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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 7  |  Issue : 3  |  Page : 151-155

Frequency of micronuclei in tobacco habitués and Non-Habitués with oral lichen planus


Department of Oral and Maxillofacial Pathology, Goa Dental College and Hospital, Bambolim, Goa, India

Date of Submission08-Nov-2019
Date of Decision17-Jun-2020
Date of Acceptance21-Aug-2020
Date of Web Publication25-Jan-2021

Correspondence Address:
Bhanu Priya
Department of Oral and Maxillofacial Pathology, Maulana Azad Institute of Dental Sciences, MAMC Complex, Bahadur Shah Zafar Marg, Delhi - 110 002
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjhr.cjhr_109_1910.4103/cjhr.cjhr_109_19

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  Abstract 


Background: Tobacco in any form is genotoxic to the oral epithelium and manifests as tobacco-associated mucosal lesions, some of which are OPMDs with a propensity for progression into OSCC (2-17%). The aberrations in the count and morphology of micronuclei in exfoliative cytological preparations have been demonstrated in all forms of oral tobacco usage and OLP independently. The more extensive these aberrations, the greater the possibility of undergoing malignant change. This study focused on assessing the extent of micronuclear damage in tobacco-habitués with OLP, thus placing them in a higher risk group as compared to non- habitués with OLP. Aim and Objectives: To evaluate the frequency of micronucleated exfoliative cells in oral lichen planus and to evaluate the effect of tobacco on the frequency of micronuclei in these patients. Material and Methods: Patients were divided into 3 groups: OLP with habit, OLP without habit and healthy controls. Exfoliated cells were obtained from the lesion, smeared on slides, stained by Papanicolaou's method and 1000 cells per slide evaluated for micronuclei according to the Tolbert et al criteria. Statistical Analysis: A statistically significant result was obtained using ANOVA and Bonferroni tests. Result: The genetic damage and increased potential for malignant transformation in OLP is compounded in tobacco habitués evidenced by the increase in the number of micronuclei. Conclusion: The Micronucleus Index was observed to be three-fold greater in tobacco habitués with OLP as compared to non-habitués with OLP.

Keywords: Genotoxicity, Micronuclei, Tobacco habitués, OLP


How to cite this article:
Priya B, Spadigam A, Dhupar A, Syed S. Frequency of micronuclei in tobacco habitués and Non-Habitués with oral lichen planus. CHRISMED J Health Res 2020;7:151-5

How to cite this URL:
Priya B, Spadigam A, Dhupar A, Syed S. Frequency of micronuclei in tobacco habitués and Non-Habitués with oral lichen planus. CHRISMED J Health Res [serial online] 2020 [cited 2021 Apr 11];7:151-5. Available from: https://www.cjhr.org/text.asp?2020/7/3/151/307813




  Introduction Top


Oral carcinomas are characterized by complex karyotypes that involve many chromosomal deletions, translocations, and structural abnormalities. Cells often have errors in chromosome segregation that leads to the formation of micronuclei.[1],[2],[3]

Micronuclei (MNi), also known as Howell–Jolly bodies, were first identified by William Howell and Justin Jolly (1970) to describe the nuclear remnants of red blood cells circulating in organs with pathological features. Micronuclei (MNi) are chromatid or whole chromosomes lagging behind in anaphase to reach spindle poles during mitosis, due to mis-repair of DNA breaks or unrepaired DNA breaks.[4] The lagging of chromatin or whole chromosome occurs due to:[5]

