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 Table of Contents  
Year : 2021  |  Volume : 13  |  Issue : 6  |  Page : 960-964  

Comparative evaluation of genotoxicity in tobacco users versus nontobacco users

1 Departments of Oral and Maxillofacial Pathology, Noorul Islam College of Dental Science, Pathamkallu, Kerala, India
2 Dr. Vivek's Dental Clinic, Thiruvananthapuram, Kerala, India
3 Prosthodontics, Noorul Islam College of Dental Science, Pathamkallu, Kerala, India
4 Public Health Dentistry, Noorul Islam College of Dental Science, Pathamkallu, Kerala, India
5 Department of Oral and Maxillofacial Pathology, Kerala State Health Services, General Hospital, Kottayam, Kerala, India

Date of Submission05-Feb-2021
Date of Decision08-Mar-2021
Date of Acceptance10-Apr-2021
Date of Web Publication10-Nov-2021

Correspondence Address:
Arun Jacob Thomas
Department of Oral and Maxillofacial Pathology, Noorul Islam College of Dental Science, Thiruvananthapuram, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpbs.jpbs_60_21

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Background: Many of the contents of cigarette smoke are genotoxic in nature, and consequently, cytogenetic injury seems to be a trustworthy biomarker for deciding the influence of exposure to chromosome damaging agents in smoke. The cytokinesis-block micronucleus assay (CBMN assay) has been proven to be an effectual tool for the study of micronuclei (MN) that will help in estimating the genotoxicity in tobacco users alone which will further help in early cancer detection. Objective: The objective is to find out whether there is pronounced contrast in genotoxicity between tobacco users and nonusers by determining MN number in peripheral blood lymphocytes using CBMN assay. Methodology: MN frequency in peripheral blood lymphocytes was estimated in 5 ml of fresh blood obtained from sixty individuals using tobacco either smoking, chewing, or combination of both and also from thirty individuals with no habit of tobacco use. All were in the age group of 20–40 years. Results: There was a significant increase in genotoxicity in tobacco users when compared to that of nontobacco users. A positive correlation was also obtained between smoking index and MN frequency in the study. Conclusion: Approximation of frequency of MN by CBMN assay can be used to evaluate the genotoxicity present in blood and helps in identifying tobacco users who are at a high risk for the presence of cancer even before the appearance of clinical changes.

Keywords: Genotoxicity, micronuclei, tobacco

How to cite this article:
Thomas AJ, Nair BJ, Oommen S, Syamkumar V, Raman RK. Comparative evaluation of genotoxicity in tobacco users versus nontobacco users. J Pharm Bioall Sci 2021;13, Suppl S2:960-4

How to cite this URL:
Thomas AJ, Nair BJ, Oommen S, Syamkumar V, Raman RK. Comparative evaluation of genotoxicity in tobacco users versus nontobacco users. J Pharm Bioall Sci [serial online] 2021 [cited 2022 Dec 7];13, Suppl S2:960-4. Available from:

   Introduction Top

Cancer is one of the main reasons for morbidity and mortality of our times. Oral cancer is the sixth most prevalent cancer, of which 95% are oral squamous cell carcinoma.[1] It is estimated that around 43% of cancer deaths occurred due to tobacco use, insalubrious diets, alcohol intake, and inactive lifestyle.[2] In recent years, aggravating number of these malignancies are accounted among younger adults.[3]

Tobacco intake is the world's most avertible risk factor of cancer as 80% of oral cancers are associated with its use.[4] The risk appears to increase with increase in duration and intensity of tobacco smoking.[5]

On intake of carcinogenic content in tobacco smoke, there is the development of covalent bonds among carcinogens and DNA creating DNA adducts which result in ineffaceable mutations in critical genes of somatic cells.[6] Therefore, cytogenetic injury seems to be a superior biomarker for finding the influence of exposure to chromosome harming agents in smoke.[7]

Cytogenetic damage can be quantitatively measured by cytogenetic biomarkers such as chromosomal aberrations, sister chromatid exchanges, and micronuclei (MN) in PBLs.[8] Cytokinesis-block micronucleus (CBMN) assay is one of the benchmark cytogenetic tests for measuring chromosomal damage in PBLs because of its simplicity, good duplicability, and dependability.[9]

We did not come across any study which estimated the level of MN in peripheral blood of tobacco users alone. Thus, the present study was conducted to find out whether there is pronounced contrast in genotoxicity in tobacco users and nonusers by CBMN assay in human peripheral blood lymphocytes by evaluating the average number of MN.

