ESR EVIDENCE FOR THE GENERATION OF REACTIVE OXYGEN SPECIES FROM THE COPPER-MEDIATED OXIDATION OF THE BENZENE METABOLITE, HYDROQUINONE - ROLE IN DNA-DAMAGE

In previous studies, we observed that Cu(II) strongly induces the oxid ation of hydroquinone (KQ), producing benzoquinone and H2O2 through a Cu(II)/Cu(I) redox cycle mechanism, The oxidation of HQ by Cu(II) also results in plasmid DNA cleavage, In this study, using ESR spectroscop y we have investigated whether this chemical-metal redox system can ge nerate reactive oxygen species which induce DNA damage. In order to se t the stage for the ESR experiments and the inhibitors to be used in t hese experiments, some preliminary O-2 consumption and plasmid DNA cle avage experiments were performed. Mixing 100 mu M HQ with 10 mu M Cu(I I) in phosphate-buffered saline (PBS) resulted in a marked consumption of O-2 and the concomitant generation of H2O2, and extensive DNA degr adation in phi X-174 RF I DNA, The presence of superoxide dismutase (S OD) or mannitol did not affect either the O-2 consumption, H2O2 genera tion or DNA damage. In contrast, the Cu(I) chelators, bathocuproinedis ulfonic acid (BCS) and glutathione (GSH), extensively inhibited the HQ /Cu(II)-mediated O-2 consumption and DNA damage. The presence of catal ase also prevented the DNA damage. Although the HQ/Cu(II)-mediated O-2 consumption increased in the presence of azide, azide markedly inhibi ted the HQ/Cu(II)-induced DNA degradation, resulting in primarily open circles. Using ESR spectroscopy, it was observed that Cu(II) strongly mediated the formation of semiquinone anion radicals from HQ in PBS, which could be blocked by BCS. alpha-(4-Pyridyl-1-oxide)-N-tert-butyln itrone (4-POBN)-spin trapping experiments showed that the interaction of HQ with Cu(II) produced 4-POBN-CH3 and 4-POBN-CH(OH)CH3 adducts in the presence of dimethyl sulfoxide (DMSO) and ethanol, respectively, s uggesting that hydroxyl radical or an equivalent reactive intermediate is generated from the HQ/Cu(II) system. The presence of catalase, BCS or GSH but not SOD completely prevented the formation of 4-POBN-CH3 a dduct from the HQ/Cu(II) plus 4-POBN/DMSO system. This indicates that both H2O2 and Cu(I) are critical for the formation of reactive oxygen from the HQ/Cu(II) system. Anaerobic conditions induced an similar to 85% decrease in the formation of 4-POBN-CH3 adduct. Reactive oxygen sc avenger experiments showed that the formation of the 4-POBN-CH3 adduct was significantly inhibited by azide but not by mannitol. Overall, th e above results indicate that through a copper-redox cycling mechanism the copper-mediated oxidation of HQ generates reactive oxygen species which may participate in DNA damage.