ROLE OF CU ZN-SUPEROXIDE DISMUTASE IN XENOBIOTIC ACTIVATION .2. BIOLOGICAL EFFECTS RESULTING FROM THE CU/ZN-SUPEROXIDE DISMUTASE-ACCELERATED OXIDATION OF THE BENZENE METABOLITE 1,4-HYDROQUINONE/

Cu/Zn-superoxide dismutase (SOD)-accelerated oxidation of the benzene metabolite 1,4-hydroquinone (HQ) results in the enhanced formation of semiquinone anion radicals, electrophilic I,4-benzoquinone (BQ), and H 2O2. We selected bone marrow stromal cells and oX-174 double-stranded plasmid DNA as model systems to investigate the cytotoxicity and DNA c leaving activity of the Cu/Zn-SOD-mediated activation of HQ. The addit ion of either Cu/Zn-SOD or Mn-SOD to the primary bone marrow stromal c ell cultures significantly enhanced HQ-induced cytotoxicity, which cou ld be completely prevented by adding reduced glutathione (GSH) or dith iothreitol but not by adding catalase. Incubation of the plasmid DNA w ith the HQ/Cu/Zn-SOD system resulted in the induction of single- as we ll as double-strand breaks, which could be inhibited by catalase and t he Cu(I) chelators, bathocuproinedisulfonic acid (BCS) and GSH. Althou gh Mn-SOD could enhance HQ-induced cytotoxicity to stromal cells, the activation of HQ by Mn-SOD did not contribute to the induction of DNA strand breaks. Similar to the HQ/Cu(ll) and H2O2/Cu(ll) systems, the D NA strand breaks mediated by HQ/Cu/Zn-SOD could not be effectively inh ibited by the hydroxyl radical scavengers, including dimethylsulfoxide , mannitol, and 5,5-dimethyl-1-pyrroline N-oxide, but could be protect ed by sodium azide. Low-temperature electron spin resonance experiment s showed that incubation of Cu/Zn-SOD with HQ resulted in the release of copper from the Cu/Zn-SOD, which could be prevented by catalase. al pha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (POBN)/spin-trapping studi es demonstrated that the interaction of HQ with Cu/Zn-SOD, but not wit h Mn-SOD, resulted in the significant formation of POBN-CH3, adduct in the presence of dimethylsulfoxide, suggesting the production of hydro xyl radical or its equivalent from this enzyme/xenobiotic interaction. The formation of the POBN-CH, adduct from the HQ/Cu/Zn-SOD could be i nhibited by catalase, BCS or GSH, indicating the important role for H2 O2 and Cu(I) in the production of reactive oxygen species. Addition of human myeloperoxidase to the HQ/Cu/Zn-SOD synergistically enhanced th e formation of BQ from HQ. This enhancement could be abolished by cata lase. Taken together, these results demonstrate that activation of HQ by either Cu/Zn-SOD or Mn-SOD results in cytotoxicity to primary bone marrow stromal cells through the formation of electrophilic BQ. Intera ction of HQ with Cu/Zn-SOD causes oxidative damage to Cu/Zn-SOD, leadi ng to the release of copper from the enzyme. The further reaction betw een the released copper and H2O2 generates reactive oxygen species tha t participate in the induction of strand breaks in plasmid DNA. The H2 O2 generated from the Cu/Zn-SOD-accelerated oxidation of HQ can also b e utilized by myeloperoxidase resulting in additional conversion of HQ to BQ.