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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 2  |  Page : 372-376

Effect of staining procedures on the results of micronucleus assay in the exfoliated buccal mucosal cells of smokers and nonsmokers: A pilot study


Department of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Udaipur, Rajasthan, India

Date of Web Publication8-Mar-2018

Correspondence Address:
Dr. Rashmi Metgud
Professor and Head of the Department, Department of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Udaipur - 313 024, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.157351

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 > Abstract 


Background: Tobacco consumption in smoke form causes severe health problems such as cancer. Micronuclei are structures that present after genomic damage in buccal mucosal cells which could be a good indicator of chromosomal alterations in cytological samples. The nuclear anomalies that are consequences of cell injury are also found in these cells.
Aim: The present study was aimed at evaluating the effectiveness of deoxyribonucleic acid (DNA)-specific stain over DNA-nonspecific stain in exfoliated buccal mucosa cells in smokers and nonsmokers for assessment of micronuclei and nuclear anomalies.
Materials and Methods: Thirty subjects were divided into three groups; Group 1 (control) nonsmokers (n = 10), Group 2 individuals with history of smoking for less than 10 years (n = 10), and Group 3 individuals with history of smoking for more than 10 years (n = 10). The exfoliated buccal mucosal cells were scrapped using wooden spatula, smeared over the glass slide, and stained with DNA-nonspecific stain, Giemsa; and DNA-specific stains, Feulgen and acridine orange.
Results: We found that the mean micronuclei score with DNA-nonspecific stain, Giemsa, was significantly higher in smokers group when compared to nonsmokers group. No such significant differences were observed with DNA-specific stains. Also, in the smokers groups, mean micronuclei were higher in Group 3 when compared to Group 2.
Conclusion: The score of MN assay and nuclear anomalies in exfoliated buccal mucosal cells of smokers and nonsmokers strongly depended on staining procedures and duration of smoking. Hence, these should be interpreted with caution to avoid false-positive results.

Keywords: Acridine orange, feulgen, giemsa, micronuclei, nuclear abnormalities


How to cite this article:
Metgud R, Neelesh BT. Effect of staining procedures on the results of micronucleus assay in the exfoliated buccal mucosal cells of smokers and nonsmokers: A pilot study. J Can Res Ther 2018;14:372-6

How to cite this URL:
Metgud R, Neelesh BT. Effect of staining procedures on the results of micronucleus assay in the exfoliated buccal mucosal cells of smokers and nonsmokers: A pilot study. J Can Res Ther [serial online] 2018 [cited 2020 Jun 1];14:372-6. Available from: http://www.cancerjournal.net/text.asp?2018/14/2/372/157351




 > Introduction Top


In recent times, lot of efforts have been made to determine the effect of genetic (deoxyribonucleic acid (DNA)) damaging lifestyle factors like smoking, alcohol consumption, diet like vitamin deficiencies and supplementations, stress, and drugs in human populations.[1] These DNA damage can be studied with cytochemical tools like micronucleus (MN) assay, sister chromatid exchange, and chromosomal aberration.[2] Micronuclei assay was first introduced in 1983, since then it is gaining popularity as a biomarker of genetic damage for the detection of increased cancer risk.[3] It is a fast, simple, economical, noninvasive method; and hence is well-accepted.[4]

MN contains genetic material which are lost during mitosis as a result of clastogenic (causing chromosomal breaks) and aneugenic (causing aneuploidy or abnormal chromosome segregation) events, which occur before any preneoplastic histopathological change becomes evident.[4] This assay is helpful in the detection of extent of DNA damage in a cell and gives us information about cancer formation in an individual.[5]

In 1991 and 1992, Tolbert et al., published two studies that suggested a new criteria for cell counts in MN test. According to him, degenerative nuclear phenomena should be counted separately at the time of MN analysis, because greater occurrence of this phenomenon, in itself is indicative of apoptosis (karyorrhexis (KH), condensed chromatin (CC), and pyknosis (PN)) and necrosis (karyolysis (KL), KH, CC, and PN), which reveal the genotoxic and cytotoxic effects, respectively, of a given exposure.[6]

