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 Table of Contents  
REVIEW ARTICLE
Year : 2012  |  Volume : 8  |  Issue : 2  |  Page : 184-191

Micronuclei assay of exfoliated oral buccal cells: Means to assess the nuclear abnormalities in different diseases


1 Department of Oral Pathology, Saraswati Dental College and Hospital, Lucknow, Uttar Pradesh, India
2 Department of Oral and Maxillofacial Surgery, Saraswati Dental College and Hospital, Lucknow, Uttar Pradesh, India

Date of Web Publication26-Jul-2012

Correspondence Address:
Bina Kashyap
Staff Quarters, Saraswati Dental College and Hospital, Faizabad Road, Near Chinhut, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.98968

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 > Abstract 

The micronuclei assay (MA) in exfoliated buccal cells is an innovative genotoxicity technique, which holds promise for the study of epithelial carcinogens. Micronuclei are suitable internal dosimeters for revealing tissue-specific genotoxic damage in individuals exposed to carcinogenic mixtures. This article reviews the MN assay with respect to oral buccal mucosa, which has been used since the 1980s to demonstrate cytogenetic effects of environmental and occupational exposures, lifestyle factors, dietary deficiencies, and different diseases along with the characteristics of micronuclei and other nuclear abnormalities.

Keywords: Carcinogens, exfoliated buccal cells, genotoxic, micronuclei


How to cite this article:
Kashyap B, Reddy PS. Micronuclei assay of exfoliated oral buccal cells: Means to assess the nuclear abnormalities in different diseases. J Can Res Ther 2012;8:184-91

How to cite this URL:
Kashyap B, Reddy PS. Micronuclei assay of exfoliated oral buccal cells: Means to assess the nuclear abnormalities in different diseases. J Can Res Ther [serial online] 2012 [cited 2019 Nov 19];8:184-91. Available from: http://www.cancerjournal.net/text.asp?2012/8/2/184/98968


 > Introduction Top


Within epidemiologic cancer research, molecular epidemiology is an area of increasingly intense activity and interest, combining molecular biology and epidemiologic methods to strengthen epidemiologic evidence. [1],[2] Molecular epidemiology research focuses on three types of biomarkers: biomarkers of exposure (eg, cytogenetic endpoints-chromosomal aberrations, micronuclei, and sister chromatid exchanges), biomarkers of susceptibility (eg, genetic polymorphisms), and biomarkers of disease (eg, tumor biomarkers). [2],[3],[4]

Genomic damage is probably the most important fundamental cause of developmental and degenerative disease. It is also well established that genomic damage is produced by environmental exposure to genotoxins, medical procedures (eg, radiation and chemicals), micronutrient deficiency (eg, folate), lifestyle factors (eg, alcohol, smoking, drugs, and stress), and genetic factors, such as inherited defects in DNA metabolism and/or repair. [5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23] It is essential to have reliable and relevant minimally invasive biomarkers to improve the implementation of biomonitoring, diagnostics, and treatment of diseases caused by, or associated with, genetic damage. The MN assay is potentially an excellent candidate to serve as such a biomarker, which detects chromosome loss or malfunction of mitotic spindle caused by aneugenic mechanisms. [12]

In the late 1800s and early 1900s, Howell and Jolly described Feulgen-positive nuclear bodies in human reticulocytes, known as Howell-Jolly bodies, and representing chromosomes separated from the mitotic spindle. [24] In the early 1970s the term micronucleus test was suggested for the first time by Boller and Schmidt and Heddle who showed that this assay provided a simple method to detect the genotoxic potential of mutagens after in vivo exposure of animals using bone marrow erythrocytes. A few years later it was shown by Countryman and Heddle that peripheral blood lymphocytes could be used for the micronucleus approach and they recommended using micronuclei as a biomarker in testing schemes.

The human micronucleus (HUMN) project (http://www.humn.org), established in 1997, is an international collaborative program aimed to standardize micronucleus assay in peripheral blood lymphocytes and to assess the effects of protocol and scoring criteria on the values obtained. This part of HUMN project was completed and the results were published in 2001. [25],[26]

The aims of this article were to provide an overview of the current status of the MN assay in oral buccal cells and to compare the strengths and limitations of the existing techniques for collection, staining, and scoring of buccal cells.


