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DNA Strand Breaks by Metal-Induced Oxygen Radicals in Purified Salmonella typhimurium DNA

Authors


Address for correspondence: Dr. Ezzatollah Keyhani, Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, 13145 Tehran, Iran. Voice: +98-21-6695-6974; fax: +98-21-6640-4680.
 e-mail: keyhanie@ibb.ut.ac.ir

Abstract

Abstract: Purified Salmonella typhimurium DNA was incubated for 1h at 37°C with various concentrations (10–100 μM) of transition metal ions (Fe2+, Fe3+, Cu2+, Ni2+, Cd2+), with various concentrations (0.1–100 mM) of H2O2, and with various concentrations of each transition metal ion in the presence of various concentrations of H2O2. Damage to DNA was assessed by electrophoresis of the reaction mixtures in 1% agarose gel. Breakage of the DNA strands would produce a series of DNA fragments resulting in a smear in the gel, while intact DNA produced a single band. Results showed that no damage to the DNA was detectable after incubation with either H2O2 alone or either of the metal ions alone. However, all of the metal ions investigated triggered DNA breakage in the presence of H2O2. The extent of breakage depended on the metal ion and on its concentration, as well as on the 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 evidenced by the thiobarbituric acid method. The most extensive damage was caused by Cu2+ followed, in decreasing order, by Fe2+, Fe3+, Ni2+, and Cd2+.

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