Smoking Genotoxicity In Peripheral Blood Lymphocytes Using The Comet Assay

Introduction

The World Health Organization (WHO) ranks tobacco smoking among the 10 greatest risks to health. Cigarette smoke contains more than 4000 chemical agents several types of toxic components including carbon monoxide, nicotine, aromatic hydrocarbons and specially free radicals and reactive oxygen (O^2-). Many of them are known to be mutagens and carcinogens. ^[1] Cigarette smoke induces cancer, emphysema, arteriosclerosis and other diseases after a delay of years ^[2]. The exact mechanism by which smoking contributes to the pathogenesis of various diseases has not yet been identified in detail. One of the methods suggested is oxidative stress.

Reactive oxygen (ROS) and nitrogen species (RNS) are continuously generated in the biological system and play an important role in a wide variety of physiological and pathological processes, including cancer and emphysema. ^[3] Cigarette smoke induces various forms of DNA damage, including singlestrand breaks (SSBs) ^[4,5] and possibly double-strand breaks (DSBs) ^[6,7]. SSBs represent the initial DNA damage and are often used as a biomarker of exposure ^[8-10] . DSBs are considered to be more biologically relevant since they can lead to chromosome translocation and cancer if repaired improperly in addition to genetic instability and cell death if left unrepaired ^[11] .

The comet assay (single-cell gel electrophoresis, SCG or SCGE) is a well-established genotoxicity test for in vitro and in vivo testing of chemicals. ^[12,13] Because of its ease of application, the comet assay is also being increasingly used in human biomonitoring. ^[14-16] The parameter often used to evaluate DNA damage is the “tail moment”, which is defined as the product of the tail length and the fraction of the total DNA content that is present in the tail.⁽¹⁷⁾ Other parameters used are "olive moment" and % DNA in the tail. Olive moment is calculated as: (tail mean - head mean) x tail % DNA/100. % DNA in tail is 100- %DNA in head.

This study was taken up to evaluate the effects of smoking on DNA assessed in the peripheral blood lymphocytes and to quantitatively assess the degree of DNA damage in the body cells.

Materials & Methods:

Results

A total of 50 patients were considered for the statistical analysis. They were divided into 2 groups: group 1 consisted of smokers (n=30) and group 2 consisted of non-smokers (n=20).

Table 1 shows the base line characteristics of the 2 groups. There was no statistically significant variation in the 2 groups. Table 2 compares the % DNA in tail, tail moment and olive moment of the 2 groups. Using the MANN-WHITNEY test, we found no statistically significant difference in the 2 groups. The difference in the DNA damage in the 2 groups was present, but this difference however was statistically insignificant (p>0.05).

Discussion

Cigarette smoke is a major source of free radicals and tobacco smoke contains oxidants and pro-oxidant agents. It is also known to contain many mutagens/ carcinogens, with polycyclic aromatic hydrocarbons (PAHs), aromatic amines, N-nitrosamines and aldehydes representing the major classes of harmful substances. Comet assay can sensitively detect DNA single strand break and alkali-labile site. It was used in this study to examine lymphocyte DNA damage of smokers.

The present study compared the DNA damage in peripheral blood lymphocytes in 30 smokers and 20 non-smokers by using the COMET assay. We compared the % DNA in tail, tail moment and olive moment of the 2 groups.

As per our studies, we did not find any statistical difference in these values among the 2 groups (p<0.05). Some studies done before by Manuel et al, Sram et al in 1998, Tates et al in 1996 have showed significant difference in the genotoxicity in the blood lymphocytes by using the COMET assay. ^[18'^20] Other studies have shown difference by using othertechniques such as MN assay.^[21] Our results are not in accordance with these studies. Perhaps our sample size was too small. When the duration of smoking was correlated with the same 3 parameters, there was no statistical significance. However, there are other studies which do support our result.. Polasa et al. compared the DNA damage in 10 smokers and 10 non-smokers by using the comet assay and also found no significant outcome (p< 0.05)^[22]

The comet assay has repeatedly been used to measure DNA damage related to tobacco smoking in various studies. However, conflicting results have been reported. It is possible that physiological, environmental and many other factors contribute to the overall variation of the comet assay. There have been no satisfactory descriptions of the effect of single factors or of the manner in which single factors interact with each other. Some possible factors that are said to cause variation in the level of DNA damage in healthy untreated individuals are: age, gender, air pollution, diet, exercise, sunlight, and smoking. These factors could lead to a change in DNA status in healthy individuals and the subjects, for which our study did not account for. Further studies with strict criteria for selection of subjects and controls are required to find the exact correlation between smoking and genotoxicity.

Conclusion

Our study concludes that smoking genotoxicity as measured by comet assay in the peripheral blood lymphocytes does not vary significantly among smokers and non-smokers. Even though various studies have showed significant damage to DNA in smokers by using the comet assay, there have also been studies which were inconclusive. Hence, better modalities which are standardized are required to detect the genomic defects. More studies must be undertaken to validate the efficacy of COMET assay. Studies with a larger sample size must be undertaken to come to a better conclusion and understanding. Our study can be used as a pilot study to further explore the relation between smoking and DNA damage.