Investigating Metal-Mediated DNA Damage Prevention by Antioxidants and Examining the Role of Metals in Fluconazole-Mediated DNA Damage

Reactive oxygen species (ROS) play an important role in the development of many diseases. Common assays to test the ability of hydrophobic compounds to prevent oxidative stress are radical scavenging assays and cell studies. However, these radical assays often do not accurately reflect biological outcomes, and the use of different cell lines limits the ability to directly compare compound efficacy (Chapter 1). Results from DNA damage prevention assays can directly compare antioxidant efficacy, but these assays have been limited to water-soluble compounds. Chapter 2 discusses the first assay that quantifiably evaluates the ability of hydrophobic compounds to prevent metal-mediated DNA damage inhibition via gel electrophoresis. This assay allows biologically relevant evaluation of compounds for their effectiveness under consistent conditions. The glutathione peroxidase mimic ebselen and its derivatives prevent copper-mediated DNA damage (IC50 values 280-450 µM), but do not significantly inhibit iron-mediated DNA damage. In combination with radical scavenging assays, these biologically relevant assays enable identification of structure-function relationships for hydrophobic antioxidant compounds and drugs. Studies presented in Chapter 3 investigate the effect of metal binding on drug properties. Fluconazole (FLC) binds both iron and copper, and stabilizes Cu+ and Fe2+ over Cu2+ and Fe3+, respectively, as measured by cyclic voltammetry. Using gel electrophoresis assays, the effects of FLC on copper- and iron-mediated DNA damage were determined. FLC does not cause DNA damage by itself, but addition of FLC lowers the concentration of Fe2+ or Cu+ needed to cause 50% DNA damage (EC50) by 50 and 40 %, respectively, increasing reactive oxygen species production. The studies described in Chapter 4 investigate the antioxidant capabilities of a series of plant-derived, procyanidin-rich condensed tannins (CTs) with different structural features for their ability to inhibit copper (IC50 162.5 ± 0.3 µM to 27% DNA damage inhibition at the highest concentration) and iron-mediated (IC50 0.75 ± 0.01 to 4.96 ± 0.01 µM) DNA damage. The activity of CTs are compared to six commercially available polyphenolic compounds. This is the first study to investigate structure-activity relationships for CTs and their abilities to prevent metal-mediated DNA damage