 > Measurement of Micronuclei Cells in Oral Exfoliated Buccal Cells Top


Micronuclei are fragments or whole chromosomes, which did not reach spindle poles during mitosis and remained encapsulated at telophase in a separate nucleus. The chromosome aberration assay detects only the genome damage, whereas micronucleus assay additionally detects chromosome loss or malfunction of mitotic spindle caused by aneugenic mechanisms. [27] Aneuploidy is an integral factor in the development of malignancies. [28] There is a hypothesis that micronuclei and chromosomal aberrations could have a predictive value for cancer and therefore substitute chromosomal aberrations as cancer risk biomarkers or provide additional information on the mechanism of action of aneugenic agents. [29]

Buccal cells form the first barrier for the inhalation or ingestion route and are capable of metabolizing proximate carcinogens to reactive products. [30],[31],[32],[33] About 92% of human cancers are derived from the external and internal epithelium, that is, the skin, the bronchial epithelium, and the epithelia lining the alimentary canal. Therefore, it could be argued that oral epithelial cells represent a preferred target site for early genotoxic events induced by carcinogenic agents entering the body via inhalation and ingestion.

The research opportunities afforded by buccal cells is that, for a given patient, regulatory mechanisms, signaling pathways, and genetic modulation can be assessed before, during, and following antitumor therapy. Buccal cells not only offer the clinician opportunities for early diagnosis and an aid for smoking cessation counseling but also provide a unique model for mutation research that permits correlating genetic alterations with histopathologic changes and for drug discovery investigations. [34]

The oral epithelium is composed of four strata of structural, progenitor, and maturing cell populations, that is, the basal cell layer (stratum basale), prickle cell layer (stratum spinosum), and the keratinized layer at the surface. A series of finger-like structures called "rete pegs" project up from the lamina propria into the epidermal layer producing an undulating basal cell layer effect. The oral epithelium maintains itself by continuous cell renewal whereby new cells produced in the basal layer by mitosis migrate to the surface replacing those that are shed. The basal layer contains the stem cells that may express genetic damage (chromosome breakage or loss) as MN during nuclear division. The daughter cells, which may or may not contain MN, eventually differentiate into the prickle cell layer and the keratinized superficial layer, and then exfoliate into the buccal cavity. Some of these cells may degenerate into cells with condensed chromatin, fragmented nuclei (karyorrhectic cells), pyknotic nuclei, or completely lose their nuclear material (karyolitic or "ghost" cells). [5] In rare cases, some cells may be blocked in a binucleated stage or may exhibit nuclear buds (also known as "broken eggs" in buccal cells), a biomarker of gene amplification. These biomarkers of genome damage (eg, MN, nuclear buds) and cell death (eg, apoptosis, karyolysis) can be observed in both the lymphocyte and buccal cell systems, and thus provide a more comprehensive assessment of genome damage then only MN in the context of cytotoxicity and cytostatic effects. [5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[35]

Casartelli et al. [71] observed MN frequencies in exfoliated buccal cells in normal mucosa, precancerous lesions, and squamous cell carcinoma. They concluded that the gradual increase in MN counts from normal mucosal to precancerous lesions to carcinoma suggested a link of this biomarker with neoplastic progression. It is notable that the first studies of Stich and Rosin, [36] conducted between 1983 and 1984 had higher baseline MN frequencies than subsequent studies. This may have been due to a lack of defined scoring criteria and a relatively small number of scored cells (in some cases less than 500). Since then, published biomonitoring studies using the MN assay in buccal mucosa cells have investigated the effects of multiple factors, including environmental and occupational exposures, radiotherapy, chemoprevention, vitamin supplementation trials, lifestyle habits, cancer, and other diseases. [36]


 > Occupational and Environmental Exposures Top


In the last 15-20 years, the MN assay has been applied to evaluate chromosomal damage for biologic monitoring of human populations exposed to a variety of mutagenic and carcinogenic chemical or physical agents, such as antineoplastic drugs used for hospital staffs, areca nut chewers, arsenic in drinking water, dioxin as fertilizers, ethylene oxide, formaldehyde, lead oxide, solvents, benzene, ozone, polycyclic aromatic hydrocarbons, chlorants, toxic gases, pesticide mixtures, toluene, hexane, acetone, methyl-ethyl-ketone, 2-trans-hexol, and all forms of tobacco. A broad range of baseline MN frequencies has been reported (0.05-11.5 MN/1000 cells) with the majority of values between 0.5 and 2.5 MN/1000 cells. There is no clear pattern of the variations among laboratories from different countries. Many studies report a statistically significant elevation of MN levels in exposed individuals compared with control groups, although the observed effects are relatively small, ranging between 1.1- and 4-fold, [37-41] and many other studies report changes that were not statistically significant. [42],[43],[44] A 12-fold increase in MN frequency was observed in a study by Suruda et al. [45] This increase was unusually large, but has been confirmed by independent analyses using a different staining procedure.


