A. Abushakra et E. Zeiger, FORMATION OF 8-HYDROXY-2'-DEOXYGUANOSINE FOLLOWING TREATMENT OF 2'-DEOXYGUANOSINE OR DNA BY HYDROGEN-PEROXIDE OR GLUTATHIONE, Mutation research. Genetic toxicology and environmental mutagenesis, 390(1-2), 1997, pp. 45-50
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.