Comparative evaluation of micronuclei in Saudi smokers and non-smokers without any visible oral lesions – A pilot study

505 ISSN Online 0719-2479 www.joralres.com © 2019 [E pu b a he ad of pr int ] Abstract: Objectives: A systematic review was conducted to evaluate effectiveness and safety of beta carotenes for the treatment of oral leukoplakia regarding clinical resolution and prevention of malignant transformation. Material and Methods: The systematic search was conducted in three electronic databases and the study’s selection was performed according to pre-set eligibility criteria. Four studies evaluating the efficacy of beta carotenes in oral leukoplakia compared to placebo were included in the review; three of which were assigned for quantitative analysis. Data were extracted, tabulated, quality assessed and statistically analyzed. Results: The metaanalysis revealed that when comparing clinical resolution the beta carotene group favored was favored compared to placebo, with statistically significant difference. However, a meta-analysis comparing beta carotene and placebo groups regarding malignant transformation as a primary outcome failed to show any significant benefit. Furthermore, results showed evidence of beta carotene safety. Conclusion: the overall quality of evidence about efficacy of beta carotene in oral leukoplakia treatment was not high. However, given the obvious safety of this agent, data suggests it could have a promising effect in clinical improvement of oral leukoplakia lesions. However, no evidence supporting its benefits in reducing risk of malignant transformation in these lesions was found. Therefore, further long term, well designed randomized clinical trials are highly recommended.


Comparative evaluation of micronuclei in Saudi smokers and non-smokers without
any visible oral lesions-A pilot study.

INTRODUCTION.
Oral cancer is a serious health problem the world over. In high risk countries such as Sri Lanka, Pakistan, India and Bangladesh, oral cancer is the most common cancer in men and may represent up to 25% of all new cases of cancer. 1 It is a multifactorial disease that occurs due to activation of oncogenes as a result of mutations in the DNA. Approximately 75% of oral cancers are related to the use of tobacco and alcohol.
In general, many oral cancers pass through clinically evident premalignant mucosal changes which indicate the presence of a risk and present an opportunity for early detection and to intercept the development of a cancerous lesion.
It is very important to prevent cancer at an early stage, as despite the best treatment, the associated morbidity is very high and is dependent on the disease stage at the time of admission. Approximately 50% of the patients are in at an advanced stage at the time of diagnosis. Oral cancer-related mass screening programs do not meet the guidelines for a successful outcome. However, there may be some benefit when focusing on high-risk groups, such as heavy smokers and alcoholabusers. There still remains a big challenge to reduce the morbidity associated with the treatment of oral cancer without compromising the survival rate.
Studies have shown that there is a marked reduction (50%) in the risk of getting oral cancer if habits such as the use of tobacco are stopped. Hence, it appears that riskfactor reduction may be a very effective tool in decreasing the morbidity and mortality associated with oral cancer. Chronic use of carcinogenic agents brings about irreversible cytogenetic changes such as chromosomal aberrations, exchange of sister chromatids, and micronuclei which can be frequently used as indicators. Micronuclei (MNi) are defined as microscopically visible, round or oval cytoplasmic chromatin mass next to the nucleus.
They originate from aberrant mitosis and consist of eccentric chromosomes, chromatid fragments or whole chromosomes that have failed to be incorporated into the daughter nuclei during mitosis. They have proved to be an important biomarker of genomic damage. 1, 2 The MNi test is one of the current rapid, efficient, economical and non-invasive tests, used as an indicator of cytogenetic damage as it provides a quantitative measure of the genotoxic action of carcinogens and mutagens. 1, 2 Extensive research has been carried out and has confirmed that micronuclei frequency is significantly increased in potentially malignant disorders as compared to healthy individuals.
However, not many studies have been carried out to know if there is an increase in the frequency of MNi in smokers without any visible oral lesions. Hence, in this study we intend to analyze and compare the frequency of micronuclei in exfoliated oral epithelial cells of smokers without any visible oral lesion and compare it to that of non-smokers.

