Development of a 32P-postlabelling method for the analysis of 2'-deoxyguanosine-3'-monophosphate and DNA adducts of methylglyoxal.
A 32P-postlabelling assay was developed for the analysis of adducts arising from the reaction of 2'-deoxyguanosine-3'-monophosphate with the 1,2-dicarbonyl compound methylglyoxal, a major mutagen in several foodstuffs, in particular, instant and brewed coffee. The 32P-postlabelling reaction was optimized by testing various parameters such as the kinetics of phosphorylation by T4 polynucleotide kinase, substrate concentration-dependent labelling efficiency and the concentration of the various ingredients of the phosphorylation reaction. The sensitivity to the 3'-monophosphate dephosphorylation activity of nuclease P1 was also studied. Four isomeric reaction products were separated by HPLC, structurally characterized and identified as 3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-6,7-dihydro-6,7-dihydro xy-6- methylimidazo[2,3-b]purine-9(8H)one. The same adducts could be detected from calf thymus DNA that had been reacted in vitro with methylglyoxal. DNA adducts were isolated after enzymatic digestion to mononucleotides followed by nuclease P1 digestion of normal nucleotides. The total level of methylglyoxal-DNA adducts obtained was 5.7 +/- 1.7 (n = 15) adducts/10(6) nucleotides. The 32P-postlabelling method was further validated by the detection of adducts of methylglyoxal in DNA from freshly isolated and stimulated human lymphocytes exposed in vitro. The concentrations of the adducts detected in these samples were 8.2 +/- 0.9 (n = 3) adducts/10(7) nucleotides and 1.5 +/- 0.1 (n = 3) adducts/10(6) nucleotides after treatment with 1.5 and 3.0 mM methylglyoxal respectively.
Vaca
C E
CE
Molecular Epidemiology Unit, NOVUM, Karolinska Institute, Huddinge, Sweden.
Fang
J L
JL
Conradi
M
M
Hou
S M
SM
D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis* / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods*; D010761:Phosphorus Radioisotopes; D011765:Pyruvaldehyde / Q000032:analysis* / Q000378:metabolism*
D018736:DNA Adducts; D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; D011765:Pyruvaldehyde; C007257:2'-deoxyguanosine 5'-phosphate; D004247:DNA