According to Thomas et al., 2009, (i) cell with MN and nuclear buds, assess the DNA damage, (ii) cell presenting KH, KL, P, and CC, make inferences about occurrences of cell death, (iii) basal cells, assess the proliferative potential of the mucosa, and (iv) binucleates cell, reveal information on defects in cytokinesis.[7]

Oral mucosal buccal cells are cells which first come in contact with different potentially carcinogenic products, like tobacco and alcohol, hence, these cells are susceptible to damage by these agents before it gets reflected in any systemic condition. As these cells are highly proliferative, they are responsible for causing >90% of the epithelial cancer. Therefore, the application of this test in epithelial cells is considered to be a sensitive tool for biomonitoring the genetic damage in human population. Screening of individuals who are at a higher risk of malignant transformation is more pivotal in preventing and reducing the number of deaths than the costly and painful treatment done later on.[4]

Hence, there is a need for an accurate counting of these cells (micronuclei + nuclear abnormalities) with specific staining procedures to avoid any false-positive test. Only little attention has been given until now, on the effect of different staining procedures on the results of micronuclei assays which are gaining popularity in the recent times. Very few studies have been done emphasizing on various staining procedure used and the duration of smoking.[8]

The aim of the present study was (i) to find out the effect of different stains on the results of micronuclei in exfoliated mucosal buccal cells, and (ii) to find out if the duration of smoking plays any role in the induction of these. Therefore, buccal mucosal cells were collected from nonsmokers, smokers with a history of smoking for less than 10 years, and smokers with a history of smoking for more than 10 years; and the mean micronuclei were comparatively evaluated with the DNA-specific stains, Feulgen and Acridine Orange; and DNA-nonspecific stain, Giemsa. Smokers were included in this study as it is a well-established fact that they are at an increased risk for oral and other forms of epithelial cancer.[8] Also, damage of epithelial cells leads to formation of nuclear anomalies other than micronuclei, such as KH, KL, PN, CC, broken eggs, and binucleates. It has been stressed that these anomalies are difficult to distinguish from classic micronuclei and may led to misinterpretations.[8] Therefore, we additionally evaluated these variables and analyzed with DNA-nonspecific Giemsa and DNA-specific stains Feulgen and acridine orange.


 > Materials and Methods Top


A total of 30 subjects who attended the outpatient department were included in the study and divided into three groups. Group 1 - control group, comprised of 10 healthy individuals, Group 2 - 10 individuals with history of smoking for less than 10 years, and Group 3 - 10 individuals with history of smoking for more than 10 years. All smokers had a history of smoking 1 pack of cigarettes (10 cigarettes in a pack) per day. All subjects were healthy and none of them suffered from any systemic disease, or had history of consuming any drugs or exposure to ionizing radiations. Ethical clearance was obtained from the institution and informed consent was obtained from all the participants.

Subjects were asked to rinse their mouth twice with water, before preparing the cytological smears. The exfoliated buccal mucosal cells were gently scrapped using wooden spatula and smeared over the precleaned glass slides and three slides were prepared from each subject. Slides were coded and stained with acridine orange, Feulgen (counter stained with fast green), and Giemsa.

From each smear, 10 microscopic fields were evaluated under ×10 and ×40 magnification for each staining procedure (for Giemsa and Feulgen - light microscope [Figure 1] (×40) and [Figure 2] (×40), respectively] and acridine orange - fluorescent microscope were used) [Figure 3] (×40)]. Micronuclei were scored in buccal cells according to the criteria defined by Tolbert et al. In addition, the mean nuclear anomalies, namely binucleation, CC, KH, KL, PN, and nuclear bud, were recorded in Feulgen and acridine orange stained slides according to Thomas et al., [Figure 4] (×40) and [Figure 5] (×40)].
Figure 1: Photomicrograph revealing micronuclei in Giemsa stain (magnification, ×40)

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Figure 2: Photomicrograph revealing karyolysis and karyorrhexis in Feulgen stain (magnification, ×40)

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Figure 3: Photomicrograph revealing condensed chromatin in Feulgen stain (magnification, ×40)

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Figure 4: Photomicrograph revealing micronuclei in Feulgen stain (magnification, ×40)

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Figure 5: Photomicrograph revealing micronuclei in acridine orange stain (magnification, ×40)

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Statistical analysis

Two-way analysis of variance (ANOVA) was used to find out the micronuclei difference among the various staining procedures and also amongst smokers and nonsmokers. Spearman's correlation was computed to assess correlations between the nuclear anomalies and mean MN in DNA-nonspecific and -specific stains.


