DNA-DAMAGE CAUSED BY REACTIVE OXYGEN SPECIES ORIGINATING FROM A COPPER-DEPENDENT OXIDATION OF THE 2-HYDROXY CATECHOL OF ESTRADIOL

It has previously been proposed that redox cycling between catechol es trogens and their quinones, mediated by cytochrome P450, could lead to the generation of free radicals that would subsequently cause oxidati ve damage to DNA and proteins that might have a role in hormonal carci nogenesis. Alternative, non-enzymatic mechanisms involving copper have been shown to participate in the oxidation of various chemicals throu gh processes that also result in the appearance of reactive oxygen spe cies and subsequent site-specific oxidative DNA damage. The goal of th e present study was to determine whether the 2-hydroxy-catechol of est radiol (2-OH-E2) can be oxidized by copper through a process which gen erates reactive oxygen species that cause oxidative DNA damage as dete cted by the appearance of strand breaks in phi X-174 plasmid DNA. Our results show that both single- and double-strand breaks are formed in the presence of Cu(II) plus micromolar concentrations of 2-OH-E2, and 4-OH-E2, in a concentration/time-dependent process. No strand breaks w ere detected in the presence of Cu(II) or 2-OH-E2 alone. The reaction of 2-OH-E2 with Cu(II) was accompanied by the reduction of Cu(II) to C u(I), the utilization of O-2, and the generation of H2O2. The utilizat ion of O-2 and the formation of strand breaks was completely blocked b y the Cu(I)-specific chelator bathocuproinedisulfonic acid (BCS) at a ratio of BCS to Cu(II) of 4:1. The appearance of strand breaks was als o blocked by catalase and inhibited by the singlet oxygen scavengers s odium azide and 2,2,6,6-tetramethyl-4-piperidone. In contrast the free hydroxyl radical scavengers mannitol and N-tert-butyl-alpha-phenylnit rone were not effective inhibitors; superoxide dismutase had no inhibi tory effect. These results are similar to what has been observed by ot hers for the formation of oxidative DNA damage by the H2O2/Cu(II) syst em and by us for the induction of strand breaks by hydroquinone/ Cu(II ). Since copper is known to be present in the nucleus, particularly in association with guanines in DNA, our results with 2-OH-E2/Cu(II) tog ether with those of others with H2O2Cu(II), discussed below, suggest a n alternate site-specific mechanism for the formation of oxidative DNA damage associated with estrogen treatment. Furthermore, the results s uggest that the oxidative damage results from the localized generation of singlet oxygen or a similar bound reactive entity rather than free hydroxyl radical.