Abstract
Reactive oxygen species (ROS) represent a number of highly reactive oxygen-derived by-products generated by the normal mitochondrial respiration and other cellular metabolic reactions. ROS can oxidize macromolecules including lipids, proteins, and nucleic acids. Under physiological condition, the cellular levels of ROS are controlled by several antioxidant enzymes. However, an imbalance between ROS production and detoxification results in oxidative stress, which leads to the accumulation of macromolecular damage and progressive decline in normal physiological functions.
Oxidative deterioration of DNA can result in lesion that are mutagenic and contribute to aging and age-related diseases. Therefore, methods for the detection of ROS and oxidative deterioration of macromolecules such as DNA in cells provide important tool in aging research. Here, we described protocols for the detection of cytoplasmic and mitochondria pools of hydrogen peroxide, and the DNA modification 8-oxoguanine, a biomarker of oxidative damage, that are applicable to cell-based studies on aging and other related areas.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Forkink M, Smeitink JA, Brock R, Willems PH, Koopman WJ (2010) Detection and manipulation of mitochondrial reactive oxygen species in mammalian cells. Biochim Biophys Acta 1797(6–7):1034–1044. https://doi.org/10.1016/j.bbabio.2010.01.022
Miller AF (2012) Superoxide dismutases: ancient enzymes and new insights. FEBS Lett 586(5):585–595. https://doi.org/10.1016/j.febslet.2011.10.048
Rhee SG (2006) Cell signaling. H2O2, a necessary evil for cell signaling. Science 312(5782):1882–1883. https://doi.org/10.1126/science.1130481
Schieber M, Chandel NS (2014) ROS function in redox signaling and oxidative stress. Curr Biol 24(10):R453–R462. https://doi.org/10.1016/j.cub.2014.03.034
Bolisetty S, Jaimes EA (2013) Mitochondria and reactive oxygen species: physiology and pathophysiology. Int J Mol Sci 14(3):6306–6344. https://doi.org/10.3390/ijms14036306
Gorrini C, Harris IS, Mak TW (2013) Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov 12(12):931–947. https://doi.org/10.1038/nrd4002
Circu ML, Aw TY (2010) Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 48(6):749–762. https://doi.org/10.1016/j.freeradbiomed.2009.12.022
Kotiadis VN, Duchen MR, Osellame LD (2014) Mitochondrial quality control and communications with the nucleus are important in maintaining mitochondrial function and cell health. Biochim Biophys Acta 1840(4):1254–1265. https://doi.org/10.1016/j.bbagen.2013.10.041
Nogueira V, Hay N (2013) Molecular pathways: reactive oxygen species homeostasis in cancer cells and implications for cancer therapy. Clin Cancer Res 19(16):4309–4314. https://doi.org/10.1158/1078-0432.CCR-12-1424
Phaniendra A, Jestadi DB, Periyasamy L (2015) Free radicals: properties, sources, targets, and their implication in various diseases. Indian J Clin Biochem 30(1):11–26. https://doi.org/10.1007/s12291-014-0446-0
Avery SV (2011) Molecular targets of oxidative stress. Biochem J 434(2):201–210. https://doi.org/10.1042/BJ20101695
Sedelnikova OA, Redon CE, Dickey JS, Nakamura AJ, Georgakilas AG, Bonner WM (2010) Role of oxidatively induced DNA lesions in human pathogenesis. Mutat Res 704(1–3):152–159. https://doi.org/10.1016/j.mrrev.2009.12.005
Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013) The hallmarks of aging. Cell 153(6):1194–1217. https://doi.org/10.1016/j.cell.2013.05.039
Thapa B, Schlegel HB (2015) Calculations of pKa’s and redox potentials of nucleobases with explicit waters and polarizable continuum solvation. J Phys Chem A 119(21):5134–5144. https://doi.org/10.1021/jp5088866
Guo C, Ding P, Xie C, Ye C, Ye M, Pan C, Cao X, Zhang S, Zheng S (2017) Potential application of the oxidative nucleic acid damage biomarkers in detection of diseases. Oncotarget 8(43):75767–75777. https://doi.org/10.18632/oncotarget.20801
Li B, Iglesias-Pedraz JM, Chen LY, Yin F, Cadenas E, Reddy S, Comai L (2014) Downregulation of the Werner syndrome protein induces a metabolic shift that compromises redox homeostasis and limits proliferation of cancer cells. Aging Cell 13(2):367–378. https://doi.org/10.1111/acel.12181
Donaldson JG (2001) Immunofluorescence staining. Curr Protoc Cell Biol. Chapter 4:Unit 4.3. https://doi.org/10.1002/0471143030.cb0403s00
Howat WJ, Wilson BA (2014) Tissue fixation and the effect of molecular fixatives on downstream staining procedures. Methods 70(1):12–19. https://doi.org/10.1016/j.ymeth.2014.01.022
Campalans A, Kortulewski T, Amouroux R, Menoni H, Vermeulen W, Radicella JP (2013) Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair. Nucleic Acids Res 41(5):3115–3129. https://doi.org/10.1093/nar/gkt025
Amouroux R, Campalans A, Epe B, Radicella JP (2010) Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions. Nucleic Acids Res 38(9):2878–2890. https://doi.org/10.1093/nar/gkp1247
Bennett BT, Bewersdorf J, Knight KL (2009) Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy. Methods 48(1):63–71. https://doi.org/10.1016/j.ymeth.2009.02.009
Bhattacharyya D, Hammond AT, Glick BS (2010) High-quality immunofluorescence of cultured cells. Methods Mol Biol 619:403–410. https://doi.org/10.1007/978-1-60327-412-8_24
Melan MA, Sluder G (1992) Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy. J Cell Sci 101(Pt 4):731–743
Soultanakis RP, Melamede RJ, Bespalov IA, Wallace SS, Beckman KB, Ames BN, Taatjes DJ, Janssen-Heininger YM (2000) Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy. Free Radic Biol Med 28(6):987–998
Funding
This work was supported by grant Nº 150-2017-FONDECYT from Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC) to JMI-P, and grant R01AG034156 form the National Institute of Aging, NIH, USA to LC.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Iglesias-Pedraz, J.M., Comai, L. (2020). Measurements of Hydrogen Peroxide and Oxidative DNA Damage in a Cell Model of Premature Aging. In: Curran, S. (eds) Aging. Methods in Molecular Biology, vol 2144. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0592-9_22
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0592-9_22
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0591-2
Online ISBN: 978-1-0716-0592-9
eBook Packages: Springer Protocols