Immunochemical detection of oxidative DNA damage in cancer and aging usinganti-reactive oxygen species modified DNA monoclonal antibody

The formation of reactive oxygen species (ROS), although a normal cellular activity, is considerably enhanced under chronic inflammatory conditions an d ischemia. These species have been implicated in various disorders, mutage nesis, carcinogenesis and aging. Of many macromolecules, DNA is the most su sceptible to hydroxyl radical, the most reactive of the ROS. The present st udy is designed to detect oxidative DNA damage in cancer patients and healt hy aged humans using an anti-ROS-DNA monoclonal antibody (mAb). Purified ca lf thymus DNA fragments (approximate size 400 bp) were modified with (OH)-O -., generated by UV-irradiation (254 nm) of hydrogen peroxide. ROS-modified DNA was characterized by UV-spectroscopy, melting temperature, alkaline su crose density gradient ultracentrifugation and ion-exchange chromatography. ROS-DNA showed single strand breaks, decrease in T-m, modification of thym ine (58.3%) and guanine (20%). The mAb generated against ROS-DNA was charac terized for antigen binding specificity by competition ELISA. Monoclonal an tibody showed strong binding to ROS-modified DNA, its modified fragments, p olynucleotides and bases. With the exception of native DNA, binding of unmo dified polynucleotides and bases was much lower. The mAb distinctly recogni zed DNA samples from lymphocytes of healthy aged humans and gave maximum in hibitions of 49, 53, 64 and 70%, while not reacting with DNA from young pop ulation. Similarly, oxidative lesions in DNA from cancer patients were also efficiently recognized by the mAb. DNA from healthy controls served as neg ative control. The studies demonstrate that the mAb, although cross-reactiv e, preferentially binds ROS-modified epitopes on DNA. High reactivity of mA b to DNA samples from cancer patients and healthy aged humans indicates inc reased oxidative stress leading to DNA damage. (C) 1998 Elsevier Science Lt d. All rights reserved.