Quantifying Etheno–DNA Adducts in Human Tissues, White Blood Cells, and Urine by Ultrasensitive 32P-Postlabeling and Immunohistochemistry
Exocyclic etheno–DNA adducts are formed by the reaction of lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE) with DNA bases to yield 1,N 6-etheno-2′-deoxyadenosine (εdA), 3,N 4-etheno-2′-deoxycytidine (εdC), and etheno-2′-deoxyguanosine. These adducts act as a driving force for many human malignancies and are elevated in the organs of cancer-prone patients suffering from chronic inflammation and infections. Here, we describe the ultrasensitive and specific techniques for the detection of εdA and εdC in tissue and white blood cell (WBC) DNA. This approach is based on combined immunopurification by monoclonal antibodies and 32P-postlabeling analysis. The detection limit is about five adducts per 1010 parent nucleotides, requiring 5–10 μg of DNA. In addition, we describe techniques for immunohistochemical detection of εdA and εdC in tissue biopsies, and the approaches for the analysis of εdA and εdC excreted in urine. The utility of these detection methods for human studies is based on: (1) high sensitivity and specificity, (2) low amounts of DNA required, (3) capability to detect “background” levels of etheno–DNA adducts in biopsies, WBC, and urine samples of healthy subjects, and (4) reliable monitoring of the disease-related increase of these substances in patients.
The described methods are useful in diagnosis and monitoring of chronic degenerative diseases, including cancer, atherosclerosis, and neurodegenerative disorders.
Key wordsEtheno–DNA adducts Ultrasensitive detection Oxidative stress Lipid peroxidation Carcinogenesis Chronic degenerative diseases Human biomonitoring
The authors greatly acknowledge the earlier contributions to method development/standardization by A. Barbin, M. Hollstein, and Y. Guichard (IARC, Lyon, France), A. Frank, Y. Yang, S. Dechakhamphu, and M. Meerang (DKFZ, Heidelberg). We thank S. Fuladdjusch for excellent secretarial help and Prof. Manfred F. Rajewsky and coworkers, Institute of Cell Biology (Cancer Research), University of Duisburg-Essen Medical School and West German Cancer Center Essen, Hufeland-Strasse 55, D-45122 Essen Germany for agreeing to provide the antibodies upon request. This article is dedicated to Jagadeesan Nair who passed away prematurely in August 2007. Without his perseverance, these achievements would have not been possible (see obituary: Dr Jagadeesan Nair, Senior Scientist at the German Cancer Research Center (DKFZ). Carcinogenesis. 2008; 29, 887–888).
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