Abstract
Reactive oxygen species (ROS) such as hydroxyl radicals (*OH), superoxide anions (O2 −) and hydrogen peroxide (H2O2) have been shown to damage to chromosomal DNA and other cellular components, resulting in DNA degradation, protein denaturation, and lipid peroxidation (1,2). We know a little about the in vivo action mechanism of ROS produced by anticancer drugs and by X-ray irradiation on chromatin DNA in the nuclei of intact cells. DNA damage induced by ROS in vivo or in the cultured cell system is classified into singleand double-strand breaks and nucleotide base oxidative modifications (2-4). The application of gel electrophoresis to the measurement of DNA doublestrand breaks has been described by some workers for DNA irradiated in vitro (5-7). Double-strand breaks are generally thought to have a greater biological consequence than single-strand DNA breaks because they can lead directly to chromosomal aberrations, and more frequently to the loss of genetic information (6,8). Ionizing radiation such as X-ray and γ-ray are, in general, thought to produce *OH radicals from water molecules in or around the target sites in the DNA, and these in turn attack DNA and break it down (1,3). In addition, the involvement of such radicals in the induction of apoptosis has been suggested in several cell lines (9-12).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bielski, B. H. J. and Gebichi, J. M. (1977) Application of radiation chemistry to biology. Free Rad. Biol. 3, 1–51.
Halliwell, B. and Aruoma, O. I. (1991) DNA damage by oxygen-derived species: its mechanism and measurement in mammalian systems. FEBS Lett. 281, 9–19.
Ward, J. F. (1988) DNA damage produced by ionizing radiation in mammalian cells. Prog. Nucleic Acid Res. Mol. Biol. 35, 95–125.
Wlodek, D. and Hittelman, W. N. (1987) The repair of double-strand breaks correlates with radiosensitivity of L-5178Y-S and L5178Y-R cells. Radiat. Res. 112, 146–155.
Yamada, T. and Ohyama, H. (1988) Radiation-induced interphase death of rat thymocytes is internally programmed (apoptosis). Int. J. Radiat. Biol. 53, 65–75.
Nevaldine, B., Longo, J. A., King, G. A., Vilenchik, M., Sagerman, R. H., and Hahn, P. J. (1993) Induction and repair of DNA double-strand breaks. Radiation Res. 133, 370–374.
Lobrich, M., Ikpeme, S., and Kiefer, J. (1994) DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing. Int. J. Radiat. Biol. 65, 623–630.
Bryant, P. E. (1984) Enzymatic restriction of mammalian cell DNA using Pvu II and Bam HI; evidence for the double strand break origin of chromosomal aberrations. Int. J. Radiation Biol. 46, 57–65.
Oberhammer, F., Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Walkeling, A. E., et al. (1993) Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679–3684.
Lagarkova, M. A., Iarovaia, O. V., and Razin, S. V. (1995) Large-scale fragmentation of mammalian DNA in the course of apoptosis proceeds via excision of chromosomal DNA loops and their oligomers. J. Biol. Chem. 270, 20,239–20,241.
Slater, A., Nobel, S., and Orrenius, S. (1995) The role of intracellular oxidants in apoptosis. Biochim. Biophys. Acta 1271, 59–62.
Clutton, S. (1997) The importance of oxidative stress in apoptosis. Br. Med. Bull.53, 662–668.
Stamato, T. D. and Denko, N. (1990) Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells. Radiat. Res. 121, 196–205.
Erixon, K. and Cedervall, B. (1995) Linear induction of DNA double-strand breakage with X-ray dose, as determined from DNA fragment size distribution. Radiat. Res. 142, 153–162.
Matsukawa, S. and Higuchi, Y. (1991) The nature of giant DNA molecules produced from nuclear chromosome DNA by active oxygen producing agents, in Oxidative Damage and Repair (Davies, K. T. A., ed.), Pergamon Press, pp. 197–201.
Higuchi, Y. and Matsukawa, S. (1997) Appearance of 1-2 Mb giant DNA fragments as an early common response leading to cell death induced by various substances which cause oxidative stress. Free Rad. Biol. Med. 23, 90–99.
Higuchi, Y. and Matsukawa, S. (1998) Active oxygen-mediated chromosomal 1-2 Mbp giant DNA fragmentation into internucleosomal DNA fragmentation in apoptosis of glioma cells induced by glutamate. Free Rad. Biol. Med. 24, 418–426.
Higuchi, Y. and Matsukawa, S. (1999) Glutathione depletion induces chromosomal giant DNA and high molecular weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells. Arch. Biochem. Biophys. 393, 33–42.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. E10–E15.
Schwartz, D. C., Saffran, W., Welsh, J., Haas, R., Goldberg, M., and Cantor, C. R. (1983) New techniques for purifying large DNAs and studying their properties and packaging. Cold Spring Harb. Quant. Biol. 47, 189–195.
Carles, G. F., Frank, M., and Olson, M. V. (1986) Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science 232, 65–68.
Pharmacia LKB Biotechnology. Instruction manual Pulsaphor system 80-1251-98. Pharmacia LKB Biotechnology, S-751.82 Upsala, Sweden. (New version, from Amersham Pharmacia Biotech, Bucks, UK.)
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Higuchi, Y. (2002). Measurement of DNA Double-Strand Breaks with Giant DNA and High Molecular-Weight DNA Fragments by Pulsed-Field Gel Electrophoresis. In: Armstrong, D. (eds) Oxidative Stress Biomarkers and Antioxidant Protocols. Methods in Molecular Biology™, vol 186. Humana Press. https://doi.org/10.1385/1-59259-173-6:161
Download citation
DOI: https://doi.org/10.1385/1-59259-173-6:161
Publisher Name: Humana Press
Print ISBN: 978-0-89603-850-9
Online ISBN: 978-1-59259-173-2
eBook Packages: Springer Protocols