Abstract
Solar ultraviolet radiation (UVR), through the formation of DNA photolesions, is the primary cause of most skin cancers. A better understanding of the mechanisms of UVR-induced DNA damage may help prevent skin cancer and this may be achieved using methods to quantify DNA damage. The immuno-slot blot (ISB) method is routinely used for detection and quantification of any heat- and alkali-stable DNA adducts for which a sufficiently specific monoclonal antibody is available. The main steps in ISB are fragmentation and denaturation of the DNA, immobilization of DNA to a nitrocellulose filter, incubation with primary antibody against a specific DNA adduct, incubation with an enzyme-linked secondary antibody and finally chemiluminescence detection and quantification of the DNA adducts.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Sinha RP, Hader DP (2002) UV-induced DNA damage and repair: a review. Photochem Photobiol Sci 1:225–236
Ravanat JL, Douki T, Cadet J (2001) Direct and indirect effects of UV radiation on DNA and its components. J Photochem Photobiol B 63:88–102
Cadet J, Sage E, Douki T (2005) Ultraviolet radiation-mediated damage to cellular DNA. Mutat Res 571:3–17
Douki T, Reynaud-Angelin A, Cadet J, Sage E (2003) Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation. Biochemistry 42:9221–9226
Cooke MS, Duarte TL, Cooper D, Chen J, Nandagopal S, Evans MD (2008) Combination of azathioprine and UVA irradiation is a major source of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine. DNA Repair (Amst) 7:1982–1989
Cadet J, Berger M, Douki T, Ravanat JL (1997) Oxidative damage to DNA: formation, measurement, and biological significance. Rev Physiol Biochem Pharmacol 131:1–87
Coohill TP, Peak MJ, Peak JG (1987) The effects of the ultraviolet wavelengths of radiation present in sunlight on human cells in vitro. Photochem Photobiol 46:1043–1050
Douki T, Perdiz D, Grof P, Kuluncsics Z, Moustacchi E, Cadet J, Sage E (1999) Oxidation of guanine in cellular DNA by solar UV radiation: biological role. Photochem Photobiol 70:184–190
Mouret S, Baudouin C, Charveron M, Favier A, Cadet J, Douki T (2006) Cyclobutane pyrimidine dimers are predominant DNA lesions in whole human skin exposed to UVA radiation. Proc Natl Acad Sci USA 103:13765–13770
Kvam E, Tyrrell RM (1997) Induction of oxidative DNA base damage in human skin cells by UV and near visible radiation. Carcinogenesis 18:2379–2384
Tyrrell RM, Reeve VE (2006) Potential protection of skin by acute UVA irradiation – from cellular to animal models. Prog Biophys Mol Biol 92:86–91
Poli G, Dianzani MU, Cheeseman KH, Slater TF, Lang J, Esterbauer H (1985) Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions. Biochem J 227:629–638
Everett SM, Singh R, Leuratti C, White KL, Neville P, Greenwood D, Marnett LJ, Schorah CJ, Forman D, Shuker D, Axon AT (2001) Levels of malondialdehyde-deoxyguanosine in the gastric mucosa: relationship with lipid peroxidation, ascorbic acid, and Helicobacter pylori. Cancer Epidemiol Biomarkers Prev 10:369–376
Dennis KJ, Shibamoto T (1989) Production of malonaldehyde from squalene, a major skin surface lipid, during UV-irradiation. Photochem Photobiol 49:711–716
Adamkiewicz J, Eberle G, Huh N, Nehls P, Rajewsky MF (1985) Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies. Environ Health Perspect 62:49–55
Muller R, Adamkiewicz J, Rajewsky MF (1982) Immunological detection and quantification of carcinogen-modified DNA components. IARC Sci Publ 463–479
Nehls P, Adamkiewicz J, Rajewsky MF (1984) Immuno-slot-blot: a highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA. J Cancer Res Clin Oncol 108:23–29
Ludeke BI (1992) An immuno-slot-blot assay for detection and quantitation of alkyldeoxyguanosines in DNA. In: Manson MM (ed.) Immunochemical Protocols. Humana Press, Totowa, NJ, p 307
Singh R, Leuratti C, Josyula S, Sipowicz MA, Diwan BA, Kasprzak KS, Schut HA, Marnett LJ, Anderson LM, Shuker DE (2001) Lobe-specific increases in malondialdehyde DNA adduct formation in the livers of mice following infection with Helicobacter hepaticus. Carcinogenesis 22:1281–1287
Acknowledgment
The authors would like to acknowledge Prof. Peter B. Farmer (University of Leicester, UK) for use of immuno-slot blot equipment and laboratory space, Dr. Rajinder Singh (University of Leicester, UK) for advice, and Prof. Lawrence Marnett (Vanderbilt University, USA) for supplying the M1dG antibody.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
Karbaschi, M., Brady, N.J., Evans, M.D., Cooke, M.S. (2012). Immuno-Slot Blot Assay for Detection of UVR-Mediated DNA Damage. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_12
Download citation
DOI: https://doi.org/10.1007/978-1-61779-998-3_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-997-6
Online ISBN: 978-1-61779-998-3
eBook Packages: Springer Protocols