THE EQUINE ESTROGEN METABOLITE 4-HYDROXYEQUILENIN CAUSES DNA SINGLE-STRAND BREAKS AND OXIDATION OF DNA BASES IN-VITRO

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choic e and continues to be one of the most widely dispensed prescriptions i n North America. In addition to endogenous estrogens, Premarin contain s unsaturated equine estrogens, including equilenin [1,3,5(10),6,8-est rapentaen-3-ol-17-one]. In previous work, we showed that the equilenin metabolite 4-hydroxyequilenin (4-OHEN) can be autoxidized to 4-OHEN-o -quinone which readily entered into a redox couple with the semiquinon e radical catalyzed by NAD(P)H, P450 reductase, or quinone reductase, resulting in generation of reactive oxygen species [Shen, L., Pisha, E ., Huang, Z., Pezzuto, J. M., Krol, E., Alam, Z., van Breemen, R. B., and Bolton, J. L. (1997) Carcinogenesis 18, 1093-1101]. As oxidative d amage to DNA by reactive oxygen species generated by redox active comp ounds has been proposed to lead to tumor formation, we investigated wh ether 4-OHEN could cause DNA damage. We treated II phage DNA with 4-OH EN and found that extensive single-strand breaks could be obtained wit h increasing concentrations of 4-OHEN as well as increasing incubation times. If scavengers of reactive oxygen species are included in the i ncubations, DNA could be completely protected from 4-OHEN-mediated dam age. In contrast, NADH and CuCl2 enhanced the ability of 4-OHEN to cau se DNA single-strand breaks presumably due to redox cycling between 4- OHEN and the semiquinone radical generating hydrogen peroxide and ulti mately copper peroxide complexes. We also confirmed that 4-OHEN could oxidize DNA bases since hydrolysis of 4-OHEN-treated calf thymus DNA a nd HPLC separation with electrospray MS detection revealed oxidized de oxynucleosides, including 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. Our data suggest that DNA single-strand breaks and oxidation of DNA b ases by 4-OHEN could contribute to the carcinogenic mechanism(s) of eq uine estrogens.