Room temperature derivatization of 5-hydroxy-2'- deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine for analysis by...

Hydroxylated DNA basesare one type of oxygen free radical-induced damage to DNA. Such damage has been implicated in the process of carcinogenesis, and the levels of hydroxylated DNA bases may serve as a marker of cancer risk in humans. Measurement of oxidative DNA damage can be hampered by the ease with which artifactual oxidative DNA damage can be induced via sample processing. In this report we describe convenient room temperature derivatization and stability of 5-hydroxy-2'-deoxycytidine (5-OHdCyd) and 5-hydroxymethyl-2'-deoxyuridine (5-OHmdU) using GC/MS analysis. The derivatization reagent was N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchloro-silane:acetonitrile, 2:1. This method avoids use of acid and is much milder than previously reported derivatization conditions which typically involve heating above 100C for at least 20 min. Although heating has been reported to be problematic, the calculated levels of 5-OHdCyd and 5-OHmdU in enzymatically-hydrolysed calf thymus DNA were very similar in our hands with and without heating the sample for 20 min. As an example of the technique, comparison of 5-OHdCyd and 5-OHmdU levels in calf thymus DNA indicated relatively higher endogenous levels of 5-OHdCyd. In DNA treated with hydrogen peroxide and ferric chloride, however, the levels of 5-OHmdU increased much more than that of 5-OHdCyd. In addition to these hydroxylated derivatives of deoxycytidine and thymidine, the method also appears to work well with 8-oxoguanine, 4,6-diamino-5-(formylamino)pyrimidine, and 5-methyl-2'-deoxycytidine. This method may therefore be useful with a variety of modified DNA bases and nucleosides.