**2.1.1 Cytokinesis-Block micronucleus assay**

The scope and the application of cytokinesis-block MN assay (CBMN) in biomonitoring has also been expanded in recent years so that in addition to scoring MN in binucleate cells, there are proposals to evaluate MN in mononucleate cells (to provide a more comprehensive assessment of DNA damage), nucleoplasmic bridges (indicative of DNA misrepair, chromosome rearrangement or telomere endfusions) and nuclear buds (a measure of gene amplification or acentric fragments). Fenech (2007), has proposed that CBMN assay can be used to measure chromosomal instability, mitotic dysfunction and cell death (necrosis and apoptosis) and has suggested the term CBMN assay. Identification of the contents of MN (e.g. presence and absence of centromeres) is now considered important in the evaluation of MN in biomonitoring studies, providing insight into mechanisms underpinning the positive results reported, i.e. to differentiate between clastogens and aneugenic responses (Battershill, et al., 2008).

The CBMN assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells and include: micronucleus (MN), nucleoplasmic bridges (NPB) and nuclear buds (NBUDs). Cytostatic effects are measured via the proportion of mono-, bi- and multinucleated cells and cytotoxicity via necrotic and/or apoptotic cell ratios (Fenech, 2002a, 2006, 2007).

MN originate from chromosome fragments or whole chromosomes that lag behind anaphase during nuclear division. The CBMN assay is the preferred method for measuring MN in cultured human and/or mammalian cells because scoring is specifically restricted to once-divided binucleated cells, which are the cells that can express MN. In the CBMN assay, once-divided cells are recognized by their binucleated appearance after blocking cytokinesis with cytochalasin-B (Cyt-B), an inhibitor of microfilament ring assembly required for the completion of cytokinesis.

The CBMN assay allows measuring chromosome breakage, DNA misrepair, chromosome loss, non-disjunction, necrosis, apoptosis and cytostasis. Also measure NPB, a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair, and to measure NBUDs, a biomarker of gene amplification.

Because of its reliability and good reproducibility, the CBMN assay has become one of the standard cytogenetic tests for genetic toxicology testing in human and mammalian cells (Fenech, 2002b, 2007).

NPB occur when centromeres of dicentric chromosomes are pulled to opposite poles of the cell at anaphase. There are various mechanisms that could lead to NPB formation following DNA misrepair of strand breaks in DNA. Typically, a dicentric chromosome and an acentric chromosome fragment are formed that result in the formation of an NPB and an MN, respectively. Misrepair of DNA strand breaks could also lead to the formation of dicentric ring chromosomes and concatenated ring chromosomes which could also result in the

MN analysis, therefore, appears to be a good tool for investigating the effects of clastogens and aneuploidogens in occupational and environmental exposure in human epidemiological studies (Ishikawa et al., 2003) and are described as a promising approach with regard to

The scope and the application of cytokinesis-block MN assay (CBMN) in biomonitoring has also been expanded in recent years so that in addition to scoring MN in binucleate cells, there are proposals to evaluate MN in mononucleate cells (to provide a more comprehensive assessment of DNA damage), nucleoplasmic bridges (indicative of DNA misrepair, chromosome rearrangement or telomere endfusions) and nuclear buds (a measure of gene amplification or acentric fragments). Fenech (2007), has proposed that CBMN assay can be used to measure chromosomal instability, mitotic dysfunction and cell death (necrosis and apoptosis) and has suggested the term CBMN assay. Identification of the contents of MN (e.g. presence and absence of centromeres) is now considered important in the evaluation of MN in biomonitoring studies, providing insight into mechanisms underpinning the positive results reported, i.e. to differentiate between clastogens and aneugenic responses

The CBMN assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells and include: micronucleus (MN), nucleoplasmic bridges (NPB) and nuclear buds (NBUDs). Cytostatic effects are measured via the proportion of mono-, bi- and multinucleated cells

MN originate from chromosome fragments or whole chromosomes that lag behind anaphase during nuclear division. The CBMN assay is the preferred method for measuring MN in cultured human and/or mammalian cells because scoring is specifically restricted to once-divided binucleated cells, which are the cells that can express MN. In the CBMN assay, once-divided cells are recognized by their binucleated appearance after blocking cytokinesis with cytochalasin-B (Cyt-B), an inhibitor of microfilament ring assembly required for the

