Chemical and biological characterization of glycidamide-adducted adenine in DNA

Acrylamide, a food contaminant, is metabolically converted to glycidamide (GA), which reacts with nucleobases in DNA. Although several GA adducts with dG, 2'-deoxyadenosine (dA), and dC are known, the mutagenic potency of each adduct remains unclear. Here, we focused on N⁶-(2-carboxy-2-hydroxyethyl)-2'-deoxyadenosine (N⁶-GA-dA), which is produced by the Dimroth rearrangement of N1-(2-carboxy-2-hydroxyethyl)-2'-deoxyadenosine. We separately prepared N1-(2-carboxy-2-hydroxyethyl)-2'-deoxyadenosine and N⁶-GA-dA and measured their Dimroth rearrangement reaction rates. Since N⁶-GA-dA is the stable final product under physiological conditions (pH 7.0, at 37°C), we examined its presence in genomic DNA isolated from GA-treated cells. N⁶-GA-dA was successfully detected by LC-MS analysis of the nucleoside components of DNA from XP2OSSV cells, at about 40 adducts per 10⁸ dA. In addition, N⁶-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine, which we have previously shown its mutagenic potency, was also detected at 5 × 10⁴ adducts per 10⁸ dG, a level comparable to N7-(2-carbamoyl-2-hydroxyethyl)guanine. To assess the biological impact of N⁶-GA-dA, we prepared oligonucleotides containing a single N⁶-GA-dA at a specific position and analyzed base-pair formation and mutagenic potential. The thermodynamic parameters of duplexes revealed that N⁶-GA-dA forms a base pair with thymidine similarly to dA. DNA polymerase ε bypassed N⁶-GA-dA by incorporating the correct dTTP opposite the lesion. Furthermore, a site-specific intracellular mutagenesis assay showed no detectable replication inhibition or mutation induction at the lesion site. In summary, we detected and quantified N⁶-GA-dA formed in GA-treated cells and investigated its mutagenic potency. This study contributes to understanding the molecular aspects of GA adducts in DNA.