Abstract
Metabolic activation of estrogens to catechols and further oxidation to highly reactive o-quinones generates DNA damage including apurinic/apyrimidinic (AP) sites. 4-Hydroxyequilenin (4-OHEN) is the major catechol metabolite of equine estrogens present in estrogen replacement formulations, known to cause DNA strand breaks, oxidized bases, and stable and depurinating adducts. However, the direct formation of AP sites by 4-OHEN has not been characterized. In the present study, the induction of AP sites in vitro by 4-OHEN and the endogenous catechol estrogen metabolite, 4-hydroxyestrone (4-OHE), was examined by an aldehyde reactive probe assay. Both 4-OHEN and 4-OHE can significantly enhance the levels of AP sites in calf thymus DNA in the presence of the redox cycling agents, copper ion and NADPH. The B-ring unsaturated catechol 4-OHEN induced AP sites without added copper, whereas 4-OHE required copper. AP sites were also generated much more rapidly by 4-OHEN. For both catechol estrogens, the levels of AP sites correlated linearly with 8-oxo-dG levels, implying that depuriniation resulted from reactive oxygen species (ROS) rather than depurination of estrogen-DNA adducts. ROS modulators such as catalase, which scavenges hydrogen peroxide and a Cu(I) chelator, blocked the formation of AP sites. In MCF-7 breast cancer cells, 4-OHEN significantly enhanced the formation of AP sites with added NADH. In contrast, no significant induction of AP sites was detected in 4-OHE-treated cells. The greater redox activity of the equine catechol estrogen produces rapid oxidative DNA damage via ROS, which is enhanced by redox cycling agents and interestingly by NADPH-dependent quinone oxidoreductase.