 > Radiation Top


Ionizing radiation plays an important role in the treatment of many neoplasias, but it also produces genetic damage. Several studies evaluated MN in buccal cells of patients undergoing radiotherapy in the head and neck region. The most striking increase in cytogenetic damage (150-300 MN/1000 cells) was observed in an early study of three patients exposed to a cumulative dose of 3400-4000 cGy. [6] Some authors reported 68 MN/1000 cells after 2000 cGy [9] and 16 MN/1000 cells after treatment with 1000 cGy for 3 weeks. [10] Moore et al. [46] in their study showed more than 16-fold increase in MN frequency shortly after the initiation of radiotherapy, followed by return to baseline 12 weeks later and 3 weeks after cessation of the treatment. In a more recent study, an increase in MN frequency in a group of head-and-neck cancer patients undergoing radiotherapy was evident both in buccal cells and peripheral lymphocytes. [47] Cao et al. [48] found that the cytokinesis-blocked MN assay in peripheral blood lymphocytes was more sensitive than the buccal MN assay. It was reported that radiation-sensitive oral tumors showed higher MN levels in exfoliated cells after radiation therapy than the more radiation-resistant ones, and the assay can be used as a predictor of tumor radiosensitivity. [49] However, further modified approach is required for the confirmation.


 > Oral Cancers Top


Oral cancer is one of the 10 most common cancers as stated by World Health Organization (WHO) and each year 5, 75, 000 new cases and 3, 20, 000 deaths occur worldwide. About half of all cases of oral cancer have associated leukoplakia. Other potentially malignant lesions or conditions include erythroplakia, lichen planus, submucous fibrosis, and chronic immunosuppression. [50] WHO distinguishes between oral precancerous lesions and oral precancerous conditions. A precancerous lesion consists of morphologically altered tissue that is more likely to be transformed into cancer than its normal counterpart, such as leukoplakia, erythroplakia, and the palatal changes associated with reverse smoking of cigarettes (chutta). A precancerous condition is a state associated with a significantly increased risk for cancer, such as syphilis, sideropenic dysplasia, and oral submucous fibrosis.

The MN in buccal mucosa cells were used to study preneoplastic effects by collecting the cells directly from the affected tissues. It was suggested that the MN in buccal mucosa may predict cancer risk for the upper aerodigestive tract, including premalignant stages, such as oral leukoplakia. [51] An increase in MN frequency in buccal cells was reported for diabetes mellitus with the patients having double the level of genetic damage in comparison to matched controls. [52] But it remains unclear whether an elevated frequency of MN in certain tissues, such as oral epithelia, would be predictive of increased risk of future cancer say only for oral cavity, limited to upper digestive tract epithelia, or may be projected for various cancers in other parts of the body. This breach of knowledge will be an important focal point for future HUMN projects.


 > Host and Lifestyle Factors Top


Many studies report the age and sex of the study subjects, but only a portion of these studies were able to ascertain a statistically significant effect by gender [14],[53] or by age. [16],[41],[54] Two of the studies showed slightly higher MN frequency of buccal cells in men than in women. The results were in contrast to data showing higher MN frequencies in lymphocytes of women and older subjects. [55] Smokers on average had almost tripled the MN frequency compared with nonsmokers. However, other publications report no difference between smokers and nonsmokers and between men and women. [56] Thus, the prospective involvement of gender and age in the frequency of MN in buccal cells warrants further research.

