Micronuclei as a Potent Biomarker

ABSTRACT: Oral Cancer has a remarkably high incidence worldwide and yet a a significant decrease in its mortality and morbidity rates has been established if it is diagnosed in early stages. There has been always a strong need to develop new, objective, non-invasive methods for its early detection. Micronucleus has come up in the recent past as a non-invasive biomarker for diagnosis of not only malignant and pre-malignant lesions but also many other significant diseases. Micronucleus (“MN") is defined as microscopically visible, round or oval cytoplasmic chromatin mass next to the nucleus. Micronuclei (“MNi”) originate from aberrant mitoses and consist of eccentric chromosomes, chromatid fragments or whole chromosomes that have failed to be incorporated into the daughter nuclei during mitosis. The MN assay has been widely accepted as an in vitro genotoxicity test and a biomarker assay for genotoxic exposure and effect in humans. An attempt has been made to review the related studies, utilizing micronucleus assay of buccal mucosal cells as a novel marker of genotoxicity in head and neck region. KEYWORDS: Micronucleus; micronuclei; micronucleus assay genotoxicity marker; biomarker. INTRODUCTION: The literal meaning of the word Micronucleus (MN) is describes it as a small nucleus in a large cell, or the smaller nuclei in cells that have two or more such structures. Schmid (1975) defined MN as a microscopically visible, round or oval cytoplasmic chromatin mass next to the nucleus. MN is the name given to the small extranucleus that forms whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division . The two basic phenomena responsible for the formation of MNi in mitotic cells are dysfunction of the mitotic apparatus and chromosome breakage. Micronuclei (MNi) are formed from the whole chromosomes or chromatid fragments that lag behind in anaphase and are separated out from the daughter nuclei in telophase. Additionally, some MNi gets originated from fragments derived from broken anaphase bridges formed due to chromosome rearrangements such as dicentric chromatids, intermingled ring chromosomes or union of sister chromatids . In the course of telophase these chromosomal regions are included in the daughter cells where they can fuse with the main nucleus or can form one or more smaller secondary nuclei . This smaller secondary nucleus is known as MN and its number can vary from one to many. Now the MN formed can either go with the daughter nucleus they derive from or the other daughter nucleus. In the former scenario, neither of the daughter cells is aneuploid, and in the latter case the micronucleated cell has gained a chromosome, while its daughter cell has lost it . MICRONUCLEUS ASSAY The Micronucleus assay is defined as an investigative procedure carried out to analyze micronuclei quantitatively. It is one of the standard cytogenetic tools implemented to assess micronuclei formation (signifying chromosomal damage) subsequent to exposure to genotoxic/cytotoxic agents . In humans, MN assay can be easily employed in lymphocytes, erythrocytes, and exfoliated epithelial cells (e.g. oral, urothelial, nasal) to obtain a measure of genome damage induced in vivo . In the earlier times, once-divided cultured cells (mostly peripheral lymphocytes) were considered ideal for expressing MNi and the procedure used was known as cytokinesis-block micronucleus (CBMN) assay. In the CBMN assay, once divided cells are identified by their MBN appearance after blocking cytokinesis with cytochalasin-B (Cyt-B) . In the last three decades, ever since the advent of MN assay on exfoliated cells, the buccal mucosal cells have completely replaced the usage of lymphocytes for the obvious reason of non-invasive collection of cells and simpler method for analyzing the MNi. The general genotoxicity results by both the methods of MN analysis are comparable . The basal cells in the oral epithelium are the cells which possess the capability of mitosis, the new cells formed gets differentiated and mature as they migrate to the surface finally replacing the cells which are shed. Thus any genetic damage (expressed as MNi formation) instigated in these basal cells is reflected in the exfoliated cells; thus analysis of MNi in these cells can be correlated with the amount of genetic damage. As the oral epithelium acts as the first barrier for the inhalation or ingestion route and approximately 90% of cancers originate from epithelial cells, therefore, these cells represent a favored target site for early genotoxic events induced by carcinogenic agents entering the body via inhalation and ingestion. The procedure for MN assay has been summarized in Tables 1-3. Through this review, we intend to highlight the application of MN assay on buccal cells by reviewing significant studies from the English literature. We also aim to emphasize the pitfalls associated and the measures to overcome the same. TABLE 1. THE PROCEDURE FOR MN ASSAY USING A CYTOSMEAR

• The subjects are asked to rinse their mouth with water and a cytobrush is used to obtain exfoliated cells from the oral mucosa.
• The samples are transferred to dry glass slides, to ensure an adequate harvest of cells.
• Smears are air dried and fixed with 95% ethanol spray.
• Smears are stained with either DNA specific or DNA non-specific stain.
• Smears are scored manually/digitally using a Tolbert (most accepted) criteria.

TABLE 2. STAINS COMMONLY USED FOR STAINING THE CYTOSMEARS FOR MN ASSAYDNA specific stains:

• Feulgen Stain
• DAPI (4’,6-diamidino-2-phenylindole)
• Acridine orange

DNA non-specific stains:

• Giemsa stain
• May Grunwald-Giemsa stain
• PAP stain
• H & E stain

TABLE 3. FOR DESIGNATING AN EXTRA NUCLEAR BODY AS MICRONUCLEUS, THE FOLLOWING CRITERIA GIVEN BY TOLBERT IS APPLIED

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