 > Results Top


[Table 1] summarizes the demographic details of the three groups (total number of subjects, age, gender, and mean duration of smoking (in years)).
Table 1: Demographic details

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[Table 2] summarizes the mean micronuclei observed in the study groups with different staining procedures. The mean micronuclei scored with Giemsa were significantly higher in smokers group when compared to nonsmokers group. On the other hand, no such significant differences were observed with DNA-specific stains, Feulgen and acridine orange. It was also noted that amongst the smoker group, high mean micronuclei was noted in Group 3 when compared to Group 2; hence, it suggests that increased duration of smoking increases the micronuclei which was statistically significant.
Table 2: Mean micronuclei in the study groups with different staining procedures

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[Table 3] reveals increased micronuclei frequencies which was significantly associated with certain nuclear anomalies, such as KH, KL, binucleates, and CC, when DNA nonspecific staining procedure was used, whereas no such associations were observed with the DNA-specific stains.
Table 3: Spearman's correlation to assess correlations between the nuclear anomalies and the micronuclei

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Spearman's correlation was used to assess correlations between nuclear anomalies and mean MN in DNA-nonspecific and -specific stains. Statistical significance was observed only with nuclear bud.


 > Discussion Top


The findings in the present study revealed that the results of micronuclei assay in exfoliated oral mucosal cells of smokers and nonsmokers strongly depended on the staining procedures. It was noted that much higher mean MN was found with DNA-nonspecific stain, Giemsa; and no such significant differences were found between acridine orange and Feulgen. These comparisons indicated that micronuclei formation in epithelial cells may be overestimated when DNA-nonspecific stains are used. Also, Nersesyan et al., in 2006 concluded that much higher micronuclei frequencies were found in smokers with the DNA-nonspecific stains Giemsa and May–Grunwald–Giemsa, with no significant differences observed with acridine orange, diaminophenylindole (DAPI), Feulgen; which was similar to our study.[8]

Singh et al., in 2012 studied the MN frequencies in oral exfoliated epithelial cells using three different stains, that is, Feulgen, Papanicolaou (PAP), and hematoxylin and eosin (H and E) stains in 45 potentially malignant disorders (oral submucous fibrosis (OSMF) and leukoplakia (L)) and 15 controls with healthy mucosa. The results revealed that Feulgen showed the least MN frequency and H and E showed the maximum count, while the value obtained with PAP was intermediate between the former two. Hence, he concluded that Feulgen being DNA-specific stain gave least count, which were similar to our study.[9]

The present study showed that micronuclei formation in buccal mucosa cells is overestimated with Giemsa-based stains and is also supported by the results reported by Casartelli et al., who compared micronuclei numbers after Giemsa and propidium iodide staining in cells from patients with different pathologies of the oral cavity (LPs and squamous cell carcinomas). They found a threefold higher frequency of micronuclei with Giemsa than with Hoechst 33258.[10]

It has been noted that mean MN showed increased results as many researchers have observed in their study that Romonswsky-based Giemsa stain, stains cell debris and many other proteins; thus, masking the counting of MN.[11] There can be many possibilities why high MN frequency were observed in DNA-nonspecific stain, which can be:

  • Misinterpretation of nuclear anomalies (NA) like KH, KL, CC, BN as MN
  • Formation of keratin granules that are found in degenerated cells with NA. These round bodies found as a consequence of cell injury do not contain DNA and may be counted as MN
  • Contamination of bacteria
  • Other - small dye granule resembling MN, but can be differentiated with its refractive index and color intensity.


