A simplified LC–MS-based method for sensitive analysis of DNA adducts utilizing accessible in vitro metabolism models

Genetic information of living cells is encoded by the specific arrangement of nucleobases in deoxyribonucleic acid (DNA). Even minor changes or modifications of these nucleobases, favored by many reactive sites, can significantly affect correct replication and DNA integrity leading to severe toxic effects. Analyzing these DNA modifications is highly complex and often requires non-specific assays for DNA damage or ³²P-postlabelling. A major limitation of these methods is their inability to provide structural information, a gap that can be addressed by instrumental analytical techniques. An additional major challenge is the selection of an appropriate biological system capable of reliable DNA adduct formation in high yields, since most compounds require metabolic activation prior to reacting with DNA. Therefore, the aim of this study was to develop a fast and simple workflow for sensitive DNA adduct analysis. In addition to highlighting the main pitfalls in sample preparation, this publication focuses on the comparison of biological systems in terms of metabolic activity, using six well-known carcinogens from different chemical classes. The combination of HepG2 cells and liver S9 fractions demonstrated comparable or even superior capabilities to primary hepatocytes and enabled the detection of DNA adducts from aflatoxin B₁, benzo[a]pyrene, methyleugenol as well as α-asarone and β-asarone, particularly after metabolic activation by the aryl hydrocarbon receptor agonist β-naphthoflavone. Notably, the adduct formation of phenylpropanoids was documented for the first time in a non-transfected cancer cell line using high-performance liquid chromatography coupled to mass spectrometry. Therefore, the method can be used to detect previously unknown DNA adducts from diverse chemical classes and provides structural insights into the formed DNA adducts.