Detection of Oxidative DNA Damage in Mammalian Cells After Radiation Exposure Using Fluorescence Microscopy and Fluorescence Activated Cell Scanning

Abstract

Astronauts on space missions are at constant risk of exposure to high doses of ionising radiation, which could lead to severe short- and long-term health consequences. It is therefore necessary to persistently develop new state-of-the-art methods to counteract and reduce these negative effects of ionising radiation and maintain the health of astronauts. Oxidative stress is a major consequence of ionising radiation. Exposure to ionising radiation causes formation of reactive oxygen species (ROS), resulting in DNA, lipid and protein oxidation. ROS also disrupt signalling pathways, contribute to aging and to cancer development. Detection of ROS has traditionally been done using fluorescent dyes. A novel detection method is the use of CellROX® Green oxidative stress detector. This fluorescent dye binds to the DNA after oxidation by ROS and permits detection of ROS in living and fixated cells. A staining protocol was therefore developed for the dye and used to study the effects of X-irradiation on ROS formation by fluorescence microscopy and FACS. Menadione which is a known chemical inducer of oxidative stress was used as model substance to induce stress in cells followed by staining with the CellROX Green dye. The assay was established with the use of A549 human epithelial cell and later tested on mouse embryonic fibroblasts (MEF). The effects of dose and time on ROS formation were studied after exposure to X-rays. X-irradiation leads to a dose-dependent increase in oxidative stress and a persistence in oxidative stress for days after irradiation. This study showed that CellROX® Green can be used to reliably measure the amount of ROS and the extent of oxidative DNA damage going on in living cells.