Synthetic estrogen induces nitric oxide deficiency via endothelial xanthine oxidase

Abstract

Estrogen protects cardiovascular health partially via upregulation of nitric oxide (NO) production. Its synthetic analog diethylstilbestrol (DES), commonly used as an androgen deprivation therapy for patients with prostate cancer or a hormone replacement agent for post menopause women, is however associated with high incidence of thromboembolic events. We sought to determine whether diethylstilbestrol causes endothelial NO deficiency. Bioavailable NO produced by aortic endothelial cells was detected by electron spin resonance (ESR), and was markedly decreased by pharmacologically relevant dose of DES (12.5 μmol/L, 24 hr). 17β-estradiol instead increased NO production. Furthermore, DES induced a potent increase in endothelial superoxide (detected by dihydroethidium and ESR assays) and hydrogen peroxide (determined by Amplex Red assay) production, which was attenuated by Tamoxifen (1 μmol/L) and abolished by oxypurinol (100 μmol/L), an inhibitor for xanthine oxidase. DES alone in a cell free system did not produce any ESR-sound superoxide signal. Pretreatment with oxypurinol also prevented DES-induced loss of NO. Of note, eNOS mRNA and protein remained unchanged in response to DES. These data suggest that DES differentially activates xanthine oxidase to produce reactive oxygen species via a receptor dependent mechanism. These findings provide insights into molecular mechanisms underlying thromboembolic events associated with DES administration. Novel approaches targeting these mechanisms may prove beneficial for patients receiving DES for treatment. This work is supported by an American Heart Association Grant (0435189N), an American Diabetes Association grant (7-04-RA-16) and NIH Grants HL077440 and HL081571 (all to H Cai).