**4. Chemopreventive activities of dietary phytochemicals**

Diet and lifestyle play crucial roles in cancer aetiology. Nowadays, the idea that prevention of any disease is preferable over treatment is accepted by all. In this context, in the last decades, several studies suggest that regular consumption of fruits, vegetables and spices have health benefits including risk reduction of developing a cancer, namely, colon cancer (Terry et al., 2001). Much of the protective effects have been attributed to phytochemicals such as polyphenols, terpenes and alkaloids, present in low levels in plants (Barth et al., 2005). For instance, flavonoids (polyphenolic compounds) have been reported to possess potential on prevention of several cancers specially cancers of gastrointestinal tract, like oral cavity and colon cancer (Kuo, 1996). The World Cancer Research Fund (WCRF) in its report about diet and prevention of cancer in 2007, mentioned that fruits and vegetables in general probably protect against cancers of the mouth, pharynx, larynx, oesophagus, lung, and stomach and there are limited evidences that suggest protective effects of fruits against cancers of the nasopharynx, pancreas, liver, and colorectum (WCRF, 2007).

The use of plants for the prevention of diseases is an ancient practice. However, it was in the last decades that scientific community started to show interest in the medicinal properties of plants. The first scientific evidences showing that vegetables and fruits might be protective against some cancers emerged in the 1990s. Nevertheless, twenty year on no consensus

DNA Damage Protection and Induction of Repair

biomarker of exposure assessed by comet assay.

**5. The comet assay** 

by Dietary Phytochemicals and Cancer Prevention: What Do We Know? 245

of dietary components is sufficient to lead to a certain biological response; biomarkers of effect, which give information about the mechanisms of action of dietary components; and biomarkers of susceptibility, which indicate which individuals are susceptible to specific dietary exposures (Davis and Milner, 2007). In this review, we will be focus in one

The comet assay, also called the single cell gel electrophoresis (SCGE) assay was first introduced by Ostling and Johanson in 1984 as a microelectrophoretic technique for the direct visualization of DNA damage in individual cells. In this assay, cells embedded in agarose are placed on a microscope slide, lysed by detergents in high salt solution and submitted to electrophoresis under neutral conditions. It usually accepted that, in neutral condition, DNA migration is due to presence of double-strand breaks (DSB). However, it was demonstrated that DSB as well as single strand breaks (SSB) were detected in this conditions (Collins *et al.*, 1997a; Gedik *et al.*, 1992; Ostling and Johanson, 1984). Singh *et al.*, (1988) introduced the electrophoresis under alkaline (pH >13) conditions for detecting DNA damage in single cells. At alkaline conditions, DNA migration is associated with the presence of strand breaks (single and/or double strand), SSB associated with incomplete excision repair sites, and alkali-labile sites (ALS). The alkaline version of comet assay had more success because it allows the detection of a wide spectrum of damages, and in fact almost all genotoxic agents

Among the several methods to measure DNA damage including classical cytogenetic tests such as chromosome aberrations, micronuclei and sister chromatid exchanges, the comet assay has become the most commonly used. This assay shows some advantages relatively to other genotoxicity assays such as: 1) evaluates DNA damage at individual cell; 2) requires a small number of cells per sample; 3) any animal tissue can be used, since single cell/nucleus suspension can be obtained; 4) proliferating or non-proliferating cells can be used; 5) detects low levels of DNA damage (high sensitivity); 6) needs small amounts of a test substance; 7) detects several classes of DNA damage such as DSB, SSB, ALS, incomplete repair of a-basic sites and cross-links; 8) low costs; 9) simple and fast tool (Hartmann et al., 2003; Speit *et al.*, 2003). Despite great advantages, some limitations have been attributed to the comet assay: it does not detect high level of DNA damage and DNA fragments smaller than 50 kb, and therefore apoptotic cells detection is very difficult (Nossoni, 2008). The comet assay done with lymphocytes is an important biomarker for early biological effects of exposure to environmental mutagenic agents (Dusinska and Collins, 2008). Angerer *et al.*, (2007) in a review about human biomonitoring refer, however, some problems that should be kept in mind when lymphocytes are used. The major difficulty is the interpretation of data, because the damage levels and capacity to repair of these cells may be different from cells of others tissues. Usually lymphocytes repair their damage very slowly and not all the damage to cells and organs are detectable using lymphocytes. Furthermore, a great intra-individual and inter-individual variability of the basal level of DNA damage it was found that is influenced by a variety of factors such as lifestyle, diet, medication, air pollution, season, climate or exercise. Lymphocytes also show limited survival in vitro, requiring incubation

