[57] Fluorometric analysis of DNA unwinding to study strand breaks and repair in mammalian cells

This chapter describes a fluorometric analysis of DNA unwinding (FADU) to study strand breaks and repair in mammalian cells. The FADU technique allows the study of DNA strand breaks in cells such as leukocytes in which DNA cannot be readily radiolabeled. The principle of the method is that, under certain conditions, double-stranded DNA in crude cellular extracts can be measured using a fluorescent dye, ethidium bromide, with relatively little interference from single-stranded DNA and RNA. Different suspensions of cells are exposed either to no treatment or to treatments such as ionizing radiation, hydrogen peroxide, or other agents that cause strand breaks. An extract of each cell suspension is exposed to alkaline denaturing conditions for a fixed period of time; the pH is then lowered to stop further unwinding, and the amount of residual double-stranded DNA is determined using the fluorescence of ethidium bromide. The FADU technique has proved to be a highly useful method for detecting and quantifying DNA strand breaks induced by a wide variety of treatments in many cell types, including granulocytes, lymphocytes, bone marrow cells, leukemia cells, endothelial cells, and fibroblasts. When semiautomated by using a computer to record, calculate, and plot data, 16 or more samples can be analyzed on a daily basis by a single experienced technician.