An improved 32P-postlabeling/high-performance liquid chromatography method for the analysis of the malondialdehye-derived 1, N2-propanodeoxyguanosine DNA adduct in animal and human tissues.
Malondialdehyde (MDA) is a major lipid peroxidation product that is mutagenic and tumorigenic. The MDA-modified DNA adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1, 2-alpha]purin-10(3H)-one (M1G), has been detected in human tissues and may be a marker of human cancer risk. In this paper, we describe an improved 32P-postlabeling/HPLC method for sensitive detection and quantitation of this MDA-modified 2'-deoxyribonucleotide adduct. Specific improvements include (i) unequivocal structural identification of the postlabeling products, both the 3', 5'-bisphosphate of M1G (MDA-3',5'-dGDP) and the 5'-monophosphate of M1G (MDA-5'-dGMP); (ii) efficient separation of the 32P-postlabeling products by HPLC; and (iii) the incorporation of a synthetically prepared MDA-modified DNA (or the 3'-monophosphate of M1G) with a known modification level as an internal standard. This improved quantitative methodology provides high intra- and inter-assay reproducibility and has been applied to the analysis of this adduct in rodent and human samples.
National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
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D000328:Adult; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018736:DNA Adducts / Q000032:analysis* / Q000138:chemical synthesis / Q000302:isolation & purification; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D005260:Female; D006153:Guanosine Diphosphate / Q000032:analysis; D006157:Guanosine Monophosphate / Q000032:analysis; D006801:Humans; D007553:Isotope Labeling; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008297:Male; D008315:Malondialdehyde / Q000032:analysis*; D051379:Mice; D010179:Pancreas / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D051381:Rats
D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; C060934:1,N(2)-propanodeoxyguanosine; D006153:Guanosine Diphosphate; D008315:Malondialdehyde; D006157:Guanosine Monophosphate; D003849:Deoxyguanosine