Measurement of oxidative DNA damage by gas chromatography–mass spectrometry: ethanethiol prevents artifactual generation of oxidized DNA bases

Abstract

Analysis of oxidative damage to DNA bases by GC-MS enables identification of a range of base oxidation products, but requires a derivatization procedure. However, derivatization at high temperature in the presence of air can cause ‘artifactual ’ oxidation of some undamaged bases, leading to an overestimation of their oxidation products, including 8-hydroxyguanine. Therefore derivatization conditions that could minimize this problem were investigated. Decreasing derivatization temperature to 23 °C lowered levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyl)uracil measured by GC–MS in hydrolysed calf thymus DNA. Addition of the reducing agent ethanethiol (5%, v/v) to DNA samples during trimethylsilylation at 90 °C also decreased levels of these four oxidized DNA bases as well as 5-hydroxyuracil. Removal of guanine from hydrolysed DNA samples by treatment with guanase, prior to derivatization, resulted in 8-hydroxyguanine levels (54–59 pmol/mg of DNA) that were significantly lower than samples not pretreated with guanase, independent of the derivatizationconditions used. Only hydrolysed DNA samples that were derivatized at 23 °C in the presence of ethanethiol produced 8-hydroxyguanine levels (56±8 pmol/mg of DNA) that were as low as those of guanase-pretreated samples. Levels of other oxidized bases were similar to samples derivatized at 23 °C without ethanethiol, except for 5-hydroxycytosine and 5-hydroxyuracil, which were further decreased by ethanethiol. Levels of 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine measured in hydrolysed calf thymus DNA by the improved procedures described here were comparable with those reported previously by HPLC with electrochemical detection and by GC–MS with prepurification to remove undamaged base. We conclude that artifactual oxidation of DNA bases during derivatization can be prevented by decreasing the temperature to 23 °C, removing air from the derivatization reaction and adding ethanethiol.