**2.1 Genotoxicity biomarkers**

As a subtype of biomarkers of effect there are biomarkers of genotoxicity, generally used to measure specific occupational and environmental exposures or to predict the risk of disease or to monitor the effectiveness of exposure control procedures in subjects to genotoxic chemicals (Manno et al., 2010).

Cytogenetic biomarkers are the most frequently used endpoints in human biomonitoring studies and are used extensively to assess the impact of environmental, occupational and medical factors on genomic stability (Barrett et al., 1997; Battershill et al., 2008) and lymphocytes are used as a surrogate for the actual target tissues of genotoxic carcinogens (Barrett et al., 1997). The evaluation of MN in PBL is the most commonly used technique, although cells such as buccal epithelium are also utilized (Battershill et al., 2008).

MN assay is one of the most sensitive markers for detecting DNA damage, and has been used to investigate genotoxicity of a variety of chemicals. MN testing with interphase cells is more suited as a cytogenetic marker because it is not limited to metaphases, and has the advantage of allowing rapid screening of a larger numbers of cells than in studies with sister chromatid exchanges or chromosomic aberrations (Ishikawa et al., 2003).

Genotoxicity Biomarkers: Application in Histopathology Laboratories 137

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

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

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,

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

only (Fenech, 2007 ; Thomas et al., 2003).

2005, 2007; Umegaki & Fenech, 2000).

al., 2007).

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