An assessment of Genotoxic Damage in Exfoliated Buccal Cells using Saliva as a tool

Sonia Jaiswal; PK Sharma

doi:10.4103/JASI.JASI_99_19

ORIGINAL ARTICLE

Year : 2019 | Volume : 68 | Issue : 3 | Page : 181-186

An assessment of Genotoxic Damage in Exfoliated Buccal Cells using Saliva as a tool

Sonia Jaiswal, PK Sharma
Department of Anatomy, Era's Lucknow Medical College, Lucknow, Uttar Pradesh, India

Date of Submission	25-Jul-2019
Date of Acceptance	04-Sep-2019
Date of Web Publication	07-Jan-2020

Correspondence Address:
Dr. Sonia Jaiswal
Department of Anatomy, Era's Lucknow Medical College, Lucknow, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JASI.JASI_99_19

Abstract

Introduction: Exfoliated buccal cells serve as an ideal site for genotoxic events since they are the first cells to come in contact with anything ingested or inhaled. Micronuclei (MNs) are also known as Howell–Jolly bodies and are a result of some aneugenic and clastogenic events. The aim of the study was to assess the severity of genotoxic damage in the exfoliated cells of participants consuming smokeless tobacco and its products and those diagnosed with oral submucous fibrosis (OSMF). Material and Methods: Individuals addicted to alcohol, pleasure drugs, or smoked forms of tobacco were excluded from the study. Saliva was collected and centrifuged; smear was prepared using the pellet, while the supernatant was used to extract DNA. The cells collected were stained with Feulgen fast green and acridine orange. Results: A progressive increase in the number of MNs was observed from the control group to those who consumed smokeless tobacco and their products, while the frequency of MNs was found to be the highest in participants with Oral Submucous Fibrosis. Discussion and Conclusion: Micronucleus assay using saliva as a tool can be useful in detecting early genotoxic damage.

Keywords: Acridine orange, DNA, Feulgen fast, micronuclei, submucous fibrosis

How to cite this article:
Jaiswal S, Sharma P K. An assessment of Genotoxic Damage in Exfoliated Buccal Cells using Saliva as a tool. J Anat Soc India 2019;68:181-6

How to cite this URL:
Jaiswal S, Sharma P K. An assessment of Genotoxic Damage in Exfoliated Buccal Cells using Saliva as a tool. J Anat Soc India [serial online] 2019 [cited 2023 Mar 27];68:181-6. Available from: https://www.jasi.org.in/text.asp?2019/68/3/181/275276

Introduction

Saliva is rapidly emerging as a multipurpose diagnostic tool since its collection is noninvasive and easy to access. It plays an important role in the early detection and prevention of many oral lesions as it is in direct contact with the oral lesion.

DNA damage can be assessed by extracting and purifying it from biofluids such as saliva. DNA was discovered in the human circulatory system in 1948;^[1] since then, various researchers have found DNA in other body fluids as well. Lo et al discovered free DNA in body fluids in the year 1997.^[2] Later on, DNA was also detected in urine^{[3],[4],[5],[6]} and saliva.^[7]

Early detection of oral lesions was possible using cytology, a method introduced by Papanicolaou in the year 1943. It is a simple method and noninvasive in nature and thus can be carried out in suspected lesions.^[8] Cytological techniques have improved over the years which have led to the development of liquid-based preparations, thus using it as an auxiliary tool for the identification of oral lesions.^[9] Genotoxins from various medical procedures, deficiencies, factors related to lifestyle, and genetic disorders produce genetic imbalance in cells.^[10]

It is essential to have biomarkers which are reliable and are minimally invasive so as to improve their diagnostic potential in lesions associated with genetic damage. An assessment of micronuclei (MNs) is a good method to detect chromosome loss or malfunction.^[11] Various studies have shown a correlation between frequency of MN and severity of genotoxic damage.

The aim of our study was to assess genotoxic damage in exfoliated buccal cells of tobacco users using liquid-based cytology where saliva was used as a tool.

Material and Methods

The study was given clearance by the ethical committee, and informed consent was taken from all the participants.