Lifestyle factors include smoking, alcohol consumption, and diet, especially vitamin deficiencies and supplementation. [57],[58] The majority of the studies with a significant increase in MN are related to a risk of oral cancer and were performed in subgroups of subjects with specific lifestyle habits, that is, chewers of betel quids (areca nut, betel leaves, slaked lime, and tobacco), reverse smokers, snuff dippers, Khaini tobacco (tobacco mixed with slaked lime), and other similar practices. [10],[58],[59],[60] Results in these studies showed overvalue of the MN frequency because both smoking and chewing of tobacco mixtures are known to cause nuclear degeneration and appearance of MN-like bodies in exfoliated cells, were confused with MN. Hence, it is important to distinguish cell death events from genome damage in viable epithelial cells both in terms of biologic dosimetry and for evaluating cancer risk.

Micronutrients and other vitamins have been shown to significantly decrease MN levels (1.4- to 4-fold) in healthy tobacco users, as well as in individuals with precancerous lesions. [7],[61],[62],[63]

Some micronutrients, such as retinol, riboflavin, zinc, and selenium, however, failed to reduce the MN frequency in a study carried out in China in areas with a high incidence of esophageal cancer. [64] One study showed decline in MN frequencies in children and women who received controlled folate supplementation, [65],[66] and in patients with diabetes. [52] Controversy exists whether the reductions in the MN frequencies were due to a decrease in chromosomal instability as a result of supplementation, or to a modified basal cell proliferation that altered the kinetics of MN expression.


 > Identification of MN Cells Top


Several factors that affect the MN in buccal cells include differences in cell collection (timing and implements used), fixation and staining techniques, selection and number of cells counted, and the scoring criteria, and other nuclear anomalies in normal and degenerated cells. Many of these methodologic factors for buccal cells overlap with the MN assay in lymphocytes, but the differences in tissues may contribute to the variability. Thus, the data reported by the HUMN on MN assay in human lymphocytes [55] can be applied to buccal cells with caution, although a special validation study for buccal cells is needed.


 > Sample Collection Top


Different diagnostic methods, such as routine histopathology, exfoliative cytology, and immunohistochemistry, are available today. Of these, oral exfoliative cytology is particularly valuable for mass screening purpose. It has been shown to have a sensitivity of 94%, specificity of 100%, and an accuracy of 95%. [67] Exfoliated buccal mucosal cells can be collected using a wooden tongue-depressor, a metal spatula, toothpicks or toothbrushes, or a cytobrush moistened with water or buffer to swab or gently scrape the mucosa of the inner lining of one or both cheeks. [17],[43],[44],[53],[68],[69],[70] Cytobrushes appear to be most effective for collecting large numbers of buccal cells. Casartelli et al. [71] observed that MN frequencies were higher when cells were collected by vigorous, rather than by light, scraping, suggesting a decreasing MN frequency gradient from basal to superficial layers of mucosa.


 > Preparation of Slide and Staining Top


In a number of studies the cytobrush used to collect buccal cells was shaken in a centrifuge tube containing saline solution (Hank's basic or other buffer solution) to release the cells, and the tube is then centrifuged to wash the cells in a buffer solution [17],[37],[41],[42],[54],[69],[72],[73],[74],[75],[76],[77],[78],[79] or a fixative. [68] This washing procedure helps to remove bacteria and cell debris, which confound the scoring. Buccal cell smears have been prepared by spreading the cells on a clean slide transferred either by careful dropping with pipette or by cytocentrifugation followed by fixation. Commonly used fixatives include 80% methanol, absolute ethanol, or a methanol-glacial acetic acid mixture.

Numerous staining methods have been used, among them DNA-specific stains are preferred for staining nuclei, MN, and other nuclear anomalies in buccal exfoliated cells. Feulgen-Fast Green staining is favored by many investigators because of its DNA specificity and a clear transparent appearance of the cytoplasm, which enables easy identification of MN. Other stains include fluorescent dyes, such as diamidino-2-phenylindoleDAPI, [64],[69],[70] acridine orange [Figure 1], [80],[81] Hoechst, [82] and propidium iodide, [15],[46],[76],[82] May-Grunwald Giemsa (Giemsa) stain [Figure 2]. [22],[51],[64],[83],[84],[85],[86]
Figure 1: Photomicrograph showing micronuclei with acridine orange stain

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Figure 2: Photomicrograph showing micronuclei with May– Grunwald Giemsa (Giemsa) stain

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Micronuclei assay in contrast to other body sites-the mouth is readily accessible and cells can be collected from:

  1. the same patient;
  2. in a noninvasive manner;
  3. easily and repetitively obtained;
  4. from normal tissue, preneoplastic lesions, and malignant tumors; and
  5. for longitudinal studies of drug discovery and assessment.


