-4 nuclei and N is the total number of viable cells scored (excluding necrotic [Nec] and apoptotic [Apo] cells).
In addition, Nec and Apo cells were observed, the NDCI was also calculated using the formula given below:
NDCI = (Nec + Apo + M1 + 2M2 + 3M3 + 4M4 )/N.
Student's t-test was used to compare the MN frequency between case and control. The P < 0.05 is considered to be statistically significant.
| Results|| |
This study is a case-control study with a sample size (n = 12), mean age of the study subjects 58.2 ± 10 are summarized in [Table 1]. The average duration of smoking 33.25 ± 10 years and the number of cigarettes/beedi smoked per day was documented as 38.83 ± 10. All the study subjects are occasional alcoholic.
Nuclear division index and nuclear division cytotoxicity index
[Figure 1] shows the comparison of NDI and NDCI in controls and cases. The NDI in control was 2.02 ± 0.01 and case was 1.49 ± 0.09, respectively. The NDCI was calculated depending on the number of Apo cells and Nec cells observed, which was found to be only in cases (1.46 ± 0.1).
|Figure 1: Nuclear division index and nuclear division cytotoxicity index observed in case– control|
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Frequency of micronucleus, nucleoplasmic bridge, nuclear buds
MN, NPB, and NBUD were found in all the cases. The frequency of these CBMN assay end points was stratified on factors such as age, duration of smoking, and the number of cigarettes smoked per day. The frequency of these end points was found to increase with increases in all of those factors. The yield of MN in binucleate cells showed a 4-fold increase in cases than controls (0.008 ± 0.004; 0.002 ± 0.001) (P < 0.0001), respectively [Figure 2]. The nucleoplasmic bud and NPB were found only in cases [Figure 3].
|Figure 2: Micronucleus frequency observed in binucleated cells from peripheral blood lymphocytes of lung cancer patient and healthy subjects|
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|Figure 3: Nuclear bud and nucleoplasmic bridge frequency observed in peripheral blood lymphocytes of lung cancer patient and healthy subjects|
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Micronuclei in mononucleated cells
For the most comprehensive analysis, the frequency of micronuclei in mononucleated cells can also be determined. Cases showed a higher number of mononucleated cells than the control, indicating DNA damage. Thereby the MN in mononucleated cells serves as an essential tool for scoring the case sample leading to even better understanding and discriminate results between cases and controls. There was no MN found in the mononucleated cells of the control sample as the relative number of mononucleated cells was also less. The frequency of MN in case samples (0.01 ± 0.006) is represented in [Figure 4].
|Figure 4: Micronucleus frequency observed in mononucleated cells from peripheral blood lymphocytes of lung cancer patient and healthy subjects|
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| Discussion|| |
In the present study, the extent of genetic instability was evaluated in individuals diagnosed with lung cancer; the CBMN assay is applied as a biomarker to assess early risk of lung cancer in individuals presenting with a history of smoking. Literature has shown that the baseline frequency of MN  and other end points of the CBMN assay in individuals with different types of cancer shows to have confounding confirmatory data in analyzing the genetic instability in cancer patients. The increased sensitivity to mutagens causes the much higher response of cancer patients to MN, NDI (mononucleated cells, binucleated cells, trinucleated cells, and tetranucleated cells), and NDCI (Apo cells and Nec cells).
The amount of DNA damage observed is dependent on the extent of other cellular events such as necrosis and/or apoptosis. This clearly indicates that the values of NDI (1.49 ± 0.09) and NDCI (1.46 ± 0.1). There was a 1-fold decrease in cases than controls (2.02 ± 0.01). The induction of reactive oxygen species or the mechanism that involves ß-adrenergic-mediated release of arachidonic acid is found to be induced by tobacco carcinogens.  Thereby the inability of damaged cells to enter into the next phase of cell cycle is a possible indication of decreased NDI.
The results of the present study showed that the cases had significantly higher levels of baseline micronuclei in mononucleated cells (0.01 ± 0.006) than the controls, indicating an increase in the level of in vivo genetic damage and cytotoxicity. It is possible that some of the lymphocytes already harbor micronuclei before they are stimulated to divide in culture. This is possible in situations of increased genomic instability or chronic exposure to carcinogens, in case of prolonged smoking habit. Furthermore, increased mononucleated cells indicate that the number of cells preceding to the second and the following cell divisions lesser in case when compared to the control. Micronuclei in mononucleated cells may also represent cells that are at a very early stage of induced cell death (either apoptosis or necrosis). Therefore, this could possibly explain the reason for increased NDCI in cases when compared to controls subjects.
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]