**3. Technical information on comet assay**

DNA damage can simply be evaluated using Comet assay that allows the measurement of DNA single- and double-strand breaks (frank strand breaks and incomplete excision repair sites) together with alkali labile sites and crosslinking. By choosing different pH conditions for electrophoresis and the preceding incubation, different levels of damage can be assessed. The degree of DNA migration can be correlated to the extent of DNA damage occurring in each single cell. *In vitro* studies can be performed on virtually with any cell type; however, the cell-type-of-choice in biomonitoring is mostly the lymphocyte because blood is easily

Fig. 1. Advantages and disadvantages of Comet assay

**Baliga et al., 2007)**. The field of antioxidants and free radicals is often perceived as focusing around the use of antioxidant supplements to prevent human disease. Currently, there is a growing interest in environmental chemicals that can cause oxidative stress. The genotoxic effects of some compounds are of particular interest for researchers as humans are exposed to these chemicals abundantly. Exposure to such chemicals may result in disturbances of several physiological processes and may lead to wide variety of degenerative diseases

First described by **Östling & Johanson (1984)**, and then modified by **Singh et al. in 1988,** the single cell gel electrophoresis assay (also known as Comet assay) is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It has since gained in popularity as a standard technique for evaluation of

Comet assay can easily detect the *in vitro* toxicity of environmental chemicals on different cell types, as well as *in vivo* toxicity in tissue samples obtained from animals. Besides, it is also a valid technique to evaluate whether antioxidants/micronutrients are able to protect the integrity of the genetic material **(Anderson et al., 1997; Heaton et al., 2002; Novotna et** 

 Sensitivity for detecting low levels of DNA damage: The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell and will lose sensitivity

Requirement for small number of cells per sample: <10,000 cells are enough to perform

 Flexibility: Comet assay is applicable to virtually any type of cell, as long as a single cell suspension is obtained. Besides, different combinations of unwinding and electrophoresis conditions and lesion-specific enzymes can be used to detect different

Studies can be conducted using relatively small amounts of a test substance **(Anderson** 

DNA damage can simply be evaluated using Comet assay that allows the measurement of DNA single- and double-strand breaks (frank strand breaks and incomplete excision repair sites) together with alkali labile sites and crosslinking. By choosing different pH conditions for electrophoresis and the preceding incubation, different levels of damage can be assessed. The degree of DNA migration can be correlated to the extent of DNA damage occurring in each single cell. *In vitro* studies can be performed on virtually with any cell type; however, the cell-type-of-choice in biomonitoring is mostly the lymphocyte because blood is easily

DNA damage/repair, biomonitoring and genotoxicity testing **(Singh et al., 1988)**.

**2. Why comet assay is a suitable tool for antioxidant research?** 

The benefits of Comet assay can be summarized as below:

above about 10,000 breaks per cell **(Olive & Banáth, 2006)**.

types and levels of DNA damage **(Wong et al., 2005)**. Low cost and ease of application **(Anderson et al., 1997).**

A relatively short time is needed to complete an experiment.

**3. Technical information on comet assay** 

The advantages and disadvantages of Comet assay are shown in **Figure 1**.

including cancer **(Soory, 2009)**.

**al., 2007)**.

the assay.

**et al., 1997)**

collected and lymphocytes have proved to be good surrogate cells. For example, lymphocytes exhibited genotoxicity caused by anticancer agents targeting several different organs **(Faust et al., 2004)**.

There are differences between laboratories in the isolation of lymphocytes, cells from organs/tissues or other specimens, or in the solutions used for electrophoresis. A simple alkaline Comet assay protocol can be performed in the following steps:


Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 417

A new technique "Comet fluorescence in situ hybridization (Comet FISH)" combines two well-established methods. The Comet assay comprises the basis of Comet-FISH and allows separation of fragmented from nonfragmented DNA and quantification of DNA damage and repair. FISH enables detection of specifically labeled DNA sequences of interest, including whole chromosomes. The combined technique of Comet-FISH is a modification of the Comet assay that inserts a hybridization step after unwinding and electrophoresis and permits the labeling of specific gene sequences or telomeres. Comet-FISH has been applied for detection of site-specific breaks in DNA regions that are relevant for development of various diseases, and has also been used to study the distribution of DNA damage and repair in the complete genome. Moreover, DNA sequence modifications can be detected in individual cells using Comet-FISH. The results from the Comet assay alone are only reflections of overall DNA damage. However, the addition of the FISH technique allows the assignment of the probed sequences to the damaged or undamaged part of the comet (tail or

A spesific illustration for alkaline Comet assay methodology is shown in **Figure 2**. Different

In this chapter, I will mainly focus on the genotoxicity of different environmental chemicals and both *in vivo* and *in vitro* protection studies by several selenocompounds, vitamins, and

There is considerable interest in developing strategies that prevent genotoxicity and cancer with minimal risk or toxicity. Trace elements like selenium (Se) are of particular interest as it

