AUTHOR=Gaertner Andrea A. E. , Perron Nathan R. , Anderholm Heeren M. G. , Whitehead Daniel C. , Brumaghim Julia L. 

TITLE=Quantifying antioxidant activity of hydrophobic compounds using metal-mediated DNA damage

JOURNAL=Frontiers in Chemical Biology

VOLUME=Volume 4 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/chemical-biology/articles/10.3389/fchbi.2025.1677610

DOI=10.3389/fchbi.2025.1677610

ISSN=2813-530X

ABSTRACT=Cellular damage and death caused by oxidative stress by reactive oxygen species play an important role in disease development. Testing the ability of hydrophobic compounds to prevent radical-generated oxidative stress typically involves radical scavenging assays; however, these oxidative stress assays often do not accurately reflect biological outcomes. We present an in vitro assay that quantifiably evaluates the ability of hydrophobic compounds to prevent DNA damage, a biological endpoint that is linked to disease development. This gel electrophoresis assay enables evaluation of a wide range of hydrophobic compounds for metal-mediated hydroxyl radical damage prevention by using high-ethanol concentrations in electrophoretic conditions, and the effects of these high-ethanol conditions on iron- and copper-mediated DNA damage are established. This assay was used to compare the effects of metal-mediated DNA damage and its prevention by polyphenols, bipyridine, and selone antioxidant compounds as well as the radical scavenger edaravone. We also demonstrated that the well-studied glutathione peroxidase mimic ebselen and a group of ebselen derivatives prevent copper-mediated DNA damage with IC50 values in the 280–450 µM range. These same compounds do not inhibit iron-mediated DNA damage under similar conditions. This DNA-damage assay allows determination of antioxidant properties for hydrophobic antioxidants and drugs using a model system based on metal-mediated oxidative DNA damage, a primary cause of cell death.