In the last decade, scientific community demonstrated an increasing interest in the alkaline version of comet assay that has brought a rapid increase in the number of papers and reports

induce more SSB and/or ALS than DSB (Fairbairn *et al.*, 1995; Tice et al., 2000).

with a mitogen such as phytohaemagglutinin (Collins et al., 2008).

exists about the real role of diet on cancer prevention, and many questions remain to be answered. Which component(s) of the diet is (are) responsible for the protective effects? Are the protective effects the result of the interactions between different components? What type of interactions exists between them (e.g. synergistic, antagonistic interaction)? What is the mechanism by which they prevent cancer?

Dietary agents have different structural features that are responsible for a great variety of biological activities such as anti-inflammatory, antioxidant, free radical scavenging, antimutagenic and enzyme modulating activities. These activities may be responsible for the possible chemopreventive effects of natural compound. Modulation of diet can be used as a possible cancer chemoprevention strategy (Heo *et al.*, 2001; WCRF, 2007).

Chemoprevention is the process of using natural or synthetic compounds to block, reverse, or prevent the development of cancers through the action on multiple cellular mechanisms. Generally, these cellular mechanisms can be grouped in two: 1) Anti-mutagenesis, that includes the inhibition of the uptake, formation/activation of carcinogens, their detoxification, the blockage of carcinogen–DNA binding, and the enhancement of fidelity of DNA repair; 2) Anti-proliferation/anti-progression, that includes modification of signal transduction pathways, inhibition of oncogene activity, promotion of the cellular differentiation, enhancement of apoptosis, inhibition of inflammation and angiogenesis, and modulation of hormone/growth factor activity (Davis, 2007; Moon Y. Yeo et al., 2001). Phytochemicals may alter multiple molecular targets within a specific biological process related with cancer and when in combination with other natural compounds can have an additive or synergistic effect as well as antagonistic interactions. Nowadays, it is accepted that the combination of foods and/or multiple natural compounds may offer increased chemoprevention against cancer as compared to isolated compounds. However, the interactions between the different compounds within the food or with other foods need to be clarified. Furthermore, active compounds of many plants remain uncharacterized, which restrict the knowledge about the role of diet on cancer prevention (Davis and Milner, 2007; Mehta *et al.*, 2010).

In chemoprevention studies several experimental models can be used. However, experience shows that the results may be different depending on the experimental model used and whether the whole plants evaluated or only isolated compounds. Data from cultured cells and animal models may not reflect the response in humans. Also, plants and their isolated compounds may not have similar biological effects (Davis and Milner, 2007; Mehta *et al.*, 2010). Chemopreventive effect of food and/or its compounds depend on absorption, metabolism, distribution and excretion of phytochemicals. Phytochemicals' absorption is dependent on source and the method of food processing. In the same plant species the phytochemicals contents may change depending on the plant genotype, the season of the year and the place where the plant was grown. Intensity and duration of the exposure to dietary components also influence the cellular response. Thus, dose and duration of exposure become fundamental considerations in interpreting findings from nutritional studies (Davis and Milner, 2007).

During the last decades, some long-term intervention studies have been performed to understand the contribution of diet on prevention of diseases. However, this type of studies has the inconvenience of the high time consumption and cost. Several biomarkers have been validated to predict cancer risk and to evaluate the potential chemopreventive effect of food and/or its compounds (Davis and Milner, 2007). In general, biomarkers can be divided in three major types: biomarkers of exposure, which allow the evaluation of whether the intake of dietary components is sufficient to lead to a certain biological response; biomarkers of effect, which give information about the mechanisms of action of dietary components; and biomarkers of susceptibility, which indicate which individuals are susceptible to specific dietary exposures (Davis and Milner, 2007). In this review, we will be focus in one biomarker of exposure assessed by comet assay.
