Comparative Evaluation of Genotoxicity in Tobacco Users versus Nontobacco Users.

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. Copyright:

INTRODUCTION

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

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.

Armamentarium

Disposable gloves

Mouth mask

Tourniquet

Spirit

Sterile gauze pieces

Syringe and needle

Mouth mirror

Probe

Tweezer

Cotton rolls.

Reagents

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.

Evaluation

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

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

Smoking index	Mean±SD	n	t	P
≤10,000	12.2±0.8	24	3.96**	0.000
>10,000	12.9±0.6	36

**Significant at 0.01 level. SD: Standard deviation

Graph 1

Scatter diagram of smoking index and cytokinesis-block micronucleus frequency

Table 2

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

Variable	Constant	B	SE	P
Smoking index	12.22	0.000021	0.482	0.000

Predicted equation for CBMN frequency using regression analysis, CBMN frequency=smokingindex × 0.00002 + 12.22, CBMN frequency-dependent variable, smoking, index-independent variable. R2=0.233. SE: Standard error, CBMN: Cytokinesis-block micronucleus

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

Group	Mean±SD	n	t	P
Tobacco users	12.6±0.8	60	12.74**	0.000
Nontobacco users	10.7±0.5	30

**Significant. SD: Standard deviation

DISCUSSION

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

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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.