Tandem base lesions are generated by hydroxyl radical within isolated DNA in aerated aqueous solution

The hydroxyl radical-mediated formation of two tandem base lesions within D NA including N-(2-deoxy-beta-D-erythro-pentofuranosyl)formylamine-8-oxo-7,8 -dihydro-2'-deoxyguanosine (d beta F-8-oxodGuo) and and 8-oxodGuo-d beta F is reported in this study. A specific enzymatic processing was developed to quantitatively release the tandem lesions as dinucleoside monophosphates f rom DNA. Then, the resulting hydrolyzed DNA samples were analyzed using liq uid chromatography coupled to electrospray ionization tandem mass spectrome try. The simultaneous measurement of the two vicinal lesions was performed in the negative mode using the accurate and sensitive multiple reaction mon itoring technique. For this purpose, two characteristic ions arising from t he fragmentation of the pseudo-molecular ion [M - H](-) were monitored. Bot h d beta F-8-oxodGuo and 8-oxodGuo-d beta F damage were found to be generat ed in gamma-irradiated DNA as a significant fraction of (OH)-O-. radical-in duced base damage. Interestingly, 8-oxodGuo-d beta F was produced in a much higher yield than the reversed sequence lesion. Indirect evidence is provi ded for the formation of other tandem lesions involving 8-oxodGuo, but that still remain to be fully identified. Insights into the mechanism of format ion of the DNA damage were gained from several experiments including DNA ph otosensitization, gamma-irradiation in the presence of iron, and exposure t o Fenton reagents. This allowed refinement of the proposed pathways for the formation of d beta F-8-oxodGuo and 8-oxodGuo-d beta F tandem base damage.