**5.1 Standard comet assay**

Among the various versions of the comet assay, the alkaline (pH of the unwinding and electrophoresis buffer >13) is the most used.The first step is to cover microscope slides with 1% of normal melting point (NMP) agarose and dry it at room temperature. A second layer composed by cells embedded in 0.5% LMP agarose (at 37ºC) is spread above the first layer, covering it with a cover glass and keeping at 4ºC during few minutes. After removing cover glass, slides are lysed at least for one hour up to 24h in lysing solution containing detergent and high molarity NaCl (2.5M NaCl, 100mM Na2EDTA, 10mM Tris Base, pH 10 plus 1% Triton X-100). This solution removes membranes and soluble cellular (300mM NaOH, 1mM Na2EDTA, pH >13) constituents, as well as histones, producing nucleoids of supercoiled DNA attached to the nuclear matrix. DNA unwinding occur in alkaline conditions (pH>13) immersing slides in alkaline buffer at 4ºC. The time required for unwinding changes based on the cells being examined. In this step, breaks in the DNA relax the supercoiling and allow DNA loops to expand. Electrophoresis occurs under alkaline condition at 0.8V/cm and 300 mA for 20 min.

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.

Among the various versions of the comet assay, the alkaline (pH of the unwinding and electrophoresis buffer >13) is the most used.The first step is to cover microscope slides with 1% of normal melting point (NMP) agarose and dry it at room temperature. A second layer composed by cells embedded in 0.5% LMP agarose (at 37ºC) is spread above the first layer, covering it with a cover glass and keeping at 4ºC during few minutes. After removing cover glass, slides are lysed at least for one hour up to 24h in lysing solution containing detergent and high molarity NaCl (2.5M NaCl, 100mM Na2EDTA, 10mM Tris Base, pH 10 plus 1% Triton X-100). This solution removes membranes and soluble cellular (300mM NaOH, 1mM Na2EDTA, pH >13) constituents, as well as histones, producing nucleoids of supercoiled DNA attached to the nuclear matrix. DNA unwinding occur in alkaline conditions (pH>13) immersing slides in alkaline buffer at 4ºC. The time required for unwinding changes based on the cells being examined. In this step, breaks in the DNA relax the supercoiling and allow DNA loops to expand. Electrophoresis occurs under alkaline condition at 0.8V/cm and 300 mA for 20 min.

**5.1 Standard comet assay** 

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

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

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 modifications will be explained below.

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

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

DNA Damage Protection and Induction of Repair

of damage (Shaposhnikov et al., 2010).

Moon *et al.*, 2006; Ross and Kasum, 2002).

and anthocyanidins (Fig.2).

**6.1.1 Polyphenols** 

and Wong *et al.*, 2005).

by Dietary Phytochemicals and Cancer Prevention: What Do We Know? 249

samples, and also when is necessary to use several repair enzymes to detect different kinds

Application of the comet assay to the study of the protective effects of diet against oxidative/alkylating DNA damage and repair will be summarized bellow. For more details see the following revisions, (Hoelzl et al., 2009; Moller and Loft, 2002; Wasson *et al.*, 2008

Prevention of DNA damage is one of the cellular mechanisms that may prevent cancer. Diet plays an important role in regulating DNA damage for instance by modulating the antioxidant/oxidant balance. The protective effect observed in many studies could be due, in part, to the presence of phenolic and/or non-phenolic constituents that have ability to act as antioxidant by free radical scavenging and chelating metal ions (Anderson *et al.*, 2000); (Ross and Kasum, 2002). They can also act as indirect antioxidant by increasing levels of antioxidants such as glutathione (GSH) and/or by increasing the activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD) (Alia *et al.*, 2005; Lima *et al.*, 2005; Lima *et al.*, 2006; Frei and Higdon, 2003). Phytochemicals can also modulate phase I (activating) enzymes and phase II (detoxifying) enzymes involved in xenobiotic metabolism (Chen and Kong, 2004; Ferguson *et al.*, 2004;

Phytochemicals are bioactive compounds present in plants where they are produced as secondary metabolites to protect themselves from several pathogenic agents. Their consumption by humans confers protection against some diseases. Most bioactive phytochemicals belong to one of 5 groups: polyphenols, carotenoids, alkaloids, nitrogencontaining compounds, and organosulfur compounds. Polyphenols and carotenoids include several hundreds of compounds and are the most studied groups. Polyphenols may be further classified into 4 groups, according with the number of phenol rings that they contain: Flavonois, phenolic acids, stilbenes, and lignans. The flavonoids may themselves be classified into the follow subgroups: flavonols, flavones, isoflavones, flavanones, flavanols

Numerous studies have shown the antioxidant and DNA protective properties of polyphenols. Quercetin is a flavonoid found in a variety of foods including apples, onions, wine and tea. Several studies, including our own showed the protective effects of quercetin against oxidative DNA damage in HepG2 cells (Lima et al., 2006; Ramos et al., 2008), Caco-2 cells (Aherne and O'Brien, 1999), HMB-2 cells (Horvathova *et al.*, 2005), human macrophage (U-937 cells) (Kanupriya et al., 2006; Moon et al., 2006); human lymphocytes (healthy volunteers) (Wilms *et al.*, 2007), and murine leukemia L1210 cells (Horvathova *et al.*, 2003). Wilms *et al.*, (2005) reported the protective effects of quercetin against the formation of oxidative DNA damage and bulky DNA adducts in human lymphocytes, induced by H2O2 and benzo(a)pyrene (B(a)P, respectively. In the same study, results obtained in an in vivo experiments showed that four weeks of quercetin-rich blueberry/apple juice mixture

**6. Application of the comet assay in chemoprevention studies** 

**6.1 Effect of diet on prevention of oxidative DNA damage** 

SBs and AP sites) to the condition treated with enzyme gives a value that correspond to the damage recognized by the enzyme and is usually referred as 'netenzyme-sensitive sites'.

#### **5.3 DNA repair assays**

Beside effects on protection against DNA damage, comet assay was also developed to assess DNA repair ability of the cells and effects of diet on DNA damage repair rates. For that, two different methodologies are frequently used:


#### **5.4 Other modifications**

The format of comet assay most used is 2 gels per slide. To large numbers of samples new formats have been developed, such as 12 gels per slide, that have several advantages: 1) reduces the number of cells required for each gel (approx. 200 cells/gel); 2) allows different conditions in the same slide (different genotoxic chemicals; enzymes or cell extracts); 3) requires low volume of solutions; 4) increases the number of samples processed at one time. This new format has been used in human biomonitoring studies with a great number of samples, and also when is necessary to use several repair enzymes to detect different kinds of damage (Shaposhnikov et al., 2010).
