MEASUREMENT OF 2,6-DIAMINO-4-HYDROXY-5-FORMAMIDOPYRIMIDINE AND 8-OXO-7,8-DIHYDROGUANINE IN ISOLATED DNA EXPOSED TO GAMMA-RADIATION IN AQUEOUS-SOLUTION

Hydroxyl radical-mediated modifications of nucleobases are among the m ain deleterious processes induced in DNA by ionizing radiation, Determ ination of the underlying mechanisms in both isolated and cellular DNA requires the development of accurate assays for the detection of modi fied bases, High-performance liquid chromatography associated with ele ctrochemical detection (HPLC-EC) and gas chromatography coupled to mas s spectrometry (GC-MS) are the two main methods used for this purpose, In the present work, HPLC-EC was applied to the measurement of 8-oxo- 7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) following enzymatic digestio n of DNA, Evidence was provided for the quantitative aspect of the lat ter step, Moreover, a GC-MS assay was designed for the detection of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), It was first show n that the widely used hot formic acid hydrolysis of DNA induced a com plete decomposition of the lesion, A similar observation was also made for 4,6-diamino-5-formamidopyrimidine (FapyAde), A milder DNA hydroly zing agent, HF in pyridine, was substituted to HCOOH for the quantitat ive release of FapyGua from DNA, The radiation-induced formation of 8- oxodGuo and FapyGua in aqueous solution of DNA was compared, Insights in the mechanistic aspects were inferred from the results of gamma-irr adiation studies carried out under different gas saturating conditions , It was clearly shown that the formation of both lesions is mediated by HO degrees. In addition, the presence of oxygen favors the formatio n of 8-oxodGuo at the expense of FapyGua.