Micronucleus Assay Among A group of Smokers in Relation to Oral Health Status

Umelbaneen J Alalawy^1*, Ahlam T Mohammed¹ and Layla Sabri Yass²

^*Correspondence: Umelbaneen J Alalawy, Department of Pedodontics and Preventive Dentistry, College of Dentistry, University of Baghdad, Baghdad, Iraq, Email:

Author info »

Introduction

Humans are exposed to a number of genotoxic compounds found in today's polluted environment in the modern world. As a result, tests are needed to determine the level of exposure and the health risks connected with it. Although there are other tests available, the Micronucleus test (MN) is one of the most popular and widely utilized [1]. Micronucleus assay is non-invasive, new methods for investigating DNA and chromosomal damage, cytokinetic defect, the regenerative ability of the tissue and cell death in the buccal cells that exfoliated by scraping [2]. It's a simple, sensitive assay does not need the use of blood or tissue biopsies, nor does it involve the formation of a cell culture [3]. The etiology of these micronuclei can be attributed to environmental pollutants such as drugs, chemicals, food and free radical injuries, as well as occupational exposures to (organic solvents, antineoplastic agents), lead containing paints solvents and arsenic contaminated drinking water, as well as ionizing radiation used to treat neoplasia (smoking, alcohol consumption, diet, vitamin deficiencies) [4-6].

The buccal micronucleus assay is preferred for a variety of reasons, including the fact that buccal tissue serves as a first barrier and absorbent of all inhaled or ingested agents, buccal tissue has limited ability to repair its DNA, allowing it to reflect age related damage and the fact that epithelium is the source of 90% of cancers [7-10].

Tobacco is the only legal medicine that kills large number of its users when used as directed by the manufacturer. According to the World Health Organization (WHO), tobacco use (including smoking and non-smoking) kills approximately six million people globally each year, with many of these deaths happening prematurely. It has been associated to an increased risk of communicable disease related death [11]. Despite its association with ill health, disability and death from non-communicable chronic diseases. Smoking can cause visual alterations in the oral cavity, such as stained teeth, discolored 'tooth-colored' restorations and dentures [12]. Furthermore, smoking is a significant risk factor for periodontal disease, since it promotes the loss of gingival attachment and increases gingival regression, resulting in increased progression periodontal inflammation [13]. Tobacco generates a complex combination of around 7000 chemical compounds in the form of gases, liquid vapours and particulate debris [14]. The mutagenicity of different compounds in cigarette smoke induced DNA strand breakage, oxidative DNA adducts, gene mutation, sister cremated exchange, chromosomal abnormality and micronuclei, all contribute to the carcinogenic effects of cigarette smoke in a wide range of systems [15].

Oral health is a fundamental but often overlooked part of general health and well-being, and oral diseases can have a significant impact on an individual's health and wellbeing by causing pain, morbidity, mortality and a loss of ability to participate in school, social and economic activities [16]. Dental caries, periodontal disease and oral malignancies are significant clinical disorders that are considered global public health issues [17]. Dental caries is a complicated, chronic, multifactorial disease that is one of the most common diseases in both developed and developing countries [18,19]. Periodontal diseases are a type of inflammatory illnesses affecting the supporting components of the teeth (gums, bone and periodontal ligament). Periodontal diseases are a type of inflammatory illnesses affecting the supporting components of the teeth (gums, bone and periodontal ligament). They're produced by a dysbiosis of the commensal oral microbiota (dental plaque), which interacts with the host's immune system, causing inflammation and disease [20]. As available knowledge from previous literatures there is only one Iraqi study about micronucleus assay and its relation to dental caries, oral cleanliness and gingival condition among smokers, but no Iraqi study about its relation to periodontitis among smokers, therefore, this study will be conducted [21].

Materials and Methods

Seventy males’ volunteers aged (30-35) years (35 were heavy smokers those who smoked at least 20 cigarettes per day for at least the past 5 years and had no period of smoking abstinence longer than 3 months in the past years and 35 were non-smokers) attending to the oral diagnosis department of specialized center for dentistry in Al Kut city. They had no systemic disease and were not take any supplements.

