Estrogen is a known risk factor for human breast cancer, although the mechanism by which estrogens induce cancer remains unestablished. We have demonstrated that DNA strand breakage is induced synergistically when pBR322 plasmid DNA is incubated in the presence of both a nitric oxide (NO)-releasing compound (diethylamine NONOate, etc.) and a catechol-estrogen (2- or 4-hydroxyestradiol or -hydroxyestrone). Either the NO-releasing compound or the catechol-estrogen alone induced much fewer strand breaks. Estradiol, estrone, O-methylated catechol-estrogens, and diethylstilbestrol did not exert such DNA damaging effects. Strand breakage induced by NO plus 2- or 4-hydroxyestradiol was inhibited by carboxy-PTIO (an NO-trapping agent) and, to a lesser extent, by superoxide dismutase. Antioxidants (e.g., N-acetylcysteine, ascorbate), but not HO. scavengers, exhibited inhibitory effects. A possible mechanism for this strand breakage would be: (1) NO mediates conversion of catechol-estrogens to quinones, (2) the quinone/hydroquinone redox system produces O2.-, and (3) O2.- reacts with NO to form peroxynitrite, which causes DNA strand breaks. Our results imply that interaction of catechol-estrogens and NO, both known to be formed in human breast and uterus, leads to production of a potent oxidant(s), which could cause damage in cells and DNA, thus playing an important role in hormonal carcinogenesis.