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Determination of DNA Damage in the Retina Photoreceptors of Drosophila   

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A brief version of this protocol appeared in:
Molecular Neurobiology
Sep 2017

Abstract

The retina is sensitive for light damages, because of direct light exposure, especially intense blue and UV light, which increase level of ROS and other toxic phototransduction products in photoreceptor cells. In our previous work (Damulewicz et al., 2017a and 2017b), we used 8-oxo-deoxyguanosine (8-OHdG) as a marker for oxidative stress to investigate the role of heme oxygenase in DNA protection against UV light. In this protocol, we showed how to determine the level of DNA damages in the retina using immunohistochemical staining.

Keywords: Oxidative stress, 8-Oxo-deoxyguanosine, UV light, Retina degeneration, DNA damage

Background

Deoxyribonucleic acid (DNA) is an essential molecule for all living organisms. It contains genetic code with instruction for proteins and other molecules necessary for structure and metabolism of an organism. DNA is composed of two strands of nucleotides. Each nucleotide is built of one of four types of nucleobases (cytosine, guanine, adenine and thymine), deoxyribose and phosphate group. Physical and chemical changes in DNA structure are classified as DNA damages. It includes one or two strands of DNA breaks, missing nucleotide or chemical changes of nucleobases. Damages can be induced by environmental factors, like chemicals or UV light exposure, or in result of metabolic processes, which produce reactive oxygen species, reactive nitrogen species, reactive carbonyl species and alkylating agents (De Bont and van Larebeke, 2004).

One of the most common DNA damage is 8-oxo-deoxyguanosine (8-OHdG), which is a product of DNA oxidation by reactive oxygen species and it serves as a marker of oxidative stress (De Souza-Pinto et al., 2001; Swenberg et al., 2011; Valavanidis et al., 2013). 8-OHdG enhances the risk of transversion mutation, because it has ability to pair with adenine and cytosine bases (Maki and Sekiguchi, 1992). In effect it plays a role in mutagenesis, cancerogenesis and aging (De Bont and van Larebeke, 2004; Kohen and Nyska, 2002). Higher 8-OHdG level was described in breast, renal and gastric tumors (Lee et al., 1998; Musarrat et al., 1996; Okamoto et al., 1994).

There are several methods to determine 8-OHdG level in a specific tissue, like immunohistochemistry (De Carvalho et al., 2013), Western blot (Kannan et al., 2006), standard HPLC (Herrero and Barja, 1999), liquid chromatography nanoelectrospray-tandem mass spectrometry (Ma et al., 2016). Here, we present a detailed protocol for DNA damage determination in the retina of fruit fly, Drosophila melanogaster using immunohistochemistry method (Damulewicz et al., 2017a and 2017b).

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Damulewicz, M. and Pyza, E. (2018). Determination of DNA Damage in the Retina Photoreceptors of Drosophila. Bio-protocol 8(3): e2708. DOI: 10.21769/BioProtoc.2708.
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