To avoid false counting of MN, these points were taken into consideration. But, still Giemsa revealed increased mean MN. Also, the reliability of many DNA-nonspecific stains has been questioned in various studies.[9]

The possible explanation for the lowest count with Feulgen and acridine orange could be its high DNA specificity and a clear transparent appearance of the cytoplasm which enables easy identification of MN. The advantage of acridine orange is that it makes the appearance of various cells to be clearer and disadvantage is evaluation with fluorescence microscope which is costlier.[5]

The present study revealed that metanucleated cells with anomalies other than micronuclei, such as KH, KL, binucleates, and CC, were significantly increased in smokers. These anomalies reflect the consequences of cell injury, cell death, and mitotic errors. Some of them (PN, CC, and KL) are accompanied by apoptosis, which may result from DNA damage. However, the same phenomenon is also seen in cells undergoing necrosis and cannot be regarded as a reliable marker for increased DNA damage and cancer risk. Only few studies have been published in which such anomalies have been recorded. In all studies including ours, KL was strongly increased compared with the control groups, whereas the pattern of induction of the other anomalies differed strongly.

Thus, we can conclude that micronuclei counts should be interpreted with caution when DNA-nonspecific stains are used. From the present study and review of the literature, it was observed that induction of micronuclei in smokers with DNA-specific stains yielded negative results and the positive results may be due to misinterpretation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Alexandresceu I, Havarneanu D, Popa D. New approaches in biomonitoring human populations exposed to genotoxic agents: Epithelial cell micronucleus assay. J Prev Med 2006;14:57-5.  Back to cited text no. 1
    
2.
Kalita H, Boruah DC, Dutta K, Devi R. Genotoxic effect on buccal epithelial cells of betel quid chewers by micronuclei assay. Asian J Exp Biol Sci 2013;4:491-4.  Back to cited text no. 2
    
3.
Khanna S, Purwar A, Singh NN, Sreedhar G, Singh S, Bhalla S. Cytogenetic biomonitoring of premalignant and malignant oral lesions by micronuclei assessment: A screening evaluation. Eur J Gen Dent 2014;3:46-2.  Back to cited text no. 3
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4.
Torres-Bugarín O, Zavala-Cerna MG, Nava A, Flores-García A, Ramos-Ibarra ML. Potential uses, limitations, and basic procedures of micronuclei and nuclear abnormalities in buccal cells. Dis Markers 2014;2014:956835.  Back to cited text no. 4
    
5.
Jyoti S, Khan S, Afzal M, Naz F, Siddique YH. Evaluation of micronucleus frequency by acridine orange fluorescent staining in bucccal epithelial cells of oral submucosus fibrosis (OSMF) patients. Egypt J Med Hum Genet 2013;14:189-3.  Back to cited text no. 5
    
6.
Tolbert PE, Shy CM, Allen JW. Micronuclei and other nuclear anomalies in buccal smears: Methods development. Mutat Res 1992;271:69-77.  Back to cited text no. 6
    
7.
Thomas P, Holland N, Bolognesi C, Volders MK, Bonassi S, Zeiger E, et al. Buccal micronucleus cytome assay. Nat Protoc 2009;4:825-7.  Back to cited text no. 7
    
8.
Nersesyan A, Kundi M, Atefie K, Schulte-Hermann R, Knasmüller S. Effect of staining procedures on the results of micronucleus assays with exfoliated oral mucosa cells. Cancer Epidemiol Biomarkers Prev 2006;15:1835-40.  Back to cited text no. 8
    
9.
Grover S, Mujib A, Jahangirdar A, Telagi N, Kulkarni P. A comparative study for selectivity of micronuclei in oral exfoliated epithelial cells. J Cytol 2012;29:230-5.  Back to cited text no. 9
[PUBMED]  [Full text]  
10.
Casartelli G, Monteghirfo S, De Ferrari M, Bonatti S, Scala M, Toma S, et al. Staining of micronuclei in squamous epithelial cells of human oral mucosa. Anal Quant Cytol Histol 1997;19:475-81.  Back to cited text no. 10
    
11.
Palaskar S, Jindal C. Evaluation of micronuclei using papanicolaou and may grunwald giemsa stain in individuals with different tobacco habits- A comparative study. J Clin Diagn Res 2010;4:3607-3.  Back to cited text no. 11
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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