The CBMN assay allows measuring chromosome breakage, DNA misrepair, chromosome loss, non-disjunction, necrosis, apoptosis and cytostasis. Also measure NPB, a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair, and to

Because of its reliability and good reproducibility, the CBMN assay has become one of the standard cytogenetic tests for genetic toxicology testing in human and mammalian cells

NPB occur when centromeres of dicentric chromosomes are pulled to opposite poles of the cell at anaphase. There are various mechanisms that could lead to NPB formation following DNA misrepair of strand breaks in DNA. Typically, a dicentric chromosome and an acentric chromosome fragment are formed that result in the formation of an NPB and an MN, respectively. Misrepair of DNA strand breaks could also lead to the formation of dicentric ring chromosomes and concatenated ring chromosomes which could also result in the

and cytotoxicity via necrotic and/or apoptotic cell ratios (Fenech, 2002a, 2006, 2007).

assessing health risks (Battershill et al., 2008).

**2.1.1 Cytokinesis-Block micronucleus assay** 

(Battershill, et al., 2008).

completion of cytokinesis.

(Fenech, 2002b, 2007).

measure NBUDs, a biomarker of gene amplification.

formation of NPB. An alternative mechanism for dicentric chromosome and NPB formation is telomere end fusion caused by telomere shortening, loss of telomere capping proteins or defects in telomere cohesion. The importance of scoring NPB should not be underestimated because it provides direct evidence of genome damage resulting for misrepaired DNA breaks or telomere end fusions, which is otherwise not possible to deduce by scoring MN only (Fenech, 2007 ; Thomas et al., 2003).

NBUD are biomarkers of elimination of amplified DNA and/or DNA repair complexes. The nuclear budding process has been observed in cultures grown under strong selective conditions that induce gene amplification as well as under moderate folic acid deficiency. Amplified DNA may be eliminated through recombination between homologous regions within amplified sequences forming mini-circles of acentric and atelomeric DNA (double minutes), which localized to distinct regions within the nucleus, or through the excision of amplified sequences after segregation to distinct regions of the nucleus. The process of nuclear budding occurs during S phase and the NBUD are characterized by having the same morphology as an MN with the exception that they are linked to the nucleus by a narrow or wide stalk of nucleoplasmic material depending on the stage of the budding process. The duration of the nuclear budding process and the extrusion of the resulting MN from the cell remain largely unknown (Fenech, 2007; Serrano-García & Montero-Montoya, 2001; Utani et al., 2007).

Most chemical agents and different types of radiation have multiple effects at the molecular, cellular and chromosomal level, which may occur simultaneously and to varying extents depending on the dose. Interpretation of genotoxic events in the absence of data on effects in nuclear division rate and necrosis or apoptosis can be confounding because observed increases in genome damage may be due to indirect factors such as inhibition of apoptosis or defective/permissive cell-cycle checkpoints leading to shorter cell-cycle times and higher rates of chromosome malsegregation. Furthermore, determining nuclear division index (NDI) and proportion of cells undergoing necrosis and apoptosis provides important information on cytostatic and cytotoxic properties of the agent being examined that is relevant to the toxicity assessment. In human lymphocytes, the NDI also provides a measure of mitogen response, which is a useful biomarker of immune response in nutrition studies and may also be related to genotoxic exposure. The cytome approach in the CBMN cytome assay is important because it allows genotoxic (MN, NPB and NBUD in binucleated cells), cytotoxic (proportion of necrotic and apoptotic cells) and cytostatic (proportion and ratios of mono-, bi- and multinucleated cells, NDI) events to be captured within one assay (Fenech, 2005, 2007; Umegaki & Fenech, 2000).

