Detection of 8-oxoguanine in cellular DNA using 2,6-diamino-8-oxopurine as an internal standard for high-performance liquid chromatography with electrochemical detection

The quantitative aspect of the electrochemical detection method to detect 8-oxo-7,8-dihydroguanine (8-oxoGua) has been improved by using an internal standard. In addition, emphasis was placed on the reduction of artifactual oxidation of DNA during isolation and hydrolysis. Nuclear DNA was isolated from rat organs and purified on an anion-exchange column following treatment with proteinase K and RNase. DNA hydrolysis to nucleobases or nucleosides was performed using either formic acid treatment or enzymatic digestion, respectively. The levels of either 8-oxoGua or 8-hydroxy-7,8- dihydro-2'-deoxyguanosine were comparable. For accurate quantification, 2,6- diamino-8-oxopurine [(NH₂)₂-OH-Pur], added prior to hydrolysis, was used as an internal standard for the high-performance liquid chromatography with electrochemical detection assay. The baseline level of 8-oxoGua in DNA of Sprague-Dawley rats was estimated to be 2 to 5 8-oxoGua residues per 10⁶ DNA bases, with slight differences depending on the tissue origin. In agreement with the results of previous observations, the level of the oxidized base in the kidney of animal treated with iron complexed to nitrilotriacetic acid (Fe-NTA) (15 mg/kg) was three- to fourfold higher than that of untreated rats or animals treated with a saline solution, while there was no change in 8- oxoGua levels in the liver and colon of these treated animals.