Five hundred participants were screened with the help of a structured questionnaire. The questionnaire was divided into two sections: one related to the oral hygiene status of the participants and their pattern of the use of tobacco and its products and the other section was dedicated to the clinical examination of oral submucous fibrosis (OSMF). Participants consuming alcohol, smoked form of tobacco, and pleasure drugs were excluded from the study. All those participants who were suffering from debilitating diseases or exposed to radiation were also excluded from the study. The participants in the study belonged to the rural areas near Lucknow. Their main occupation was farming.

After a careful evaluation, 240 participants were selected and divided into four groups with 60 participants each in the following categories: those consuming tobacco, those who consumed gutkha (a preparation of areca nut with tobacco), a group comprising participants with OSMF, and a control group.

Collection of saliva

Saliva samples were collected early in the morning; participants were asked to rinse their mouths to remove any residual debris. Saliva was collected in a test tube through a passive drool method, in unstimulated conditions. We used sterilized glass test tubes to collect the samples from Group 1 (control group) and Group 2 (tobacco). We had limited Oragene saliva collection kits and hence used them to collect saliva samples from Group 3 (gutkha) and Group 4 (OSMF).

Preparation of smear and staining

The collected sample was centrifuged at 1500 rpm for 5 min. The supernatant was kept aside. A drop of normal saline was added to the pellet and vortexed to get a homogeneous mixture of the sample. Fifty microliters of diluted pellet was placed on clean slides with a drop of fixative solution. The smear was subsequently stained with acridine orange and Feulgen fast green.

Assessment of micronuclei

One thousand cells per individual were assessed using the parameters laid down by Tolbert et al.^[12] who state that the diameter of the MN must be at least one-third of the diameter of the nucleus. The staining intensity and texture should be similar to the nucleus, and the MNs should lie in the same focal plane as the nucleus.

Purification and extraction of DNA from saliva

The reagents used were Prep IT and L2P. The sample was mixed with DNA Genotek kit. The procedure was done according to the protocol manual. The DNA pellet obtained was quantified using a NanoDrop.

Results

Micronuclei in buccal cells

The mean frequency/cell of MN formation was found to be highest in the submucous fibrosis group (2.37 ± 1.10), followed by the group of gutkha users (2.03 ± 1.16) and tobacco users (1.49 ± 0.77).

The mean frequency/cell of MN formation was minimum (1.02 ± 0.17) in the group of normal individuals.

According to the Kruskal–Wallis test, the difference in mean frequency/cell of MN formation among various groups is found to be highly significant (P< 0.001) [Figure 1], [Table 1].

Figure 1: Intergroup comparison of micronucleus frequency/cell in exfoliated buccal cells

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Table 1: Intergroup comparison of micronucleus frequency/cell in exfoliated buccal cells

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Results using Feulgen fast and acridine orange

Feulgen fast staining technique

The DNA stained magenta and the cytoplasm light green when the cells were stained with Feulgen fast [Figure 2]. In this technique, there is a mild hydrolysis done with 1 molar hydrochloric acid at 60^°C breaking the purine-deoxyribose bond. Then, a reaction with Schiff's reagent gives DNA a magenta color. An analysis of MNs was easier with this nuclear-specific stain since the cytoplasm was clear.

Figure 2: Cells stained with Feulgen fast green stain, arrow marked shows the micronuclei at ×40 magnification viewed under a light microscope

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Acridine orange staining technique

When acridine orange intercalates into double-stranded DNA it emits green fluorescence on the contrary it emits red when there is an interaction with single-stranded DNA (apoptotic cells have a larger fraction of DNA in the denatured form and displays an intense red fluorescence [Figure 3]. Green emission was seen in the control group and tobacco chewer [Figure 4] group, and red emission was seen in the gutkha group [Figure 5] and Oral submucous fibrosis group [Figure 6].

