**4. Protection studies using comet assay**

416 Gel Electrophoresis – Advanced Techniques

**f.** After electrophoresis, slides are rinsed three times for 5 min with neutralization buffer (0.4 M Tris-HCl, pH 7.4), and stained with ethidium bromide (20 μg/ml) in PBS. Ethidium bromide is an intercalating agent commonly used as a fluorescent nucleic acid stain in molecular biology. There are a number of alternative stains to ethidium bromide, including acridine orange, propidium iodide, YOYO-1 iodide stain, SYBR Gold nucleic acid gel stain, SbYR Green I stain, TOTO-3 stain and silver (for non-

**g.** For quantification, a fluorescence microscope can be used which can be connected to a

**h.** The extent of DNA damage was determined after electrophoretic migration of DNA

**i.** For each condition randomly selected comets (50/100/200) on each slide can be scored, and % head DNA, % tail DNA, tail length, tail moment and comet length can be determined. Usually, % tail DNA and tail moment are preferred for assessing the DNA

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

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

charge-coupled device (CDC) and a computer-based analysis system.

Na2-EDTA, pH 13) for 30 min to allow DNA unwinding. **e.** Electrophoresis is then performed at 25 V/300 mA for 30 min.

fluorescent staining).

damage.

few hours.

fragments in the agarose gel.

and clinical studies **(Engelward Lab, 2011)**.

immersed in freshly prepared alkaline electrophoresis buffer (300 mM NaOH, 1 mM

### **4.1 Prevention of genotoxicity by selenocompounds**

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 is the key component of antioxidant enzyme systems.

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

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

Fig. 2. Different protocols of Comet assay in research field

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 419

factor in cancer risk **(Esworthy et al., 1985)**. Other modes of action, either direct or indirect, may also be operative, such as the partial retransformation of tumor cells and the inactivation of oncogenes. However, the effects of Se in the physiological dosage range are not attributable to cytotoxicity, allowing Se to be defined as a genuine nutritional cancerprotecting agent **(Yu et al., 1990)**. On the other hand, selenocompounds such as selenodiglutathione, methylselenol, selenomethionine (SM), and Se-methylselenocysteine might affect the metabolism of carcinogens, thus preventing initiation of carcinogenesis **(Gopalakrishna & Gundimeda, 2001)**. These compounds might also restrict cell proliferation by inhibiting protein kinases and by halting phases of the cell cycle that play a central part in cell growth, tumor promotion, and differentiation **(Brinkman et al., 2006)**. A further possible mechanism of action is enhancement of the immune system by stimulating the cytotoxic activities of natural killer cells and lymphokine activated killer cells to act against cancer cells **(Combs, 1998)**. The anticarcinogenic effects of Se are counteracted by Se-

For maximal utilization of its cancer-protective potential, Se supplementation should start early in life and be maintained over the entire lifespan **(Schrauzer & White, 1978; Persson-Moschos et al., 1998; Schrauzer, 2000**). In addition, exposure to Se antagonists and carcinogenic risk factors should be minimized by appropriate dietary and lifestyle changes **(Schrauzer, 1976; Schrauzer, 1977)**. Because geographical studies done in the 1970s reported a possible inverse association between Se and cancer mortality, epidemiological studies have focused on investigating the anticarcinogenic properties of this nutrient **(Brinkman et al., 2006)**. Two key findings that emerged from these early studies were the inverse association between Se and cancer seemed to be both sex and organ specific **(Li et al., 2004)**.

Fig. 3. Alkaline Comet assay methodology

antagonistic compounds, and elements (**Schrauzer, 2000)**.

Fig. 3. Alkaline Comet assay methodology

Fig. 2. Different protocols of Comet assay in research field

factor in cancer risk **(Esworthy et al., 1985)**. Other modes of action, either direct or indirect, may also be operative, such as the partial retransformation of tumor cells and the inactivation of oncogenes. However, the effects of Se in the physiological dosage range are not attributable to cytotoxicity, allowing Se to be defined as a genuine nutritional cancerprotecting agent **(Yu et al., 1990)**. On the other hand, selenocompounds such as selenodiglutathione, methylselenol, selenomethionine (SM), and Se-methylselenocysteine might affect the metabolism of carcinogens, thus preventing initiation of carcinogenesis **(Gopalakrishna & Gundimeda, 2001)**. These compounds might also restrict cell proliferation by inhibiting protein kinases and by halting phases of the cell cycle that play a central part in cell growth, tumor promotion, and differentiation **(Brinkman et al., 2006)**. A further possible mechanism of action is enhancement of the immune system by stimulating the cytotoxic activities of natural killer cells and lymphokine activated killer cells to act against cancer cells **(Combs, 1998)**. The anticarcinogenic effects of Se are counteracted by Seantagonistic compounds, and elements (**Schrauzer, 2000)**.

For maximal utilization of its cancer-protective potential, Se supplementation should start early in life and be maintained over the entire lifespan **(Schrauzer & White, 1978; Persson-Moschos et al., 1998; Schrauzer, 2000**). In addition, exposure to Se antagonists and carcinogenic risk factors should be minimized by appropriate dietary and lifestyle changes **(Schrauzer, 1976; Schrauzer, 1977)**. Because geographical studies done in the 1970s reported a possible inverse association between Se and cancer mortality, epidemiological studies have focused on investigating the anticarcinogenic properties of this nutrient **(Brinkman et al., 2006)**. Two key findings that emerged from these early studies were the inverse association between Se and cancer seemed to be both sex and organ specific **(Li et al., 2004)**.

