Determination of malonaldehyde-modified 2'-deoxyguanosine-3'-monophosphate and DNA by 32P-postlabelling

Abstract

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-β-D-erythro-pentafuranosyl)pyrimido [1, 2-α] 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]makonaldehyde. DNA adducts formed in vitro were isolated after enzymatic digestion to mononudeotides followed by HPLC fractination 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.