In vitro investigation and mass spectrometric characterization of novel DNA adducts of sesquimustard

Sesquimustard (Q), a potent bifunctional alkylating vesicant, is a significant toxicological concern due to its environmental persistence and capability to induce severe genotoxic effects. It can create several metabolites, some of which can be utilized as biomarkers, and it can extensively alkylate key macromolecules in organisms, including DNA. Although prior research has primarily focused on glutathione and albumin adducts as retrospective biomarkers of Q exposure, no prior studies have identified DNA adducts of Q. This study aimed to fill this critical gap by characterizing, for the first time, Q-induced DNA adducts using advanced mass spectrometric techniques. Three novel DNA adducts -hydroxyethylthioethylthioethyl-adenine (HETETE-Ade), hydroxyethylthioethylthioethyl-guanine (HETETE-Gua), and guanine-ethylthioethylthioethyl-guanine (Gua-ETETE-Gua)- were identified in Q-treated calf thymus DNA via liquid chromatography–high-resolution mass spectrometry (LC-HRMS) and structurally confirmed through high-resolution product ion spectra. Their formation was further demonstrated in a human keratinocyte (HaCaT) cell model using liquid chromatography–tandem mass spectrometry (LC-MS/MS), revealing dose-dependent increases in adduct signal intensities. Compared to protein-based biomarkers, DNA adducts may offer potential advantages in workflow simplicity and direct indication of genotoxic damage. This study provides first molecular insight into Q-induced DNA adducts and lays the groundwork for their future use in toxicological assessment, forensic verification, and biomarker development.