Measurement of oxidative damage and antioxidative protection on nucleic acid-based biosensors

As an oxygen-breathing organism, the formation of reactive oxygen species (ROS) is inevitable in the human body. Among these species, the most reactive is the hydroxyl radical (•OH), which causes non-selective oxidative damage to biomacromolecules such as DNA, lipids, and proteins. ROS are not harmful as long as they are balanced by antioxidants, either enzymatic or dietary. However, a disruption of this balance in favor of oxidants can lead to various neurodegenerative damages. In vitro investigation of these effects using realistic probes provides valuable information for health. In this thesis, oxidative damage induced by •OH, obtained by the Fenton method (Fe(II)-EDTA/H2O2), on DNA bases was investigated. For this purpose, oxidative damage induced on guanine, adenine, cytosine, and thymine was determined using colorimetric (CUPRAC, ABTS), chromatographic (HPLC, GC-MS/MS), and electroanalytical (differential pulse voltammetry) methods. Following the determination of oxidative damage, the protective effects of specific antioxidants (gallic acid, ascorbic acid, glutathione, and N-acetyl cysteine) on oxidative damage were investigated. All techniques except GC-MS/MS attempted to determine the total damage products without examining the species released as a result of oxidative damage. As stated in the literature, in addition to the existence of nearly dozens of different types of oxidative damage products, most of these species are extremely unstable. Another drawback is that some species easily interconvert. Considering all these factors, it is understood that monitoring damage based on a single product is unrealistic. In this thesis, total damage products were determined using spectroscopic methods and HPLC. The GC-MS/MS method was used to separately identify potential damage products. This method allows for the simultaneous determination of many stable damage products, not just one type. For the colorimetric CUPRAC method, different concentrations of four DNA bases were taken and subjected to oxidative damage via the Fenton reaction. While the bases themselves were not CUPRAC reactive, the damage products exhibited a CUPRAC reaction. A direct proportionality was determined between the increasing concentration of the DNA base and the CUPRAC absorbance. In the ABTS method, the oxidative damage products released turn colorless ABTS reagent to a green cationic radical. Again, increasing base concentrations and color intensity were found to be directly proportional. Colorimetric methods were also repeated in the presence of four different antioxidants, and both methods determined that gallic acid provided the most effective protection. In the HPLC method, two injections were made for different concentrations of each base, one before and one after the Fenton oxidation. The difference between the peak areas obtained for the two injections was then plotted against increasing base concentrations. A linear relationship was also observed here. In experiments conducted with GC-MS/MS, the highest concentration of the damage product for guanine and adenine was alloxan, while the highest concentration for cytosine was 5-hydroxy-cytosine, and for thymine, 5-hydroxy-5-methyl-hydantoin. These results are also consistent with the literature. With the voltammetric method, damage to guanine and adenine, the more electroactive species, was detected even at low concentrations of these bases. However, while thymine, a pyrimidine base, showed reactivity at very high concentrations, no peak was observed in the voltamatogram for cytosine.