FORMATION OF 8-HYDROXY-2'-DEOXYGUANOSINE FOLLOWING TREATMENT OF 2'-DEOXYGUANOSINE OR DNA BY HYDROGEN-PEROXIDE OR GLUTATHIONE

We have demonstrated that free radicals generated by hydrogen peroxide (H2O2), in the presence of divalent iron (Fe2+) and a chelator (EDTA) , oxidize 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHd G). The 8-OHdG formed by this reaction was isolated and quantitated us ing reverse-phase HPLC with UV and electrochemical detection. A 1-h in cubation of dG with H2O2 caused a 50% increase in 8-OHdG over backgrou nd, which increased to 100% after 2 h. However, when an H2O2-generatin g system [glutathione (GSH), Fe2+, EDTA] was used there was no increas e in 8-OHdG yield after the 1-h incubation, but up to a 50% increase o ver background was observed with GSH after 2-h incubation. Attempts to detect increased levels of 8-OHdG after H2O2- or GSH-treatment of pur ified calf thymus or rat DNA, or purified Salmonella typhimurium DNA w ere not successful. This may have been because the treatment procedure s used generated 8-OHdG in the control samples at sufficiently high le vels to mask any H2O2-induced responses that may have been present. Th is artifactual production of 8-OHdG has presented a problem in all in vitro studies to date. In contrast, treatment of Salmonella cells (str ain TA104) with increasing concentrations of H2O2, caused a doubling i n the 8-OHdG yield. GSH-treatment of strain TA104 cells under the same conditions did not result in an increase of 8-OHdG. The study present ed here: shows that the ubiquitous molecule H2O2 can play a major role in DNA oxidation, mutation, and damage.