Simulation of redox processing involving nucleic acids

DNA damage has important implications in human health and may be responsible for the development of different diseases including cancer. Oxidation represents a key step in the alteration of the genetic material responsible for mutagenesis, genotoxicity, and/or cancerogenesis. Oxidation reactions produce reactive electrophiles that covalently bind to nucleic acid molecules. One class of reactive electrophiles are reactive oxygen species (ROS); the other ones are (bio)activated small molecules. A number of in vitro tests using different bacterial and mammalian cell lines and in vivo tests mainly using rodents are available to identify compounds that induce genetic damage. Despite considerable success, problems regarding sensitivity are often encountered with these assays due to insufficient metabolic activation, side reactions with biological material (e.g. proteins), transport effects or DNA protection. To overcome these limitations we want to develop a "pure" instrumental method based on on-line electrochemistry (EC) - liquid chromatography (LC) - mass spectrometry (MS) for testing the reactivity of synthetic nucleic acids with chemicals including tentative mutagenic compounds and ROS. In the envisioned setup EC will be used to mimic biooxidation processes; formed reaction products will be separated by LC, which facilitates their consecutive detection and characterization with MS. A clear advantage of the envisioned EC/LC/MS technology is its inherent speed of data generation. Expected run times will be as low as a few minutes. Furthermore, the assay will be fully automated. Nucleic acids tested will include nucleosides and nucleotides as well as small oligomers. Thus, EC/LC/MS should even be capable to study site-selectivity of reaction, necessary to discover mutational hotspots. Transformation products will be characterized with high performance MS and tandem MS to elucidate their structure. Identified lesions will represent putative biomarkers for studying the relationship between exposure and the development of diseases. We expect to gain new insights into the mechanisms guiding the reactivity between nucleic acids and (activated) molecules. The derived reaction principles will be beneficial in medical, pharmaceutical and toxicological research for risk assessment and disease prevention and might stimulate the development of new drugs (e.g. antitumor drugs, chemopreventive antioxidants).

DNA damage has important implications in human health and is known to be involved in the development of different diseases including cancer. Oxidation represents a key step in the alteration of the genetic material responsible for mutagenesis, genotoxicity, and/or cancerogenesis. Oxidation reactions produce reactive electrophiles that covalently bind to nucleic acid molecules. One class of reactive electrophiles are reactive oxygen species (ROS); the other ones are (bio)activated small molecules. As part of this project, new and advanced techniques for the fast, efficient and convenient characterization of the impact of oxidation reactions on genetic material were developed. Nucleobases, nucleosides, nucleotides and small oligomers were used as sample set. Oxidation was accomplished by electrochemical methods. Chromatographic separation in combination with mass spectrometry allowed in-depth characterization of the reaction mixtures obtained. New and exciting insights into reactivity and stability of the different nucleic acids constituents were gained. The derived reaction principles are of major importance in medical and toxicological research and will enable the development of new strategies for risk assessment and disease prevention. One competent approach for protecting the genetic material from oxidative stress might involve the use of antioxidants. Antioxidants are a class of molecules that inhibit the oxidation of other molecules. As part of this project, we could demonstrate that electrochemical techniques are very useful for the fast and efficient characterisation of antioxidants. Particularly the combination of electrochemistry with mass spectrometry enables the simultaneous identification and valuation of antioxidants part of complex mixtures, such as different kinds of wine samples.