P-32 POSTLABELING ANALYSIS OF A DNA ADDUCT, AN N-2-ACETYL DERIVATIVE OF GUANINE, FORMED IN-VITRO BY METHYLGLYOXAL AND HYDROGEN-PEROXIDE IN COMBINATION

Methylglyoxal is a direct-acting mutagen in Salmonella typhimurium TA1 00 and its mutagenicity is markedly enhanced in the presence of hydrog en peroxide. In addition, a mixture of methylglyoxal and hydrogen pero xide reacts with 2'-deoxyguanosine to form N-2-acetyl-2'-deoxyguanosin e. We examined whether the guanine residues in DNA were acetylated by methylglyoxal in the presence of hydrogen peroxide using the P-32-post labeling method. First, N-2-acetyl-2'-deoxyguanosine 3'-monophosphate and N-2-acetyl-2'-deoxyguanosine 3',5'-diphosphate were chemically syn thesized as standard compounds for the analysis, Then calf thymus DNA (3.24 mu mol) was treated with methylglyoxal (64.8 mu mol) at pH 7.4 f or 3 h at 37 degrees C, and subsequently with hydrogen peroxide (64.8 mu mol) at 37 degrees C for 2 h. The adduct formation was analyzed usi ng HPLC in combination with the P-32-postlabeling method under the sta ndard conditions. N-2-Acetyl-2'-deoxyguanosine was detected at levels of 2/10(6) nucleotides in double-stranded DNA and 1/10(5) nucleotides in single-stranded DNA. The estimated limit of detection by our method was 3 per 10(8) nucleotides.