Figure 3: a) bright red emission b) green emission using acridine orange stain viewed under a fluorescent

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Figure 4: Tobacco chewer

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Figure 5: Gutkha chewer

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Figure 6: Oral submucous fibrosis

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The purity of extracted DNA can be measured by calculating the absorbance ratio A260/A280. This ratio assesses the contamination of protein solutions because proteins absorb light at 280 nm. The absorbance ratio with commercial kits is around 1.6–1.9. The method of collection may, however, affect the purity of the extracted DNA. The purity of DNA collected in sterile tubes was lower compared to the purity of extracted DNA collected with commercial kits such as Oragene. The A260/A280 absorbance ratio obtained shows that saliva results in DNA of the highest integrity which can be used for genome sequencing, etc. [Table 2]

Table 2: Quantification of salivary DNA

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Discussion

MNs originate from whole chromosome or chromosomal fragments that fail to reach the pole during anaphase or telophase in the course of nuclear division. MNs can be easily assessed in erythrocytes, lymphocytes, and exfoliated cells, for example, oral epithelium, urothelium, and nasal epithelium to obtain a measure of genome damage induced in vivo. MN assesses the level of genomic damage in a cell, and an evaluation can be done from exfoliated apart from lymphocytes and can be done in vivo, whereas ex vivo assay can be performed after cell culture and measuring sister chromatid exchange.

Frequency of micronuclei in tobacco users

The application of MN assay is to measure tobacco-induced genotoxic damage, which was demonstrated by Stich et al.,^[13] and subsequently, further evidence was put forward by Stich et al.^[14] who used the MN assay in individuals belonging to Bihar. They particularly measured the genotoxicity in khaini-induced buccal cells.

Patel et al. in 2009^[15] conducted a study on tobacco chewers, and they measured genotoxic damage by performing a MN assay and also by evaluating sister chromatid exchange.

A study conducted by Sellappa et al.^[16] assessed individuals who were regularly consuming tobacco and areca nut with paan. The MN frequency was increased in such individuals; hence, they concluded that tobacco is hazardous for oral and systemic health. Dash et al. in 2018 found the mean number of MNs to be 18.28 ± 10.0 in smokeless tobacco users,^[17] while in our study, the frequency of MNs in tobacco chewers when compared with healthy controls was 1.49/cell, with a standard deviation of 0.771.

Frequency of micronuclei in gutkha chewers

According to Palaskar and Jindal, various studies from 1985 till date have shown that the frequency of MNs was increased in tobacco consumers as compared to healthy individuals.^[18] Our study shows a higher frequency of MNs among gutkha users; this was also proved by previous studies by Nair et al. in 1991, Gandhi and Kaur in 2000.^[19],[20] Siddique et al. in 2008 and Nair et al. in 1999 found that carcinogens in gutkha are derived from areca nut alkaloids; there is also a formation of tobacco-specific nitrosamines due to chewing of gutkha that leads to exposure of buccal cells to volatile nitrosamines derived from areca nut alkaloids.^[21],[22] According to Wenke et al. in 1984, the saliva of gutkha chewers shows that the nitrite and nitrate reductase activities are increased.^[23] Murdia et al. in 1982 and Nair et al. in 1986 found that the acidic pH in the stomach causes nitrosation of secondary and tertiary amines once the quid is swallowed; therefore, the levels of nitrosoproline are increased in the urine 4–6.5 times in gutkha chewers.^[24],[25] Chakradeo et al. in a study in 1994 found that there is a release of reactive oxygen due to aqueous extracts of areca nut which causes damage to the buccal mucosa.^[26] The frequency of MN depends on the ingredients of the quid and how many times quid is consumed and for what duration. Nair et al. in 1985 found that the frequency of MN is also affected by certain lifestyle factors.^[25] In our study, we found that the frequency of MNs per cell in gutkha chewers was 2.03, with a standard deviation of 1.160.

Micronuclei and oral lesions

Many studies have shown that there is a formation of oxygen reactive species which is responsible for the formation of fibrous band in the buccal mucosa. The alkaloids present in areca nut also contribute to the formation of collagen bands. In OSMF patients, there was an increase in the total serum protein and iron. Although the mechanism of formation of collagenous band is not fully understood, some workers suggest the role of flavonoids, guvacoline, and tannins in areca nut. The formation of collagen bands increased with smoking. OSMF occurrence was more common in individuals who used both smoked and smokeless forms of tobacco.