  1. Mitotic spindle failure: the absence or inappropriate attachment of spindle microtubules to chromosome kinetochores, such as synthetic (both sister chromatids are attached to the same spindle pole), monotelic (only one kinetochore is attached leaving the second sister chromatid unattached), or merotelic (one kinetochore is attached to both spindle poles), results in lack of tension, thus making the bond sensitive to dissociation, and leads to inappropriate segregation of chromosomes.
  2. Centromeric and paracentromeric region DNA hypomethylation: It is the main mechanism of MN formation originated from chromosome missegregation. These hypomethylated regions lead to decreased tension in kinetochores, thus creating a wrong connection between microtubules of the mitotic spindle and chromosomes. This wrong connection results into the formation of micronuclei due to unequal segregation of nuclear material.
  3. Tubulin depolymerization: Chromatids/chromosomes are unable to segregate due to spindle tubulin depolymerization.
  4. Kinetochore damage: Absence of kinetochore or centromeric defects also lags chromosomes behind at telophase.
  5. Dysfunctional homologous recombination (HR) or defects in enzymes of the nonhomologous end joining (NHEJ) pathway: Dicentrics are results of either HR between complementary DNA sequences of different chromosomes or NHEJ between two chromosomes that suffer from DNA double-strand breaks (DSBs). If the level of DSBs exceeds the repair capacity of dividing cells due to mis-repaired DNA, cell presents with increased MNi.
  6. Defects in the cell cycle control system: The breakage-fusion-bridge (BFB) cycles are the feature of chromosomal instability presented during anaphase as an initial event. In BFB, uneven breakage of nucleoplasmic bridges leads to the formation of two daughter nuclei. Such broken chromosomes usually do not contain telomeric zones and therefore can fuse with their replica during the next mitotic event, repeating the cycle for the next couple of rounds.


An increased MNi count is used as a genomic biomarker for the assessment of DNA damage, which is associated with the amplification of genes near the break point via a BFB cycle.[5] Tolbert et al.'s criteria identifies micronuclei based on their staining intensity, shape, and size.

Oral exfoliated cells present with increased numbers of micronuclei due to a variety of substances, including genotoxic agents and carcinogenic compounds in tobacco, betel nut, and alcohol.[1],[6],[7],[8] The induction of micronucleated (MN) cells by carcinogens and mutagens is evidence of the genotoxic effect of such substances. In case of oral squamous cell carcinoma (OSCC), genotoxic agents such as tobacco lead to an increase in chromosomal aberrations and increased frequency of micronuclei. Various studies show that non-tobacco-associated potentially malignant disorders such as oral lichen planus (OLP) also present with increased numbers of micronuclei.[9],[10]

OLP, a common chronic inflammatory mucocutaneous disorder (global: 1%–2%, Indians: 2.6%) affecting females,[11] predominantly between the third and sixth decades of life also shows an increased MNi frequency.[12],[13] Patients with OLP show significantly higher frequencies of micronuclei, nuclear buds, and binucleated cells, indicative of genotoxic damage. The malignant potential of OLP is 0.4%–5.3%, due to increased chromosomal instability and DNA damage.[14] OLP has an intense inflammatory response within the lamina propria characterized by the overexpression of inducible nitric oxide synthase (iNOS) and NO and reactive nitrogen species, which in turn leads to unrepaired DNA damage in the epithelium followed by carcinogenesis.

Aim

The present study aimed at assessing the evidence of DNA damage in the exfoliated cells in patients with OLP.

Objectives

  1. To evaluate the frequency of micronuclei in the exfoliated cells from OLP patients, with and without the habit.
  2. To assess the cumulative effect of OLP and tobacco on frequency of micronuclei.



  Materials and Methods Top


A prospective study comprised samples collected from 40 subjects in 1 year. The subjects were divided into two categories as under:

  • Category I: OLP with habit
  • Category II: OLP without habit


Category III consisted of 10 healthy controls (This category comprised persons not habituated to any form of tobacco and with no clinical evidence of any oral mucosal lesions).

Relevant history inclusive of oral habits was recorded, and written, informed consent was obtained from the patients for the subsequent procedures. Patients with biopsy-proven diagnosis of OLP were included in the study, and patients with any other oral potentially malignant disorders (OPMDs) were excluded.

The patient's data were arranged according to the age group and gender [Table 1].
Table 1: Distribution of study subjects according to age group and gender

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Subjects were asked to rinse their mouth gently with water. Oral mucosal cells were scraped from lesional/normal tissue located on the buccal mucosa of patient/control groups, respectively, using a moistened wooden spatula. The cells were immediately smeared onto precleaned microscopic slides. Just before drying, the smears were fixed with 95% ethyl alcohol for 30 min.[1],[2] All the cytological smears were stained by the Papanicolaou's technique. The slides were mounted with cover glass using DPX mountant. From each slide, ~1000 cells were examined under a light microscope using low magnification (×100) for screening and high magnification (×400) for counting of micronuclei. The counting record sheets were used to record MNi in 1000 cells. The most commonly used zigzag method was followed for screening of slides. The following criteria were considered for selection of cell and the micronuclei count:[15]

A. Parameters for inclusion of cells to be scored:

  1. Intact cytoplasm and relatively flat cell position on the slide
  2. Little or no overlap with adjacent cells
  3. Little or no debris
  4. Nucleus normal and intact, nuclear perimeter smooth and distinct.


