In previous studies, we observed that Cu(II) strongly induces the oxidation of hydroquinone (HQ), 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 spectroscopy we have investigated whether this chemical-metal redox system can generate reactive oxygen species which induce DNA damage. In order to set the stage for the ESR experiments and the inhibitors to be used in these experiments, some preliminary O2 consumption and plasmid DNA cleavage experiments were performed. Mixing 100 microM HQ with 10 microM Cu(II) in phosphate-buffered saline (PBS) resulted in a marked consumption of O2 and the concomitant generation of H2O2, and extensive DNA degradation in chi X-174 RF I DNA. The presence of superoxide dismutase (SOD) or mannitol did not affect either the O2 consumption, H2O2 generation or DNA damage. In contrast, the Cu(I) chelators, bathocuproinedisulfonic acid (BCS) and glutathione (GSH), extensively inhibited the HQ/Cu(II)-mediated O2 consumption and DNA damage. The presence of catalase also prevented the DNA damage. Although the HQ/Cu(II)-mediated O2 consumption increased in the presence of azide, azide markedly inhibited 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-butylnitrone (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, suggesting 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 adduct 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 approximately 85% decrease in the formation of 4-POBN-CH3 adduct. Reactive oxygen scavenger experiments showed that the formation of the 4-POBN-CH3 adduct was significantly inhibited by azide but not by mannitol. Overall, the 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.