Formation of the oxidative damage marker 8-hydroxy-2′-deoxyguanosine from the nucleoside 2′-deoxyguanosine: parameter studies and evidence of Fe(II) binding

Abstract

High-performance liquid chromatography (HPLC) with UV absorption detection was employed to measure the amounts of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) produced from the nucleoside 2′-deoxyguanosine (dG) under varying reaction conditions using iron and H 2O 2. The results indicate that 8-OH-dG produced from the reaction of iron and H 2O 2 with dG can undergo reaction with free (i.e., unchelated) Fe(III) and that adding the chelating agent ethylenediaminetetraacetic acid (EDTA) after the reaction prevents this from occurring. It also appears that the free radical species generated by iron-EDTA chelates in pH 7.4 N-(2-hydroxyethyl)piperazine- N′-(2-ethanesulfonic acid) (Hepes) buffer is either not formed or unstable in unbuffered aqueous solution. Finally, 8-OH-dG levels are significantly larger when Fe(II) is allowed to bind to the nucleoside dG prior to addition of H 2O 2. However, production of 8-OH-dG from unbound Fe(II) is also relevant. The results of this work show that differing reaction conditions in vivo, especially at the cellular level, will affect significantly the measured yields of 8-OH-dG. These results also have implications for studies involving DNA and the ability to distinguish between 8-OH-dG produced from free iron and iron bound to both phosphate groups and the DNA base guanine.