B. Parameters for identifying micronucleus by Tolbert et al.:

  1. Rounded smooth perimeter suggestive of a membrane
  2. Less than a third the diameter of the associated nucleus, but large enough to discern shape and color
  3. Staining intensity similar to that of the nucleus
  4. Texture similar to that of nucleus
  5. Same focal plane as nucleus
  6. Absence of overlap with the nucleus.


Only those structures fulfilling the above-mentioned criteria were recorded as MNi. The total number of MNi observed in 1000 intact epithelial cells was calculated as the MNi frequency.

Statistics

The data obtained were statistically analyzed. ANOVA and Bonferroni tests were used for multiple group comparisons. A P ≤ 0.05 was considered as statistically significant.


  Results Top


The micronucleus index (MI) = micronuclei count/number of cell screened from each slide, observed in the three groups, was 0.23 ± 0.02, 0.08 ± 0.02, and 0.02 ± 0.004, respectively. MI recorded in the decreasing order for Category I, Category II, and Category III. The mean number of micronuclei in Category I, Category II, and Category III was 230 ± 21.12, 87.91 ± 23.74, and 29.40 ± 4.094, respectively [Table 2]. The mean number of micronuclei and MI was increased in Category I, i.e., OLP with habit, followed by Category II (OLP without habit) and minimum in Category III (healthy control). The intercategory comparison of micronuclei and MI was done using the Bonferroni test. After calculation, the data showed statistically significant results.
Table 2: Micronucleus index and mean micronuclei for all three categories using ANOVA test

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A statistically significant result was obtained using ANOVA and Bonferroni tests. Statistically significant ANOVA test (P = 0.001).


  Discussion Top


The incidence of head and neck carcinoma has increased in recent years, with approximately 14/100,000 cases (16%–40% of all malignancies).[16],[17],[18] The increasing rate of incidence globally is probably due to the use of tobacco on a large scale. Around 500,000 new oral and pharyngeal carcinoma cases are diagnosed annually, and three quarters of these are seen in the developing world, including about 65,000 cases reported from India.[19] Deaths from tobacco-related diseases will rise in India, from 1.4% of all deaths in 1990 to 13.3% of all deaths in 2020.[20]

Oral carcinogenesis is known as a multistep process of accumulated genetic damage, leading to cell dysregulation with disruption in cell signaling, DNA-repair, and cell cycle events, which are fundamental to homeostasis.[21]

Tobacco consumption is a known genotoxic factor responsible for increased incidence rate of OSCC. Non-habit associated OPMDs such as actinic cheilitis, OLP, and dyskeratosis congenita are also associated with increased genetic damage and malignant transformation.

Oral exfoliative cytology has been used extensively for screening cellular alterations, in particular, the assessment of micronuclei in oral exfoliated cells is considered as one of the markers of chromosomal damage caused by genotoxic agents. Exfoliated cells also demonstrated certain nuclear anomalies which may mimic micronuclei: broken eggs or cell with nuclear buds, microorganisms, karyorrhexis, and binucleated cells [Figure 1]. Hence, close examination as well as conformity with Tolbert et al.'s criteria is required, for accurate identification as micronuclei. The incidence of micronuclei has been analyzed by various studies in normal patients, OPMDs, and OSCCs. Increased MNi frequency showed a correlation with DNA double-strand breaks (DSBs) and DNA recombination events, via a NHEJ pathway.[4] An increased micronuclei count is indicative of unrepaired DNA damage, due to various factors, such as occupational and environmental exposures, radiotherapy, chemoprevention, lifestyle habits, and cancer.[22],[23]
Figure 1: Structures resembling micronuclei in the exfoliated cells (PAP). (a) Microorganisms (×400). (b) Cell with nuclear buds or nuclear broken eggs appearance (×400) (c) Karyorrhexis (×400). (d) Binucleated cell (×400)

