Determination of malonaldehyde-modified 2'-deoxyguanosine-3'-monophosphate and DNA by 32P-postlabelling.
The 32P-postlabelling assay was used to determine adducts arising upon the reaction of malonaldehyde with 2'-deoxyguanosine-3'-monophosphate. The adducts formed were isolated, structurally characterized and identified as 3-(2-deoxy-beta-D-erythro-pentafuranosyl)pyrimido[1,2-alpha] purin-10(3H)-one. The kinetics of phosphorylation by T4 polynucleotide kinase was studied using 500 fmol of the synthesized standard and found to reach its maximum after 1 h of incubation. A 60% labelling efficiency was obtained at low concentrations of substrate. The adducted substrate was detected at the sub-femtomolar level. Sensitivity of the adducts towards nuclease P1 3'-dephosphorylation was also tested. The same adduct could be detected from calf thymus DNA that had been reacted in vitro with malonaldehyde, and in DNA isolated from mice treated with [14C]malonaldehyde. DNA adducts formed in vitro were isolated after enzymatic digestion to mononucleotides followed by HPLC fractionation or nuclease P1 digestion of normal nucleotides. A combination of the two procedures proved to be the method of choice for the isolation of the malonaldehyde-DNA adducts formed in vivo prior to applying the 32P-postlabelling assay.
Vaca
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Molecular Epidemiology Unit, NOVUM, Karolinska Institute, Huddinge, Sweden.
Vodicka
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Hemminki
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K
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D008297:Male; D008315:Malondialdehyde / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010761:Phosphorus Radioisotopes
D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; D008315:Malondialdehyde; D004247:DNA