   Methodology Top

Study setting

This study was conducted in the outpatient department, PMS College of Dental Sciences and Research, Trivandrum, and laboratory procedures were carried out in “Genitika” (an ISO certified laboratory), Pettah, Thiruvananthapuram, over a duration of 1½ years.

Study subjects

All the patients were briefly informed about the test procedure before sample collection, and informed consent was obtained. Study group consisted of sixty individuals using tobacco either smoking, chewing, or combination of both. The criteria for deciding the tobacco smokers were based on the definitions given by the US Centre for Disease Control and Prevention.[10]

  • Never smokers
  • Former smokers
  • Nonsmokers
  • Current smokers.

The classification of tobacco chewers[11]

  • Current tobacco chewers
  • Tobacco chewing quitters
  • Ever tobacco chewers
  • Never-Tobacco-Chewers.

Control group consisted of thirty individuals with no habit of tobacco use (smoking or chewing) and normal oral mucosa.

The study was reviewed and approved by the institutional ethical committee of the institution.

Inclusion criteria

  • Patients with a habit of tobacco use and having clinically normal mucosa
  • Patients aged 20–40 years
  • Patients with no other reported systemic illness.

Exclusion criteria

  • Patients with clinically detectable changes in the oral mucosa
  • Patients below 20 years and above 40 years
  • Patients with confirmed systemic illness
  • Exposure to cytotoxic drugs, chemicals, or radiation therapy.

Sample size

Sample size was calculated based on the equation:

where, n – Sample size, σ – Standard deviation, δ – Difference in mean, and α and β are Type I and Type II errors.


  • Disposable gloves
  • Mouth mask
  • Tourniquet
  • Spirit
  • Sterile gauze pieces
  • Syringe and needle
  • Mouth mirror
  • Probe
  • Tweezer
  • Cotton rolls.


  • Lymphoprep, sterile 500 ml
  • Hank's balanced salt solution, sterile, with calcium and magnesium, without phenol red
  • Roswell Park Memorial Institute 1640 medium, without L-glutamine
  • Fetal bovine serum, heat-inactivated
  • L-Glutamine solution
  • Sodium pyruvate solution
  • Cytocalasin-B
  • Dimethyl sulfoxide
  • Phytohemagglutinin
  • Isoton II
  • Zapoglobin
  • Depex
  • 1% (w/v) hypochlorite solution
  • Diff-Quik staining set
  • Methanol
  • Isotonic saline
  • 4.25 g sodium chloride
  • 500 ml Milli-Q water
  • Acetic acid.

Sample collection

All participants completed a questionnaire which gave their demographic data, medical status, smoking and drinking habits, prior or current exposure to medication, or environmental agents affecting MN frequency.

Five milliliter of fresh peripheral blood was drawn from all volunteers by venepuncture. Two milliliters sent for routine blood examination to rule out systemic changes, remaining 3 ml was transferred into heparinized vacutainers and sent to the laboratory within a few hours after collection of samples.

CBMN assay procedure was then carried out.


Each of the slides was coded before scoring and examined at ×100 magnification. The number of MN with no <1000 binucleated cells was scored, and the distribution of MN among binucleated cells was recorded.

The MN frequency between tobacco users and nontobacco users were then compared.

Statistical analysis

Data were analyzed using the computer software, Statistical Package for the Social Sciences (SPSS), Version 16, manufactured by (International Business Machines Corporation company, United States of America) for Windows Operating System.