The requirement for Se and its beneficial role in human health have been known for several decades. Se is an essential trace element commonly found in grains, nuts, and meats and many years of research showed that that low, non-toxic supplementation with either organic and inorganic forms could reduce cancer incidence following exposure to a wide variety of

Along with its important role for the cellular antioxidant defense, Se is also essential for the production of normal spermatozoa and thus plays a critical role in testis, sperm, and reproduction **(Flohé, 2007)**. In the physiological dosage range, Se appears to function as an antimutagenic agent, preventing the malignant transformation of normal cells and the activation of oncogenes (**Schrauzer, 2000**). Although most of its chemopreventive mechanisms still remain unclear, the protective effects of Se seem to be primarily associated with its presence in the glutathione peroxidases (GPxs), which are known to protect DNA and other cellular components from damage by oxygen radicals **(Negro, 2008)**. Low activity of another important peroxidase, GPx4, can lead to reduction in reproduction **(Flohé, 2007)**. Selenoenzymes are known to play roles in carcinogen metabolism, in the control of cell division, oxygen metabolism, detoxification processes, apoptosis induction and the functioning of the immune system oncogenes (**Schrauzer, 2000**). Several studies have determined the low activity of Se-containing cytosolic GPx, known as GPx1, as a substantial

head, respectively) **(Schlörmann & Glei, 2009)**.

**4. Protection studies using comet assay** 

**4.1 Prevention of genotoxicity by selenocompounds** 

is the key component of antioxidant enzyme systems.

carcinogens **(El-Bayoumy, 2004)**.

protocols of Comet assay in research field are given in **Figure 3**.

isothiocyanates (ITCs) against the toxicity of these compounds.

immersed in freshly prepared alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2-EDTA, pH 13) for 30 min to allow DNA unwinding.


Rather than making use of the cell's own repair enzymes to reveal damage, we can achieve greater specificity and higher sensitivity by treating the DNA with purified repair enzymes which will convert particular lesions into breaks. Thus, Comet assay protocol can also be performed using different base or nucleotide excision repair enzymes **(Collins et al., 1997)**. The most commonly used repair enyme is formamidopyrimidine DNA glycosylase (Fpg) which recognizes and removes 8-oxodeoxyguanosine (8-oxoGua) and other oxidized purines. 8-oxoguanine glycosylase (OGG1) also recognizes 8-oxoGua. Endonuclease III (Endo III) deals with oxidized pyrimidines; and T4 endonuclease V is able to incise at sites of pyrimidine dimers. Digestion with these enzymes is carried out after the initial lysis step. The excision repair pathways act more slowly than strand break rejoining **(Collins & Horvathova, 2001)**, and samples should be taken over a period of a few hours.

Different versions of Comet assay are also used for different puposes. Neutral Comet assay is usually used for assessing double strand DNA breaks in sperm cells. On the other hand, a "Comet Chip" protocol, first introduced by Massachusetts Institute of Technology (MIT) Engelward Lab**,** is nowadays gaining significant importance as a high throughput DNA damage analysis platform. This new method is also used for evaluating DNA strand breaks, sites of DNA modification and interstrand crosslinks. A limitation of the traditional assay is that each sample requires a separate glass slide and image analysis is laborious and data is intensive, thus reducing throughput. This new technique uses microfabrication technologies to enable analysis of cells within a defined array, resulting in a >200 fold reduction in the area required per condition. Each well of a 96-well plate contains patterned microwells for single cell capture and DNA damage quantification. The "CometChip" can be used to analyze dozens of conditions on a single chip. The newly developed automated image analysis software is used for detection of DNA damage, thus greatly reducing analysis time. This new technology will enable the researchers to conduct both large scale epidemiological and clinical studies **(Engelward Lab, 2011)**.

A new technique "Comet fluorescence in situ hybridization (Comet FISH)" combines two well-established methods. The Comet assay comprises the basis of Comet-FISH and allows separation of fragmented from nonfragmented DNA and quantification of DNA damage and repair. FISH enables detection of specifically labeled DNA sequences of interest, including whole chromosomes. The combined technique of Comet-FISH is a modification of the Comet assay that inserts a hybridization step after unwinding and electrophoresis and permits the labeling of specific gene sequences or telomeres. Comet-FISH has been applied for detection of site-specific breaks in DNA regions that are relevant for development of various diseases, and has also been used to study the distribution of DNA damage and repair in the complete genome. Moreover, DNA sequence modifications can be detected in individual cells using Comet-FISH. The results from the Comet assay alone are only reflections of overall DNA damage. However, the addition of the FISH technique allows the assignment of the probed sequences to the damaged or undamaged part of the comet (tail or head, respectively) **(Schlörmann & Glei, 2009)**.

A spesific illustration for alkaline Comet assay methodology is shown in **Figure 2**. Different protocols of Comet assay in research field are given in **Figure 3**.

In this chapter, I will mainly focus on the genotoxicity of different environmental chemicals and both *in vivo* and *in vitro* protection studies by several selenocompounds, vitamins, and isothiocyanates (ITCs) against the toxicity of these compounds.
