**1. Introduction**

412 Gel Electrophoresis – Advanced Techniques

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conditions in expression of *Lentinula edodes* manganese peroxidase, *Breeding Research, Proceedings of Japanese Society of Breeding 2004 105th Conference*, Vol.6, No.1, Oxidation-reduction reactions, simply referred as "redox" reactions, describe all the chemical reactions in which atoms have their oxidation state changed. This can either be a simple redox process like the oxidation of carbon (C) to carbon dioxide (CO2) or the reduction of C by hydrogen (H) to yield methane (CH4). However, in biology redox reactions are rather complex and 'redox biology' is fundamental to aerobic life **(Peters et al., 2008; Baliga et al., 2007)**. The simplest example to give is the oxidation of glucose to CO2 and water in photosynthesis **(Halliwell, 2006)**.

Aerobes are constantly subject to free radicals, but modulate their actions by synthesizing antioxidants. Free radicals are atoms, molecules, or ions with one or more unpaired electrons on an open shell configuration **(Gutteridge & Halliwell, 2000)**. The simplest form is the atomic H. There are many types of free radicals in living systems, but both nitrogen (N) and oxygen (O) radicals are the main concern for the researchers of several fields as they are suspected to be the underlying factors of several conditions and diseases **(Halliwell, 2006)**. O2 toxicity was suggested to be due to the inactivation of a variety of enzymes (particularly of antioxidant enzymes) by targeting the thiol group of cysteine residues. In the last decades, molecular biology techniques established that the toxic effects of O2 are directly linked to its reactive forms, the reactive oxygen species (ROS), acting on cellular components. Oxidative stress is a serious imbalance between the generation of ROS and antioxidant protection in favor of the former, causing excessive oxidative damage **(Dröge, 2002; Halliwell, 2011)**. Oxidative stress and ROS can account for changes that may be detrimental to the cells **(Dröge, 2002)**. ROS are shown to contribute to cellular damage, apoptosis and cell death **(Dalton et al., 1999; Finkel, 1998)**. The link between O2 toxicity and many pathologies, e.g. pulmonary diseases, **(Frankl, 1991)**, and its effect on swelling of the blood–gas barrier **(Drath et al., 1981)**, retina defects **(Geller et al., 2006)**, bowel disease **(Grisham, 1994)** neurodegeneration **(Wang et al., 2006)**, cancer **(Cerutti, 1994)**, diabetes **(Seet et al., 2010)** and ageing **(Irminger-Finger, 2007)** is very well-established. Besides, in the last decade a relationship between obesity and ROS was demonstrated **(Seet et al., 2010; Halliwell, 2011)**.

Antioxidant is a molecule that protects a biological target against oxidative damage **(Halliwell, 2011)**. Accumulating data implicate that both low antioxidant status and genetics may contribute to the risk of several types of malignancies **(Peters et al., 2008;** 

Protection Studies by Antioxidants Using Single Cell Gel Electrophoresis (Comet Assay) 415

collected and lymphocytes have proved to be good surrogate cells. For example, lymphocytes exhibited genotoxicity caused by anticancer agents targeting several different

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

a. The slides that will be used in the study should be covered with agarose (1%) the day

b. In the basic alkaline Comet assay, for primary and other cell cultures, after exposing small number of cells to a physical or chemical agent, the cells are trypsinized, centrifuged, washed, and resuspended in PBS. Because of the flexible application of the technique, the cells used can be isolated lymphocytes, cells isolated from bone marrow, cells isolated from solid organs or tissues or cells from primary or other cell cultures. Lymphocytes can be isolated from whole blood using different isolation solutions and centrifugation. Cells from bone marrow can be obtained by perfusing femur in cold mincing solutions and centrifugation. Solid organs or tissues must be minced into fine pieces, later be suspended in cold mincing solutions and centrifugated. Blood-rich organs like liver and kidney have to minced into larger pieces, the mincing solution can be aspirated and fresh mincing solution should be added. Mincing solution can be Hank's Buffered Salt Solution (HBSS, with 20 mM

**c.** Usually 50 μl of the cells obtained from either cell cultures blood or organs/tissues should be mixed with 450 μl solution of low melting point agarose (0.6% in PBS), and

**d.** Cells are lysed (in 2.5 M NaCl, 0.5 MNa2-EDTA, 10 mM Tris, 1% sodium lauryl sulfate, 1% Triton X-100, 10% DMSO, pH 10) at 4°C in dark for 1 h. After lysis, cells were

100 μl of the solution is spread on microscope slides covered with agarose.

alkaline Comet assay protocol can be performed in the following steps:

Fig. 1. Advantages and disadvantages of Comet assay

organs **(Faust et al., 2004)**.

before the experiment.

EDTA and 10% DMSO).

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

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