Parvathi et al. in 2011^[27] found an increase in the frequency of micronucleated cells, it was 0.06% in normal, in precancerous lesions it was 0.12%, and in cancerous lesions it was 0.45% thus showing cytogenetic damage within the epithelium. Halder et al. in 2004^[28] found the MN frequency in precancerous lesions to be 0.63%, and in cancerous lesions, it was 1.36%. Palve and Tupkari in 2008 also concluded that the frequency of micronuclei was found to be 0.21% in normal cells while it was 1.84% in cancerous cells.^[29] Casartelli et al. in 2000^[9] compared MN frequencies in exfoliated cells from normal mucosa, in precancerous lesions, and squamous cell carcinoma and concluded that there was an increase in the frequency of MNs in malignant lesions when compared with normal buccal mucosal cells, thus indicating a shift toward malignancy.

Kalita et al. in 2013^[30] found the MN count to be 4.2 ± 0.96 in females and 6.6 ± 1.95 in males consuming tobacco quid. Smita et al. in 2013^[31] used acridine orange to measure MNs in oral OSMF cases and found the count raised when compared with normal individuals. In a study conducted by Kiran et al. in 2018, the number of MNs was found to be 60/500 cells in participants with epithelial dysplasia,^[32] while in our study, the frequency of MNs per cell in participants with OSMF was 2.37, with a standard deviation of 1.100.

Nuclear-specific stains and micronuclei

Staining procedures affect the micronuclei count and according to the results of the detailed survey done during the eighth workshop of the HUMN (Human Micronucleus Project) more than 50% of the laboratories which participated in the survey used Feulgen stain and only one of the laboratories had used H and E stain. According to the same survey, the second most commonly used stain was May–Grünwald–Giemsa (MGG) stain; however, Ayyad et al.^[33] found better results for counting of MNs using Pap stain as compared to MGG stain.

We used Feulgen fast and acridine orange staining procedures in our study. We found that identification and counting of MNs were much easier with Feulgen fast green stain since it is a nuclear-specific stain. Using acridine orange was a cumbersome procedure since the slides can be viewed only under a fluorescent microscope and slides cannot be preserved for a long time.

Purity of extracted DNA from saliva

Source of genomic DNA in saliva is not only from epithelial cells but also from white blood cells.^[34] When extracting DNA from saliva, bacterial DNA can also be recovered if methods such as oral swabs are used.^[35] In our study, the absorbance ratio was higher when we used commercial kits such as Oragene, where the bacterial DNA content is much lower owing to its special design. Most scientists prefer large amounts of DNA for performing various applications, but with advanced technology available, even small amounts of DNA can be used for performing single-nucleotide polymorphism genotyping, etc., Collecting DNA from saliva is a noninvasive procedure. Daksis and Erikson^[36] state that acquiring DNA from oral samples is clearly advantageous over blood samples and opens the path for point-of-care testing.

Clinical implications

Saliva is a versatile biofluid which is a storehouse of a variety of biomarkers including tumor markers. Participants readily agree to give saliva samples but are hesitant to give blood samples since it is an invasive procedure. We successfully extracted and purified salivary DNA which can give a variety of information about the well-being of the participant
Smears prepared using liquid-based cytology have less cellular clumping, a clear background with distinct cellular features
A progressive increase in the number of MNs from cells obtained from the control group to the ones obtained from participants with oral lesions shows that changes can be detected at cellular level rather than waiting for a full-blown lesion for obtaining biopsy. Hence, a cellular grading will help in the early detection of genotoxic events [Figure 4], [Figure 5], [Figure 6]
Staining techniques such as acridine orange show apoptosis at 480–490 excitation in cells of participants with oral lesions and gutkha chewers. It is a proven fact that areca nut is a major etiological factor in submucous fibrosis. A bright red fluorescence shows a larger fraction of DNA in the denatured form. This can also be proved by a clinical evaluation of OSMF which is a premalignant lesion.

Conclusion

Researchers and clinicians around the world prefer working with saliva since it is a high-performance biofluid, which is easy to collect and handle. Using liquid-based cytology, we collected not only epithelial cells for evaluation of MNs but also extracted and purified DNA; therefore, we conclude that saliva can be used as a tool for mass-screening purposes. From the present study, we also conclude that the number of MNs per cell increases in participants with oral lesions. The technique is simple and noninvasive. It can become a valuable tool and a prognostic indicator in patients who are suffering from potentially malignant disorders.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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