   Results Top

In our study, a novel attempt was made to correlate smoking index with MN frequency in PBLs. We noted a strong significant positive correlation between smoking index and MN frequency [Table 1]. Using linear regression analysis [Graph 1], we were able to deduce that 23.3% variability in MN frequency was attributed to smoking index [Table 2], and thus by making the use of this regression model, we were able to predict the MN frequency in PBLs from that of smoking index in tobacco users. The regression coefficient of smoking index (0.000021) indicates that, as smoking index increases by 10,000 units, the CBMN frequency increases by 0.2 units. The predicted model is significant (P < 0.001). Thus, it can be concluded that the average CBMN frequency increases by 0.2 units for every 10,000 units of smoking index (roughly three cigarettes/day for 10 years). R2 of the regression analysis is 0.233. It means that 23.3% of variation in CBMN frequency is explained by smoking index. The implication of the result is that CBMN frequency is significantly influenced by smoking index [Table 2].
Table 1: Comparison of micronuclei frequency based on the smoking index

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Table 2: Prediction of cytokinesis-block micronucleus frequency using smoking index (linear regression)

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The MN frequency of both tobacco users and nontobacco users was statistically compared. The mean MN frequency in tobacco users was found to be 12.6 ± 0.8 and in nontobacco users was 10.7 ± 0.5 [Table 3]. Results revealed that there was a statistically significant increase (t = 12.74, P < 0.01) in number of MN among tobacco users when compared with nontobacco users.
Table 3: Comparison of micronuclei frequency between tobacco users and nontobacco users

Click here to view

   Discussion Top

Results revealed that there was a statistically significant increase (t = 12.74, P = 0.000, P < 0.01) in number of MN among tobacco users when compared with nontobacco users, emphasizing the fact that a pronounced higher level of genetic injury and cytotoxicity exist among tobacco users. The above result obtained is in consensus with studies done by El-Zein et al.,[12] Zamani et al.[13] and Guttikonda et al.[14]

El-Zein et al. in their study analyzed MN in mononucleated cells along with other variables in lymphocytes of lung cancer patients to improve the predictive value of CBMN assay. They showed that CBMN cytome assay is an exquisite and explicit predictor of lung cancer risk.[12]

Studies which showed a boost in MN frequency in different types of cancer patients were done by Jianlin et al.,[15] Venkatachalam et al.,[16] and Milosevic-Djordjevic et al.[17] Milosevic-Djordjevic also observed that gender, smoking, and cancer sites did not have any influence on MN frequency in untreated cancer patients.[17]

Guttikonda et al. evaluated DNA injury levels in PBLs of tobacco inveterate patients with clinically normal mucosa and patients with oral carcinoma using Single Cell Gel Electrophoresis (SCGE) and deduced that DNA damage in PBLs in both groups were statistically significant and that DNA damage in the former group was exhibited much before clinical alterations were evident. It was also found that, by this method, potential malignancy, predisposition, and cancer development may be predicted in susceptible individuals.[14]

Calderon-Ezquerro et al. study indicated a pronounced lesser number of MN in smoker group. A probable cause for the lesser MN frequency in smokers could be that severely injured cells might have waned to divide or died during their ex vivo culture, so it was not feasible for the induction of MN. In this study, values between the number of cigarettes/day and nicotine densities were less and trivial and this may be due to fake information provided by smokers, with a probability of being belittled in some cases and exaggerated in others.[18]

In our study, we also found that MN frequency increased with duration of tobacco use. Similar result was obtained in the study done by Naderi et al.[19] However, the result obtained was statistically not significant. They proposed that chromosomal alterations caused by smoking are possibly not linked to the time of smoking as cigarette has the capacity to exhibit chromosomal modifications from the initial period itself. However, our results showed a statistically significant correlation (F = 10.78, P = 0.000, P < 0.01) between duration of tobacco use and MN frequency.

   Conclusion Top

There was a statistically pronounced increase in the MN frequency in tobacco users when compared to nontobacco users. MN number also had a linear correlation with duration of tobacco use and smoking index.

The results deduced the fact that the genotoxicity in tobacco users was profoundly higher by comparison to that of nontobacco users. The results deduced the fact that we can predict the Micronuclei frequency in an individual by obtaining the duration and frequency of his/hertobacco usage. This in turn would allow us to evaluate the genotoxicity in tobacco users at an initial stage even before the clinical signs of cancer. This would further help in conducting tobacco cessation programs among high-risk individuals for cancer.

A higher sample size with longer duration follow-up is the need to further verify the correlation between the number of MN in peripheral blood lymphocytes and tobacco use and its use in cancer risk prediction in individuals.


