# Chemical and biological characterization of glycidamide-adducted adenine in DNA

## TL;DR

This study examines how glycidamide, a metabolite of acrylamide, forms DNA adducts and investigates their mutagenic potential.

## Contribution

The study identifies and quantifies N6-GA-dA in DNA and evaluates its mutagenic impact for the first time.

## Key findings

- N6-GA-dA was detected in genomic DNA at about 40 adducts per 108 dA.
- N6-GA-dA forms base pairs with thymidine and does not induce mutations during DNA replication.
- DNA polymerase ε correctly incorporates dTTP opposite N6-GA-dA without replication inhibition.

## Abstract

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.