Although the formation of cells with karyorrhexis, karyolysis, binucleates, and condensed chromatin are difficult to interpret and may be classified as micronuclei with nonspecific stains. Another possible confounding factor in micronuclei studies is the formation of keratin granules that are found in degenerated cells with nuclear anomalies. [87]


 > Factors Affecting the Scoring of MN Cells Top


A few studies reported the possibility that cellular structures, such as keratohyalin granules or bacteria, resembling MN, can lead to false-positive results. [82],[87] Keratohyalin granules are reported to occur in cells of the granular layer of interfollicular epidermis of the skin, [88] and are predominantly composed of the 400-kDa protein profillagrin. MN scoring can be interfered by the bacteria that are commonly found in the mouth. [76],[89] Bacteria can be differentiated from MN by their characteristic shape, smaller size, color, staining intensity, and their presence upon and between buccal cells on the slide. Small dye granules may sometimes resemble MN but usually have a slightly different refractility and color intensity.


 > Scoring Criteria Top


Heddle [90] initially described the well-established basic criteria for MN. However, the criteria for identifying cells for inclusion into the MN frequency count were not provided. Later Tolbert et al. [5],[10] developed the criteria for choosing the cells and this is being most widely applied. It consists of the following parameters:

  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.


The suggested criteria for identifying MN are as follows: (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) Feulgen positive, that is, pink in bright field illumination; (4) staining intensity similar to that of the nucleus; (5) texture similar to that of nucleus; (6) same focal plane as nucleus; and (7) absence of overlap with, or bridge to, the nucleus.

Tolbert et al. [10] also recommended the scoring of at least 1000 cells, with an increase to 2000-3000 if fewer than 5 micronucleated cells were observed after counting 1000 cells. The majority of the published studies have scored between 1000 and 3000 cells, although it has been suggested that 10,000 cells may be needed to observe a statistically significant, 50% increase, in the MN frequency. [91]


 > Rationale to Study and Examine Slide Preparation and Scoring Variables Top


The HUMN project serves as an example for developing a strategy for understanding and validating the buccal cell MN procedures. The variability of the human lymphocyte MN assay was developed by the HUMN validation project; it was evident that, in this assay, there is only a very small fraction of unexplained variance (3.1%) in the MN frequency. [55] These values were obtained through the use of a careful study design, which took the main explanatory variables into consideration and also because of prior agreement and standardization of the scoring procedures among the participating laboratories. This interlaboratory agreement was possible because scoring criteria with detailed photos illustrating each endpoint were prepared for the participants before the exercise and later published along with its results. [36],[55]

HUMN committee had recently sent out an invitation to the prospective collaborators to contribute to HUMN XL project (XL subscript was designed to emphasize the focus of this HUMN initiative on the MN assay in exfoliated buccal cells). The initial stage of assay validation will focus on (1) addressing the effect of different staining methods on the results, and (2) determining the extent of variability in slide scoring among scorers in the same laboratories, and among laboratories.

The present goal in many research laboratories is to develop screening strategies indicating individual cancers with certain biomarkers. Biomarkers are instruments of individual tumor prevention and help to detect high-risk patients. Early detection of a premalignant or cancerous oral lesion would improve the survival to a greater extent and also will reduce the morbidity associated with the treatment to a considerable extent. [92]


 > Summary Top


Although many studies have consistently shown a statistically significant increase in the buccal cell MN frequency in human populations exposed to genotoxic agents, or a decrease as a result of micronutrient supplementation or chemoprevention, the magnitude of changes is usually relatively small. Different confounding factors influencing the MN frequency in peripheral lymphocytes, such as gender, age, and lifestyle habits, have been considered for the buccal cell MN assay. Despite the considerable potential of the buccal MN assay for biomonitoring, the diversity of possible methodologic variables, and their impact on assay performance, could hinder consistency among laboratories with regard to measuring the effects of dietary, lifestyle, and genetic factors. Simplicity, accuracy, multipotentiality, and large tissue applicability of the MN technology made it attractive in the past and will ensure a key role in the evaluation of mutagenicity and primary prevention in the future.

 
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