All the authors would like to explicit their profound indebtedness to all the data collectors and participants for giving consent to participate in this research.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Johnson N. Tobacco use and oral cancer: A global perspective. J Dent Educ 2001;65:328-39.  Back to cited text no. 1
Petersen PE. Oral cancer prevention and control – The approach of the World Health Organization. Oral Oncol 2009;45:454-60.  Back to cited text no. 2
Neville BW, Day TA. Oral cancer and precancerous lesions. Cancer J Clini 2002;52:195-215.  Back to cited text no. 3
Brawley AW. Avoidable cancer deaths globally. Cancer J Clin 2011;61:67-8.  Back to cited text no. 4
Warnakulasuriya S, Dietrich T, Bornstein MM, Casals Peidró E, Preshaw PM, Walter C, et al. Oral health risks of tobacco use and effects of cessation. Int Dent J 2010;60:7-30.  Back to cited text no. 5
Hecht SS. Cigarette smoking: Cancer risks, carcinogens, and mechanisms. Langenbecks Arch Surg 2006;391:603-13.  Back to cited text no. 6
Bonassi S, Neri M, Lando C, Ceppi M, Lin YP, Chang WP, et al. Effect of smoking habit on the frequency of micronuclei in human lymphocytes: Results from the Human MicroNucleus project. Mutat Res 2003;543:155-66.  Back to cited text no. 7
Hagmar L, Bonassi S, Stromberg U, Brogger A, Knudsen LE, Norppa H, et al. Chromosomal aberrations in lymphocytes predict human cancer: A report from the European study group on cytogenetic biomarkers and health (ESCH). Cancer Res 1998;58:4117-21.  Back to cited text no. 8
Fenech M. The in vitro micronucleus technique. Mutat Res 2000;455:81-95.  Back to cited text no. 9
CDC. 2011 National Health Interview Survey (NHIS) Public Use Data Release. Hyattsville, MD: US Department of Health and Human Services, CDC, National Centre for Health Statistics; 2011. Available from: [Last accessed on 2013 Nov 01].  Back to cited text no. 10
Joshi U, Modi B, Yadav S. A study on prevalence of chewing form of tobacco and existing quitting patterns in urban population of Jamnagar, Gujarat. Indian J Community Med 2010;35:105-8.  Back to cited text no. 11
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El-Zein RA, Fenech M, Lopez MS, Spitz MR, Etzel CJ. Cytokinesis-blocked micronucleus cytome assay biomarkers identify lung cancer cases amongst smokers. Cancer Epidemiol Biomarkers Prev 2008;17:1111-9.  Back to cited text no. 12
Zamani AG, Durakbasi-Dursun HG, Demirel S, Acar A. Evaluation of smoking genotoxicity in Turkish young adults. Indian J Hum Genet 2011;17:7-12.  Back to cited text no. 13
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Guttikonda VR, Patil R, Kumar G. DNA damage in peripheral blood leukocytes in tobacco users. J Oral Maxillofac Pathol 2014;18:S16-20.  Back to cited text no. 14
Jianlin L, Jiliang H, Lifen J, Wei Z, Baohong W, Hongping D. Measuring the genetic damage in cancer patients during radiotherapy with three genetic end-points. Mutagenesis 2004;19:457-64.  Back to cited text no. 15
Venkatachalam P, Paul SF, Mohankumar MN, Prabhu BK, Gajendiran N, Kathiresan A, et al. Higher frequency of dicentrics and micronuclei in peripheral blood lymphocytes of cancer patients. Mutat Res 1999;425:1-8.  Back to cited text no. 16
Milosevic-Djordjevic O, Grujicic D, Vaskovic Z, Marinkovic D. High micronucleus frequency in peripheral blood lymphocytes of untreated cancer patints irrespective of gender, smoking and cancer sites. Tohoku J Exp Med 2010;220:115-20.  Back to cited text no. 17
Calderon-Ezquerro C, Guerrero-Guerra C, Sansores-Martinez R, Calderon-Segura ME, Alobos-Pietrini R, Amador-Munoz O, et al. Genotoxicity in lymphocytes of smokers living in Mexico City. Rev Int Contam Ambient 2010;26:47-63.  Back to cited text no. 18
Naderi NJ, Farhadi S, Sarshar S. Micronucleus assay of buccal mucosa cells in smokers with the history of smoking less and more than 10 years. Indian J Pathol Microbiol 2012;55:433-8.  Back to cited text no. 19
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  [Table 1], [Table 2], [Table 3]


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