Smoking status of the volunteers was assessed by means of self-reported questionnaire which include demographic information about the volunteer, oral health status, smoking status and the cytopathological results.

The oral examinations include the plaque, calculus, DMFS, bleeding on probing, pocket depth and clinical attachment loss indices using CPI modified probe [22-24].

The cytopathological brush should be used to obtain an oral smear from normal buccal mucosa. The stains were laid on a labeled, clean, dry glass slide and spread out. The patient's name and code were written on each slide. The slides fixed at once by 95% ethanol for 20 minutes and then stained by pap stain. The slides were assessed under 40x magnification using a light microscope; 1000 cells per slide were examined in a zig-zag pattern for the presence of micronuclei and other abnormalties (Micronucleated cells (MN), Binucleated cells (BN), Pyknotic cells (PK), Karyorrhectic cells (KR), Karyolysis cells (KL), Nuclear Buds (NB) and Cells with Condensed Chromatin (CC)) [25].

Data analysis was conducted by application of SPSS program (SPSS version 26) using means, standard deviation, percentage, independent sample t-test, ch-isquare test and pearson’s correlation coefficient test.

Results

The participants in the study sample all had various numbers of positive micronucleus expression (Table 1 and Figure 1). There was a statistically significant difference in micronuclei and other abnormalities between smokers and non-smokers, no cells with condensed chromatin were observed in the research group or in the control participants, therefore no statistical difference was found.

Table 1: Comparison in micronuclei expression between study and control groups.

Figure 1: Epithelial cells with micronuclei stained by pap stain at X 40; (a) Normal epithelial cell; (b) With micronuclei; (c) Binucleated epithelial cells; (d) With pyknotic nuclei; (e) Karyolytic epithelial cells; (f) With karyorrhectic nucleui; (g) with nuclear buds.

The mean of Plaque Index (PI) which was significantly higher in smokers than that in non-smokers, while the mean Calculus Index (CI), Bleeding on Probing (BOP) and DMFS were higher among smokers but statistically there were no differences (Table 2).

Table 2: PI, CI, BOP and DMFS between study and control group.

The percentages of Pocket Depth (PD) and Clinical Attachment Loss (CAL) scores among smokers were significantly higher than that in non-smokers when using Chi-square test (Table 3).

Table 3: PD and CAL between study and control group.

Statistically significant weak positive correlations were detected between PI and MN, between DMFS and KR, between MN and score 1 of CAL and between NB and sore 1 of PD. Also weak significant negative correlations were detected between MN and score 0 of PD, between Mn and score 0 of CAL, between PK and score 0 of PD and between NB and score 0 of PD. No statistical significant correlations detected (P ≥ 0.05) between other oral health status variables with other micronuclei expression (Tables 4 and 5).

Table 4: Correlations between (DMFS, PI, CI and BOP) and micronuclei expression.

Table 5: Correlations between (PD and CAL) and micronuclei expression stained by PAP stain.

Discussion

Tobacco smoking has a well-documented deleterious impact on oral health, which encompasses both common and unusual problems ranging from benign to life threatening diseases [26]. The buccal micronucleus assay is used to detect DNA damage and cell death in exfoliated buccal cells. By evaluating mean frequencies of micronuclei, binucleated cells, nuclear buds, karyolysis, karyorrhexis, pycknosis and condensed chromatin, it provides a powerful tool for detecting genotoxicity.

The current study was originally designated to detect the changes of micronuclei expression in Iraqi male heavy smokers as a biomarker for the oral epithelial cells genomic damage which is the significant cause in oral disease and cancers, the relation of smoking to oral health status variables and the relation of these variables to micronucleus assay since they may participate in micronuclei and other abnormalities formation.

