**5. The comet assay**

244 Selected Topics in DNA Repair

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

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

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;

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

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

possible cancer chemoprevention strategy (Heo *et al.*, 2001; WCRF, 2007).

mechanism by which they prevent cancer?

Mehta *et al.*, 2010).

studies (Davis and Milner, 2007).

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

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

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

DNA Damage Protection and Induction of Repair

scoring (Collins et al., 2008; Nossoni, 2008).

modifications will be explained below.

**5.2 The use of lesion-specific enzymes** 

by Dietary Phytochemicals and Cancer Prevention: What Do We Know? 247

During electrophoresis, DNA loops containing breaks migrate towards the anode forming in the end DNA structures like a comet, with a head (the nuclear region) and a tail that contain DNA loops that extended during electrophoresis due to breaks. DNA migration is dependent of several parameters, such as, concentration of agarose in the gel, pH, temperature and duration

After electrophoresis, slides are neutralized using neutralization buffer, stained with fluorescent agent (e.g. ethidium bromide, SYBRGold), and analyzed (scored) using a fluorescent microscope. The scoring may be done by visual scoring or by computer programs. In visual scoring the researcher scores at least one hundred comets using the following classification: 0 to comet without DNA in tail; 1, 2 and 3 with increasing amount of DNA in tail and 4 to comet were DNA is almost all in the tail. In the end the score of each sample changes between 0 and 400 (arbitrary units). An alternative methodology are the computer programs that allow to measure different parameters of the comets such as tail intensity, tail length, intensity of head, and tail moment. Tail intensity corresponds to the percentage of DNA in the tail of the comet and is the most used parameter. Intensity of tail fluorescence indicates the extent of damage. It is important to use positive and negative controls as well as to blind

Comet assay under standard conditions reflects endogenous DNA damage such as single and double strand breaks and apurinic/apyrimidinic (AP) sites in almost any eukaryotic cell population. There are other modifications that make it even more sensitive and allow to measure oxidised pyrimidines and purines and alkylation DNA damage. These

Alkaline version (described above) measures strand DNA breaks and AP sites (that are converted to strand breaks). However, genotoxic agents not only induce breaks and AP sites, but also DNA damage such as base oxidation and others base modifications, that are generated in large scale in cells. Several DNA repair enzymes recognize damaged bases, introducing breaks at sites of the base damage. Thus, inclusion of an extra step of nucleoid DNA digestion with lesion-specific enzymes following lysis, allow detection of modified bases increasing the sensitivity and specificity of the comet assay (Collins, 2009; Hoelzl et al., 2009). Endonuclease III (EndoIII) was the first enzyme used to recognize oxidized pyrimidines in DNA and to remove them, leaving an AP site that is subsequently converted in breaks at pH13. These breaks that occur at sites of base oxidation increase comet tail intensity (Collins *et al.*, 1993). Formamidopyrimidine DNA glycosylase (FPG) recognizes and breaks modified purines as well as 8-oxoguanine and also ring-opened purines, or formamidopyrimidines (Fapy) (Dusinska and Collins, 1996). T4 endonuclease V recognise UV-induced cyclobutane pyrimidine dimers (Collins *et al.*, 1997b). AlkA is a bacterial repair enzyme whose main substrate is the N3-MeA, an alkylated base and converts it to AP sites (Collins *et al.*, 2001a). The use of repair enzymes has been particularly useful in estimating oxidative damage of certain pollutants and drugs in several experimental models and in biomonitoring studies, for example to evaluate the role of dietary agents in protection against oxidative DNA damage. However, the specificity of the enzymes is limited, for instance FPG recognizes 8-

oxoGua but also detects alkylation damage (N7 MeG) (Speit *et al.*, 2004).

After lysis, in parallel with a slide incubated with a lesion-specific enzyme, a slide incubated without enzyme (only with buffer) is used as a control. Subtraction of control (which contain

of alkaline unwinding, temperature, voltage, and duration of electrophoresis.

published using this assay. Comet assay is now used in different research areas such as human and environmental biomonitoring, mechanistic studies of DNA repair, genetic toxicology, nutrition and clinical studies. Below is a detailed description of the standard comet assay and new modifications to detect different DNA damages and DNA repair capacities (fig.1).

Fig. 1. General steps of the standard comet assay and its modifications.