Click here to view


The acquisition of the tobacco habit at an early age potentiates the genotoxicity induced by tobacco which is manifested by an increase in the frequency of MNi.[28]

In the progression from tobacco-associated mucosal lesions to OPMDs to OSCC, this MNi count increases[9] substantially to find clinical application as a quantitative indicator of nuclear damage and chromosomal instability.[21] Oulu et al., 1997; Patel et al., 2009; and Alaska et al. 2011, observed an increased frequency of micronuclei in tobacco habitués. In 2007, Buajeeb et al. studied the combined effect of tobacco on OPMDs.[27] They reported that the mean MI count in tobacco users without any lesion was 2.230, in potentially malignant cases was 4.85, and in malignant cases was 8.3. Hence, the mean MI was less in potentially malignant cases as compared to the malignant cases.

In our study, MI for OLP with tobacco habit, OLP without habit, and healthy control was recorded as 0.23 ± 0.02, 0.08 ± 0.02, and 0.02 ± 0.004, respectively, which showed a high MI in the tobacco habit-associated group. Thus, there is no standard range for MI due to multifactorial effects associated with its formation, rather an increased MI in a given that population is suggestive of increased DNA damage.

On the other hand, the onset of OLP occurs in the 3–6 decades with a lesser level of genotoxicity as expressed in the relatively mild increase in MNi count as compared to that in the tobacco-induced aberrations.

The present study identifies a subset of tobacco habitués who acquire OLP or present with both (OLP with tobacco habit). Individuals without the tobacco habit (Category II) showed lower mean MNi as compared to habitués (Category I) but higher mean MNi as compared to healthy controls.

Sangle et al. observed a gradual increase in micronuclei count[1] from normal mucosal to OLP to carcinoma, which suggested a link of micronuclei with neoplastic progression.[25],[26] Similar results were reported by Casartelli et al.

In this study, the frequency of MNi in OLP with the tobacco habit was shown to be three times greater than non-habitués and healthy controls, signifying a cumulative effect of genotoxic alterations in tobacco habitués with OLP.

In case of OLP-mediated carcinogenesis, prolonged chronic inflammation leads to switching of HR repair pathway into NHEJ and activation of ras and myc oncogenes by altering DNA repair mechanisms, whereas in tobacco-associated OLP, the secondary inflammation-induced genotoxic response enhances the primary carcinogenic[25] influence of prolonged exposure of the tissues to tobacco.

Decreased MNi in habitués who have since quit the tobacco habit was reported in a comparative micro-assay study on 30 patients by Sherashiya et al.,[24] whereas a marked reduction in MNi was noticed 3 months after treatment of atrophic and/or erosive OLP.

The present study evaluated the mean micronuclei and MI in OLP with habit (Category I), OLP without habit (Category II), and healthy control (Category III). The results showed that the overall level of MI and mean micronuclei were higher in Category I as compared to Category II and Category III. This observation was similar to that reported by Patel et al. and Palaskar and Jindal. The increasing micronuclei counts from normal healthy controls to OLP patients to OLP with habit suggested a link of this genomic biomarker with neoplastic progression. A cumulative effect of tobacco usage on the malignant potential in OLP patients was thus concluded from this study.


  Conclusion Top


Our study compared the MNi count in tobacco habitués and non- habitués with OLP with healthy controls. The Micronucleus Index was observed to be three-fold greater in tobacco habitués with OLP as compared to non-habitués with OLP, which shows a cumulative effect of tobacco and OLP on genotoxicity. Given that the tobacco habit, is further related to increased genomic instability and activation of various oncogenes following their transposition or amplification, the risk or malignant transformation of OLP in habitués is significantly greater. In addition to being a non invasive tool for the early detection of and a prognostic indicator for assessing malignant transformation of OLP, the Mean Micronuclei count can be factored into the diagnostic workup and for monitoring of chemotherapeutic treatment protocols for OLP in habitués along with tobacco cessation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Sangle VA, Bijjaragi S, Shah N, Kangane S, Ghule HM, Rani SA. Comparative study of frequency of micronuclei in normal, potentially malignant diseases and oral squamous cell carcinoma. J Nat Sci Biol Med 2016;7:33-8.  Back to cited text no. 1
    
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