Finding of this study reported that the mean of DMFS was higher in smoker than the non-smokers but statistically there was no significant differences (p>0.05) which agree with al-Deen, but disagree with several studies [27-31]. Many factors that contribute to the development of dental caries should be examined, including age, oral hygiene practices, dietary habits, preventive dental visits, overall health standards and dental care education. As a result, identifying the precise degree of dental caries as a result of smoking is challenging [32].

Significant difference was found in plaque index between smokers and non-smokers group i.e. more plaque accumulation in heavy smokers group than non-smokers group, this agree with Mokeem, et al. and Nanakaly, et al. [33,34]. This may be due to the influence of smoking on both the composition of the biofilm and the host response to this colonization, as reported by a positive correlation between proinflammatory cytokine levels and commensal bacteria in smokers but not in nonsmokers [35].

There is no significant difference in calculus creation between smokers and nonsmokers. Jenkins, et al. studied the link between smoking tobacco and the formation of calculus and found no evidence of a correlation [36]. Oral hygiene practices, availability to professional care, nutrition, age, ethnic origin and interval since last tooth cleaning, systemic disease and prescription drug use all have an impact on the amount of calculus and where it forms in different populations [37].

This study found that control groups had less numbers of sites with bleeding on probing than disease groups but significantly there were no differences. This result disagree Giannopoulou, et al. Jassim and Sura who found that BOP were significantly less in smokers than nonsmokers. Non-significant difference was found in this study might be explained by the high level of education and good oral hygiene in majority of non-smoker group [38-52].

Conclusion

According to results of this study, there was significant difference in Probing Pocket Depth (PPD) and Clinical Attachment Loss (CAL) between non-smokers and smokers groups. This actual increase in PPD and CAL in smokers compared to non-smokers was compatible with several prior studies that indicated smokers had considerably more sites with increased probing depths than non-smokers. Several factors contribute to the deterioration of periodontal health in heavy smokers, including the release of chemical mediators that initiate inflammation, changes in fibroblast proliferation and a suppressed immune response to pathogens that cause periodontitis due to changes in neutrophil function. In the present study, we had significant elevation in Mn and other nuclear anomalies also like BN, NB, PK, KL and KR in the smokers than non-smokers. Cigarette smoking and other forms of tobacco have been found in several studies to increase the frequency of micronuclei in exfoliated buccal epithelial cells. Smoking contains a complex mixture of genotoxic and carcinogenic chemicals that affect oral epithelial cells, such as polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, heavy metals, noxious gases and pesticide residues. Nitrosamines found in tobacco, such as N-nitrosonor nicotine, are classified carcinogens. In a variety of systems, these materials activated in diverse organs, causing DNA strand breakage, oxidative DNA adducts, gene mutation, sister cremated exchange, chromosomal abnormality and micronuclei. But there are also contrasts, Stich and Rosin noticed an increase in MN frequency in alcoholics who smoked 2-4 packs of cigarettes per day, but those who smoked cigarettes alone did not exhibit any significant increase in MN frequency.

There were positive correlation between the (plaque index, calculus index) and micronucleus assay, when the plaque increased the micronucleus expression increased also, that disagree with Damayanti, et al. found no significant correlation between micronucleus expression and oral hygiene index (plaque and calculus). They concluded that the Genotoxic effect of smoking result in increasing in micronuclei expression and in poor oral health. Also there were positive correlation between the increasing in pocket depth, clinical attachment loss and micronucleus expression and there were negative correlation between micronucleus expression and increasing number of healthy periodontal tissue (PD and CAL less than 4 mm). That agree with Zamora, et al. finding that identified an increase in MN numbers in buccal mucosa cells from patients with periodontal disease and they assumed that the DNA damage is a critical event not only in the initiation of periodontal disease but also in the promotion and progression. So the MN test's efficacy as a cytogenetic marker for the development of a variety of oral diseases.

Ethical Approval

All experimental protocols were approved by the college of dentistry, university of Baghdad. All experiments were carried out following the approved guidelines (Ref no. 274 on 25/3/2021).

Financial Support

There was no financial disclosure.

Conflict of Interest

The authors declare that there are no conflicts of interest.

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