Background: Tobacco use continues to be prevalent in our society. It contains many substances which are genotoxic in nature. The genotoxicity of these substances can be studied in human peripheral blood lymphocytes by estimating the average number of micronuclei (MN) by cytokinesis block micronuclei assay (CBMN assay).
Aims and objectives: To find out whether there is a difference in genotoxicity between tobacco users and non tobacco users.
Materials and methods: Five ml of fresh blood was obtained from 15 persons with no habit of tobacco use and 15 persons using tobacco either smoking, chewing or combination of both with clinically normal mucosa in the age group of 20-40 years reporting to the out patient department of PMS College of Dental Sciences & Research, Thiruvanathapuram. Cytokinesis block micronuclei assay was performed in all the samples and frequency of micronuclei was estimated.
Results: Results showed a significant increase in the number of micronuclei among tobacco users when compared to that of non tobacco users.
Conclusion: Estimation of frequency of micronuclei by CBMN assay can be used to assess the genotoxicity present in blood and helps in identifying tobacco users who are at a high risk for development of cancer.
The wide spread use of tobacco products worldwide continues to be the greatest threat to global health. In developing countries, the tobacco companies are targeting the youth and the prevalence is said to be rising among men and women alike. Besides the systemic problems created by tobacco products, it also causes many oral health problems. It can vary from staining of the dentition, gingival diseases and periodontal diseases to major life threatening conditions like oral cancer.1
Tobacco is used in many ways, mostly smoked (cigarettes) and smokeless tobacco. Cigarette contains about 80 cancer causing agents which include noxious chemicals like benzaanthracene, furan, N-nitrosodimethylamine, 2-Toluidine, formaldehyde, acetaldehyde, phenol, isoprene, benzene etc.2 Smokeless tobacco comes in two ways, chewing tobacco and snuff. Moist snuff is taken orally while dry snuff is powdered tobacco that is mostly inhaled through the nose. One of the sites of highest risk for cancer due to smoking is the lung, followed by oral cavity and larynx. The risk increases with increase in number of cigarettes smoked.3
In the Human Micronucleus (HUMN) project conducted by Stefano Bonassi et al in 2003 to analyse the effect of smoking habit on the frequency of micronuclei in human lymphocytes using cytokinesis block micronuclei(CBMN) assay, a significant increase in micronuclei frequency was observed only in heavy smokers who were not exposed to other genotoxic agents.6
Micronuclei ('small nucleus') are cytoplasmic chromatin masses that originate from chromosome fragments or whole chromosomes that fail to engage with the mitotic spindle, lag behind and not carried to the opposite poles during the anaphase of cell division. These chromosome fragments develop nuclear membranes and form a third nucleus in the cytoplasm and are referred to as micronuclei.7This would further help in implementation of tobacco cessation programmes among high –risk individuals for cancer.
Quantification of frequency of micronuclei in the lymphocytes of peripheral blood using cytokinesis –block micronucleus assay is one of the standard cytogenetic tests for measuring chromosomal damage because of its good reproducibility and reliability.7,8 The assessment of increased frequency of micronuclei helps in identification of high risk group of individuals susceptible to cancer.8,9
The Human Micronucleus (HUMN) project was conducted by Stefano Bonassi et al in 2003 to analyse the effect of smoking habit on the frequency of micronuclei in human lymphocytes using cytokinesis block micronuclei(CBMN) assay. The study was done by re- analysing the pooled data of HUMN database (24 databases from 16 countries). In their analysis, only weak associations were obtained between cigarette smoking and micronuclei frequency. But in most of these studies, tobacco use overlapped with old age, alcohol consumption, chronic infections, confirmed systemic illness, history of malignancy and exposure to cytotoxic drugs, chemicals or ionising radiation. A significant increase in micronuclei frequency was observed only in heavy smokers who were not exposed to other genotoxic agents.6
In our study we have ruled out all the possible variables that can affect the frequency of micronuclei and restricted our study to tobacco and non tobacco users aged between 20 and 40 years with no visible oral changes. CBMN assay was conducted with the objective of assessing the difference in micronuclei frequencies in the peripheral blood of the study subjects with that of the control subjects. By this, genetic damage caused by tobacco alone can be identified at a very early stage, even before the appearance of clinical signs of cancer. This would further help in implementation of tobacco cessation programmes among high –risk individuals for cancer.
Materials and methods
After obtaining institutional ethical committee clearance, the study was conducted in patients reporting to the outpatient department, PMS College of Dental Sciences & Research, Thiruvananthapuram. The laboratory procedures was carried out in ‘Genitika’ (an ISO ceritified laboratory), Thiruvananthapuram. Control group consisted of 15 persons with no habit of tobacco use (smoking as well as tobacco chewing) and having clinically normal oral mucosa and study group consisted of 15 persons using tobacco either smoking, chewing or combination of both. (Current smokers – CDC classification).
The criteria for selecting the tobacco smokers, was based on the definitions given by US center for disease control and prevention ( CDC ).
Patients above 20 years and below 40 years, patients with clinically detectable changes in the oral mucosa, patients with alcoholism, confirmed systemic illness or with history of malignancy, patients who reported of infections within 3 months of study and who were exposed to cytotoxic drugs, chemicals, or radiation therapy were excluded from the study.
The number of micro nuclei was considered as the dependent variable while age, sex and disease status were the independent variables.
For the CBMN assay, 5 ml of fresh blood was taken after obtaining written informed consent from each subject by venipuncture and transferred to heparinised vacutainers and lymphocytes isolated using lymphoprep.