Source of data
The study was done on male patients attending the dental OPD, College of Dentistry, Jouf University, Sakaka from September 2016 to March 2017.
Inclusion criteria included Non-smokers with apparently healthy mucosa, for the control group; Smokers with apparently healthy mucosa for the study group.
Exclusion criteria included Patients with a history of treatment for any potentially malignant disorder such as leukoplakia, oral verrucous carcinoma, keratoacanthoma, dyskeratosis congenita and oral submucous fibrosis; Smokers and former smokers with oral lesions.

Sampling technique
The samples for the present study were selected by using the systematic random sampling technique until desired sample size was achieved. Fifteen patients with a smoking habit for over 10 years without having any visible oral lesion would constitute Group I, and 15 age-matched normal healthy non-smokers would constitute Group II (control).
This study followed the guidelines of Strengthening the Reporting of Observational studies in Epidemiology (STROBE), and the STROBE checklist was utilized in the preparation of this manuscript. 3 Study design After obtaining informed consent from all the patients, they were asked to rinse their mouth with water to remove food debris, and buccal scrapings were obtained using a sterile wooden stick. The cells were then directly spread on a clean microscopic slide to prepare a smear. Then samples were allowed to dry and were fixed with alcohol. The smears were stained with haemotaoxylin and eosin and examined under low magnification (x100) for screening and under higher magnification (x400) for counting the MNi (Figure 1).
A total of 500 cells were assessed for the presence of micronuclei and compared between the groups. The results were then subjected to statistical analysis.

Statistical Analysis
The results between the Group I and Group II were compared with a Student's t-test to evaluate the statistical significance between the two groups.

RESULTS.
The number of MNi in smokers ranged between 89 to 159 (MNi cells) per 500 assessed cells. An average of 100 cells was found to be the highest frequency. (Figure  2) In case of non-smokers the frequency was found to be 30 (MNi cells) per 500 counted cells. (Figure 3) A statistically significant difference was found between group I and group II with respect to the number of micronuclei present. (Table 1)

DISCUSSION.
Micronuclei (MNi) denote small, additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis. MNi rates, therefore, indirectly reflect chromosome breakage or impairment of the mitotic apparatus. Throughout the last few years, MNi in oral exfoliated epithelial cells have been widely used as biomarkers of chromosomal damage, genome instability and cancer risk in humans.
The mean MNi frequency noted in the present study was significantly higher (p<0.05) in smokers than in non-smokers. These results are in accordance with the findings of Grover et al., 1 and Anila et al. 4 The results of our study are in contrast to the findings in a similar study carried out by Nerseseyan et al., 5 in which a statistically significant difference in MNi counts between smokers and nonsmokers was detected, only with DNA nonspecific stains but not with DNAspecific stains.
Casartelli et al., 6 determined that the gradual rise in MNi frequency from normal mucosa to precan-cerous lesion to carcinoma represents a link of this biomarker with neoplastic progression. Oliveira et al.,7 demonstrated that the action of genotoxic agents (tobacco and alcohol) causes alterations in the frequency of micronuclei and metanucleated anomalies. According to Samanta et al., 8 the various possible mechanisms for MNi formation in preneoplastic conditions include chromosomal aberrations, chromosome loss/breakage, mitotic apparatus dysfunctions, aneuploidy, and genetic instability. First, MNi formation is generally considered as a manifestation of genetic damage or chromosomal breakage.
However, many factors such as radiation, drugs, pollutants, even the normal aging process may be responsible for MNi formation. 9,10 Selection of buccal mucosa site for smear collection is justified as this site is more vulnerable to external insult from possible carcinogens. Limitations of the present study include small sample size and that the presented results are confined to a male population only. The study helped us in identifying changes at a cellular level, before lesions become apparent, creating awareness, and motivating and educating the patients about the ill effects of smoking, and to some extent to advocate for the cessation of the habits associated with these cellular changes, to prevent oral cancer before its onset.

CONCLUSION.
A statistically significant difference was found between smokers without visible oral lesions and non-smokers regarding the number of micronuclei present. There is evident damage to the cell nucleus in smokers although there is no clinical evidence of a white lesion.