Abstract
DNA adduct is a piece of DNA covalently bond to chemicals. Adducts activate repair processes and, unless repaired prior to replication, may lead to nucleotide substitutions, deletions, and other rearrangements. As alterations of the genetic material can cause severe diseases including cancer, inflammation, and neurodegenerative disorders, tests on mutagenicity/genotoxicity have become an integral part of risk assessment during the development of any new chemical. A number of in-vitro tests using different cell lines and in vivo tests mainly using rodents are available to identify hazardous compounds. The majority of these tests are time-consuming, labor-intensive and hardly automatable. So in search for alternative methods, the usefulness of electrochemistry (EC)-liquid chromatography (LC)-mass spectrometry (MS) was evaluated. Generally, EC represents a powerful tool to study in vitro biological oxidation processes of a number of different compounds, including drugs, peptides and proteins. Here, we used EC to initiate adduct formation. The obtained reaction products were separated by LC and detected by MS. Tandem MS experiments were used for structural confirmation. In a proof of principle study acetaminophen was selected as model compound. Covalent adduct formation was observed for electrochemical activated mixtures of acetaminophen and guanosine. Mechanistic studies revealed that adduct formation will only start at electrochemical potentials sufficiently high to initiate oxidation of both acetaminophen and guanosine. The stringent necessity of coactivation sheds a new light on the mechanism of adduct formation, because according to the generally accepted theory activation of the adduct forming agent should be sufficient. Chromatographic separation enabled the differentiation of four isomeric forms. Their connection to acetaminophen was proven in dose-response experiments. EC/LC/MS represents a fast, simple, convenient and functional tool to study DNA adduct formation which has great potential to complement the existing battery of mutagenicity/genotoxicity tests.
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