In conclusion, the CBMN method has evolved into an efficient "cytome" assay of DNA damage and misrepair, chromosomal instability, mitotic abnormalities, cell death and cytostasis, enabling direct and/or indirect measurement of various aspects of cellular and nuclear dysfunction such as: unrepaired chromosome breaks fragments and asymmetrical chromosome rearrangement (MN or NPB accompanied by MN originating from acentric chromosomal fragments); telomere end fusions (NPB with telomere signals in the middle of the bridge and possibly without accompanying MN); malsegregation of chromosomes due to spindle or kinetochore defects or cell-cycle checkpoint malfunction (MN containing whole chromosomes or asymmetrical distribution of chromosome-specific centromere signals in the nuclei of BN cells); nuclear elimination of amplified DNA and/or DNA repair

Genotoxicity Biomarkers: Application in Histopathology Laboratories 139

Ideally, repeat sampling, at least once every 7 days after acute exposure, should be performed for 28 days or more so that the kinetics and extent of biomarker induction can be thoroughly investigated. In the case of chronic exposure due to habitual diet or alcohol consumption or smoking it is recommend that multiple samples are taken at least once

The uniformity of sampling is one of the many aspects to consider; therefore a circular expanding motion is used with toothbrush sampling to enhance sampling over a greater area and to avoid continual erosion in a single region of the BM. This is performed on the inside of both cheeks using a different brush for sampling left and right areas of the mouth to maximize cell sampling and to eliminate any unknown biases that may be caused by sampling one cheek only. It is important to note that repeated vigorous brushing of the same area can lead to increased collection of cells from the less differentiated basal layer. About transportation, in some investigations buccal cells may have to be collected from a distant site which may cause sample deterioration. About cell fixation, there are many possible alternatives of fixatives such as methanol: glacial acetic acid (3:1), 80% methanol or ethanol: glacial acetic (3:1). The staining technique recommend is Feulgen because is a DNAspecific stain and because permanent slides can be obtained that can be viewed under both transmitted and/or fluorescent light conditions. There are many false-positive results in MN frequency as a result of using Romanowsky-type stains such as Giemsa, May-Grunwald Giemsa and/or Leishmann's which leads to inaccurate assessment of DNA damage. Romanowsky stains have been shown to increase the number of false positives as they positively stain keratin bodies that are often mistaken for MN and are therefore not appropriate for this type of analysis. For these reasons, it is advisable to avoid Romanowsky stains in favour of DNA-specific fluorescent-based stains such as propidium iodide, DAPI,

The criterion of scoring is originally based in the described by Tolbert et al. that are intended for classifying buccal cells into categories that distinguish between "normal" cells and cells that are considered "abnormal" on the basis of cytological and nuclear features, which are indicative of DNA damage, cytokinetic failure or cell death. Therefore, some definitions of

Normal "differentiated" cells have a uniformly stained nucleus, which is oval or round in shape. They are distinguished from basal cells by their larger size and by their smaller nucleus-to-cytoplasm ratio. No other DNA-containing structures apart from the nucleus are observed in these cells. These cells are considered to be terminally differentiated relative to

Cells with MN are characterized by the presence of both a main nucleus and one more smaller nuclear structures called MN. The MN are round or oval in shape and their diameter should range between 1/3 and 1/16 of the main nucleus. MN has the same staining intensity and texture as the main nucleus. Most cells with MN will ontain only one MN but it is possible to find cells with two or more MN. Baseline frequencies for micronucleated cells in the BM are usually within the 0.5-2.5 MN/1000 cells range. Cells with multiple MN are rare in healthy subjects but become more common in individuals

Cells with nuclear buds contain nuclei with an apparent sharp constriction at one end of the nucleus suggestive of a budding process, i.e. elimination of nuclear material by budding.

every 3 months to take into account seasonal variation (Thomas et al., 2009).

Feulgen, Hoechst 33258 or Acridine Orange (Thomas et al., 2009).

basal cells, as no mitotic cells are observed in this population.

the cytological findings are (Thomas et al., 2009):

exposed to radiation or other genotoxic events.

complexes (NBUD); chromosomal instability phenotype and breakage-fusion-bridge cycles (simultaneous expression of MN, NPB and NBUD); altered mitotic activity and/or cytostasis (NDI) and cell death by necrosis or apoptosis (ratios of necrotic and apoptotic cells) (Fenech, 2007).