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 421

consumer and personal care products and medical devices **(Doull et al., 1999)**. The biological effects of DEHP are hence of major concern but so far elusive. Although, the main mechanism underlying hepatocarcinogenicity of phthalates is not fully elucidated, ROS are thought to be associated with the mechanism of tumorigenesis by PPs, including DEHP. This assumption is based to a fact that various proteins that are induced by DEHP in liver parenchymal cells (peroxisomes, mitochondria and microsomes) are prone to formation of H2O2 and other oxidants. Besides, activation of metabolizing enzymes and peroxisome proliferator-activated receptor α (PPARα) might be other substantial factors leading to high intracellular ROS production **(O'Brien et al., 2005; Gazouli et al., 2002)**. However, the mechanisms by which phthalates and particularly DEHP exert toxic effects in reproductive system are not yet fully elucidated. Irreversible and reversible changes in the development of the male reproductive tract like vimentin collapse of Sertoli cells as well as apoptosis of germ cells, effects on sex hormones (mainly on testosterone) as well as follicle stimulating hormone (FSH) and luteinizing hormone (LH), histopathological changes in testis and sperm anomalies were observed with phthalate exposure **(Corton & Lapinskas, 2005; Foster et al., 2001; Erkekoglu et al., 2011a; Erkekoglu et al., 2011b; Kasahara et al, 2002; Noriega et al., 2009)**. Most of the toxic effects were related to its antiandrogenic potential **(Ge et al., 2007)**. A PPARα-mediated pathway based on its peroxisome proliferating (PP) activity **(Gazouli et al., 2002)**, and activation of metabolizing enzymes have also been suggested **(O'Brien et al., 2005)**. While the induction of an oxidative stress may represent a common mechanism in endocrine disruptor–mediated dysfunction, especially on testicular cells **(Latchoumycandane et al., 2002)**, recent studies are also providing supporting evidences for such an effect with DEHP and its major metabolite, mono(2-ethylhexyl)phthalate (MEHP) **(Erkekoglu et al. 2010a; Erkekoglu et al. 2010b; Erkekoglu et al. 2011c; Fan et al. 2010)**. Thus, the primary targets for the DEHP and MEHP are the Sertoli and Leydig cells of testis. In several studies, it was shown that DEHP caused disruption in the function of both cell types. In fact, **Richburg and Boekelheide (1996)** demonstrated histopathological disturbances and alterations of cytoplasmatic distribution of vimentin in Sertoli cells in testis of 28-day-old Fisher rats after a single oral dose of MEHP (2000 mg/kg). Administration of MEHP to Wistar rats at a single oral dose (400 mg/kg bw) was toxic to Sertoli cells and caused detachment of germ cells **(Dalgaard et al., 2000). Tay et al. (2007)** reported vimentin disruption in MEHP-treated C57Bl/6N mice, and gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. We have also reported that in DEHP-treated rats, significant disruption and collapse of vimentin filaments and disruption of seminiferous epithelium in Sertoli cells was observed **(Erkekoglu et al., 2011b)**. Among several others, an earlier data has demonstrated the increase of ROS generation and depletion in antioxidant defenses by DEHP treatment in rat testis **(Kasahara et al., 2002)**. Our recent studies on MA-10 Leydig **(Erkekoglu et al., 2010b)** and LNCaP human prostate cells **(Erkekoglu et al., 2010a)** have also produced comprehensive data suggesting that at least one of the mechanisms underlying the reproductive toxicity of DEHP is the induction of intracellular ROS. The data of **Fan et al. (2010)** have also suggested oxidative stress as a new mechanism of MEHP action on Leydig cells steroidogenesis *via* CYP1A1-mediated ROS stress. On the other hand, in rats exposed to 1000 mg/kg DEHP for 10 days, we observed that this particular phthalate induced oxidative stress in rat testis, as evidenced by significant decrease in GSH/GSSG redox ratio (10-fold) and marked increase in TBARS

levels **(Erkekoglu et al., 2011d)**.

A larger difference in the reduced death rates was reported for men than for women in regions with high levels of Se, and mortality was significantly lower for some types of cancer **(Shamberger et al., 1976; Clark et al., 1991)**. Higher blood levels of Se have been associated with a lower risk of many types of neoplasia, including prostate, lung, colorectal, and possibly bladder, although the data are inconsistent. A significant 39% decreased risk of bladder cancer associated with high levels of Se by combining results from seven epidemiologic studies, conducted in different populations, which applied individual levels of Se measured in serum or toenails **(Brinkman et al., 2006)**.

Supra-physiological levels of sodium selenite (SS) in the presence of polythiols have oxidative properties that might have an anticancer effect by increasing the vulnerability of cancer cells to destruction. It was stated that Se, independent of type (organic/inorganic), can alter several genes to prevent cancer. High doses of Se might upregulate phase II detoxification enzymes, some Se-binding proteins, and some apoptotic genes, and downregulate phase I activating enzymes and cell proliferation genes **(El Bayoumy & Sinha, 2005)**. Inhibition of carcinogen–DNA adducts formation and induction of apoptosis by high doses of Se suggests that protection occurs at both the initiation and post-initiation phases of carcinogenesis **(El Bayoumy & Sinha, 2005)**. However, at lower physiological doses, Se prevents apoptosis, and induces DNA repair **(Longtin, 2003)**.

The literature agrees on the protective effect of Se evaluated with the Comet assay towards a variety of chemical or physical toxic agents. However, it remains inconclusive which is/are the most suitable Se compound/s to prevent DNA damage and which doses should be used to observe protection. In this chapter, the protective effects of both inorganic and organic selenocompounds, against phthalate and radiation toxicity will be discussed.
