Study of DNA damage via the comet assay and base excision repair activities in rat brain neurons and astrocytes during aging Umakanta Swain a,b , Kalluri Subba Rao c, * a Department of Biochemistry, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 500046, Andhra Pradesh, India b Dr. D.S. Kothari Post Doctoral Fellow of University Grants Commission, New Delhi, India c Centers for Biotechnology and Innovative Research, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad 500085, Andhra Pradesh, India 1. Introduction DNA damage could be defined as any modification of DNA that changes its coding properties therefore in the normal information transfer fidelity (Lindahl, 1993; Rao, 1993). Damage to the native structure of DNA can occur through two main mechanisms: spontaneous damage caused by sources within a cell and damage caused by external sources such as chemicals and radiation. Protracted oxidative, hydrolytic, deamination or alkylation reac- tions can modify DNA bases, or even sometimes cause a complete loss of bases within DNA, resulting in strand breakage. Similarly, cellular DNA can be damaged by external sources such as ultraviolet or ionizing radiation (X-rays, g-rays, a particles and cosmic rays) and an array of chemical substances can induce interstrand and intrastrand cross-links, DNA–protein cross-links, bulky DNA adducts, single strand breaks (SSBs) and double strand breaks (DSBs) (Rao, 1990, 2002, 2003; Rao and Loeb, 1992; Reddy and Vasquez, 2005; Martin, 2008). As the multitude of ways in which DNA can be damaged increases, equally diverse repair mechanisms have been evolved by nature (Misteli and Soutoglou, 2009). No matter how and what type of structural modification is induced in DNA and no matter through which type DNA repair pathway this structural alteration is corrected, the initial point of the whole recovery process is to recognize the damage and its nature to activate the appropriate repair pathway. It is therefore, obvious that accurate assessment of damaged DNA, irrespective of its molecular nature, would itself is a valuable information to weigh the health status of the cell or tissue. We have been using the post mitotic brain cells as a model system to examine the relationship between DNA damage, DNA repair and aging. Making use of a biochemical approach Mandavilli and Rao (1994) had assessed the number of SSBs and DSBs through nick translation of free 3 0 -OH groups in DNA with Escherichia coli polymerase I and addition of nucleotides at the terminal 3 0 -OH by calf thymus terminal deoxynucleotidyl transferase respectively from young, adult and old rat brain neurons and astrocytes. Measurement of DNA damage in any cell or tissue is an important pointer for a stable or unstable nature of the genomic apparatus which would reflect physiological health status of a cell and the whole organism. Thus, this parameter could be a valuable one for assessing the genomic stability. However, this biochemical method is time consuming and requires elaborate laboratory facilities thereby making unsuitable for analysis of the large number of Mechanisms of Ageing and Development 132 (2011) 374–381 A R T I C L E I N F O Article history: Available online 11 May 2011 Keywords: Aging Comet assay DNA damage Base excision repair Brain A B S T R A C T Earlier we have used biochemical approach to assess the number of single (SSBs) and double (DSBs) strand breaks in brain cellular DNA. However, a quick method to obtain a reliable measure of DNA damage in cells was in need for population studies. Therefore, single cell gel electrophoresis technique (popularly known as ‘‘comet’’ assay) has been standardized using the Trevigen protocol. DNA damage was assessed in isolated neurons and astrocytes from the cortex of young (7 days), adult (6 months) and old (2 years). Marked increase is seen in DNA damage in terms SSBs and DSBs in both types of cells by 6 months of age, which increased further by 2 years of age. The number of 8-oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) sensitive sites also increased in DNA with age with the simultaneous decrease in OGG1, UDG and AP endonuclease (APE1) activities. Thus the comet assay adapted to our lab conditions has proven to be useful for a quick assessment of DNA damage in a large number of samples that constitute our future studies. ß 2011 Elsevier Ireland Ltd. All rights reserved. Abbreviations: SSBs, single strand breaks; DSBs, double strand breaks; BER, base excision repair; U, uracil; 8-oxoG, 7,8 dihydro-8-oxoguanine; OGG1, 8-oxoguanine DNA glycosylase 1; UDG, uracil DNA glycosylase; APE1, AP endonuclease; pol b, DNA polymerase b; AP site, apurinic/apyrimidinic site; THF, tetrahydrofuran; ALS, alkali-labile sites; TBE, Tris–borate EDTA buffer; CHAPS, (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate). * Corresponding author. Tel.: +91 40 23051214/8729; fax: +91 40 23058729. E-mail addresses: ksrsl@yahoo.com, ksrbrain@gmail.com (K. Subba Rao). Contents lists available at ScienceDirect Mechanisms of Ageing and Development jo ur n al ho mep ag e: www .elsevier .c om /lo cate/m ec hag ed ev 0047-6374/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2011.04.012