32P-Postlabeling analysis of a DNA adduct, an N2-acetyl derivative of guanine, formed in vitro by methylglyoxal and hydrogen peroxide in combination

Abstract

Methylglyoxal is a direct-acting mutagen in Salmonella typhimurium TA100 and its mutagenicity is markedly enhanced in the presence of hydrogen peroxide. In addition, a mixture of methylglyoxal and hydrogen peroxide reacts with 2'-deoxyguanosine to form N2-acetyl-2'-deoxyguanosine. We examined whether the guanine residues in DNA were acetylated by methylglyoxal in the presence of hydrogen peroxide using the 32P-postlabeling method. First, N2-acetyl-2'-deoxyguanosine 3'-monophosphate and N2-acetyl-2'-deoxyguanosine 3,5'-diphosphate were chemically synthesized as standard compounds for the analysis. Then calf thymus DNA (3.24 micromol) was treated with methylglyoxal (64.8 micromol) at pH 7.4 for 3 h at 37 degrees C, and subsequently with hydrogen peroxide (64.8 micromol) at 37 degrees C for 2 h. The adduct formation was analyzed using HPLC in combination with the 32P-postlabeling method under the standard conditions. N2-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.