Visit our Expo - Redox and Inflammation signaling 2012

Session XV : Reactive oxygen species and cell signaling Poster XV, 2
Production of DNA strand breaks by metal-induced oxygen radicals
Ezzatollah Keyhani(1,2), Farnoosh Attar(1), Fatemeh Abdi-Oskoui(1), Jacqueline
Keyhani(2)
(1)Institute of Biochemistry and Biophysics, University of Tehran, 13145 Tehran, and
(2)Laboratory for Life Sciences, Tehran 19979, Iran. E-mail: keyhanie@ibb.ut.ac.ir
Elevated levels of reactive oxygen species (ROS) have been implicated in the etiology of
cancer, neurodegenerative and cardiovascular diseases, as well as in the aging process and as
a condition promoting breast cancer metastases. H2O2, a product of the cell metabolism, is a
potential source of ROS. In small concentrations (10-6 M), H2O2 is a signaling molecule
capable of inducing chemotactic activity, stimulating the synthesis of cytoskeleton elements,
and causing changes in cytosolic calcium concentrations. H2O2 is normally disposed of by
specialized enzymes such as catalases and peroxidases. However, various physiologic
perturbations of cellular homeostasis may lead to a dramatic increase in the amount of H2O2
within a cell. In the presence of transition metals, H2O2 is rapidly converted to the highly
reactive and highly toxic hydroxyl radical. The latter is responsible for lipid peroxidation,
oxidative damage to proteins and breakage of DNA strands. In this study, the extent of DNA
damage caused by H2O2 in the presence of various transition metals was evaluated in vitro.
Purified Salmonella typhimurium DNA was exposed to 0.1 to 100 mM H2O2 and either
Fe2+, Fe3+, Cu2+, Ni2+, or Cd2+ in increasing concentrations from 0.01 to 0.1 mM.
Damage to DNA was assessed by electrophoresis of the DNA preparations in 1% agarose gel.
Breakage of the DNA strands would produce a series of DNA fragments of various sizes
resulting in a smear in the gel after electrophoresis while intact DNA would produce a single
band. Results showed that all of the metals investigated triggered DNA breakage in the
presence of H2O2. The extent of breakage depended on the metal ion, its concentration, and
H2O2 concentration. Addition of either EDTA or catalase to the reaction mixture completely
inhibited the DNA degradation, confirming the involvement of both the metal ion and the
H2O2 in the breakage of DNA strands. Production of the hydroxyl radical when H2O2 and a
metal ion were both present in the reaction mixture was verified by the thiobarbituric acid
method. The extent of DNA breakage caused by the metal ions was as follows:
Cu2+>Fe2+>Fe3+>Ni2+>Cd2+.
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