**1. Introduction**

132 Biomarker

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Most cancers results from man-made and natural environmental exposures (such as tobacco smoke; chemical pollutants in air, water, food, drugs; radon; and infectious agents) acting in concert with both genetic and acquired characteristics. It has been estimated that without these environmental factors, cancer incidence would be dramatically reduced, by as much as 80%-90% (Perera, 1996). The modulation of environmental factors by host susceptibility was rarely evaluated. However, within the past few years, the interaction between environmental factors and host susceptibility factors has become a very active area of research (Perera, 2000). Molecular biology as a tool for use in epidemiological studies has significant potential in strengthening the identification of cancers associated with environmental exposures related to lifestyle, occupation, or ambient pollution. In molecular epidemiology, laboratory methods are employed to document the molecular basis and preclinical effects of environmental carcinogenesis (Portier & Bell, 1998).

Molecular epidemiology has become a major field of research and considerable progress has been made in validation and application of biomarkers and its greatest contribution has been the insights provided into interindividual variation in human cancer risk and the complex interactions between environmental factors and host susceptibility factors, both inherited and acquired, in the multistage process of carcinogenesis (Perera, 2000).

The possibility to use a biomarker to substitute classical endpoints, such as disease incidence or mortality is the most promising feature and one that is most likely to affect public health. The use of events that are on the direct pathways from the initiation to the occurrence of disease to surrogate the disease incidence is a very appealing approach, which is currently investigated in different fields (Bonassi & Au, 2002).

Biological monitoring of workers has three main aims: the primary is individual or collective exposure assessment, the second is health protection and the ultimate objective is occupational health risk assessment. It consists of standardized protocols aiming to the periodic detection of early, preferably reversible, biological signs which are indicative, if compared with adequate reference values, of an actual or potential condition of exposure,

Genotoxicity Biomarkers: Application in Histopathology Laboratories 135

in a biological system or sample. The traditional, generally accepted classification of biomarkers into three main categories - biomarkers of exposure, effect, and susceptibility;

A biomarker can potentially be any substance, structure or process that could be monitored in tissues or fluids and that predicts or influences health, or assesses the incidence or biological behaviour of a disease. Identification of biomarkers that are on causal pathway, have a high probability of reflecting health or the progression to clinical disease, and have the ability to account for all or most of the variation in a physiological state or the preponderance of cases of the specified clinical outcome, have largely remained elusive

A biomarker of exposure is a chemical or its metabolite or the product of an interaction between a chemical and some target molecule or macromolecule that is measured in a

A biomarker of effect is a measurable biochemical, structural, functional, behavioural or any other kind of alteration in an organism that, according to its magnitude, can be associated with an established or potential health impairment or disease. A sub-class of biomarkers of

A biomarker of susceptibility may be defined as an indicator of an inherent or acquired ability of an organism to respond to the challenge of exposure to a chemical (Manno et al.,

Although the different types of biomarkers are considered for classification purposes, as separate and alternative, in fact it is not always possible to attribute them to a single category. The allocation of a biomarker to one type or the other sometimes depends on its toxicological significance and the specific context in which the test is being used (Manno et

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

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,

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

although cells such as buccal epithelium are also utilized (Battershill et al., 2008).

chromatid exchanges or chromosomic aberrations (Ishikawa et al., 2003).

depending on their toxicological significance (Manno et al., 2010).

compartment or a fluid of an organism (Manno et al., 2010).

effect is represented by biomarkers of early disease (Manno et al., 2010).

(Davis et al., 2007).

2010).

al., 2010).

**2.1 Genotoxicity biomarkers** 

chemicals (Manno et al., 2010).

effect or susceptibility possibly resulting in health damage or disease. These signs are referred to as biomarkers (Manno et al., 2010).

There has been dramatic progress in the application of biomarkers to human studies of cancer causation. Progress has been made in the development and validation of biomarkers that are directly relevant to the carcinogenic process and that can be used in large-scale epidemiologic studies (Manno et al., 2010).

There are many important aspects to consider when a biomonitoring study is designed. For instance, there is needed a detail information on genotoxin exposure, e.g. type of toxin, duration of exposure, commencing date of exposure relative to sampling date of buccal cells, in order to achieve a meaningful interpretation of data. It will also helps to identify key variables affecting the observed frequency of biomarkers, like age, gender, vitamin B status, genotype and smoking status (Thomas et al., 2009).

Based on the impact on genotoxicity biomarkers in peripheral blood lymphocytes on the design of biomonitoring studies, Battershill et al. (2008) study have considered a strong/sufficient correlation between micronucleus (MN) frequency and increasing age. The effect is more pronounced in females than in males, with the increase more marked after 30 years of age. There are studies that also demonstrated a strong correlation between age and MN frequency and suggested that chromosome loss is a determining factor in this increase.

In what concern to gender, is also documented a gender difference in the background incidence of MN in peripheral blood lymphocytes (PBL), with the frequency being consistently higher in females. A study that assessed MN, chromosomal aberrations and sister chromatid exchange showed highly significant elevations in MN in lymphocytes of women (29% when adjusted for age and smoking) whereas chromosomal aberrations and sister chromatid exchange remained unchanged. This may reflect aneuploidy detected in MN assays (Battershill et al., 2008).

In respect to smoking, although the link between smoking and cancer is strong and exposure to genotoxic carcinogens present in tobacco smoke has been convincingly demonstrated, interestingly the same convincing association is less apparent when assessing biomonitoring studies of genotoxicity. HUMN project study about tobacco smoke, the majority of the laboratories showed no significant differences between smokers and nonsmokers and the pooled analysis, interestingly, indicated an overall decrease for all smokers compared to controls (Battershill et al., 2008).

It was verified a weak/insufficient evidence for association with genotoxicity end points and alcohol consumption. Alcohol consumption has been causally associated with cancer at a number of sites (e.g. head and neck cancer). Alcoholic beverages have not been reported to induce mutagenic effects in rodents. The evidence regarding an effect of drinking alcoholic beverages on increased MN or substitute for chromosomal aberrations formation in PBL is inconclusive (Battershill et al., 2008).
