Considerable work has shown that the major metabolites of estradiol and estrone are those hydroxylated at either the C-2 or the C-16a positions, although forms hydroxylated at the C-4 and C-15a are present, but in relatively lesser amounts (FIGURE 1). There exists a complete divergence in the biological properties of the 2- and 16a-hydroxylated metabolites of estradiol.

C-2 metabolites are essentially devoid of peripheral biological activity, as shown in studies on uterine weight, gonadotrophin secretion, and cell proliferation {1}. 2-Hydroxyestrogen has even been found to exert a modest anti-estrogenic effect {2,3}, and has been called "the good estrogen" {4}. The 16a-hydroxylated metabolites, 16a-hydroxyestrone (16OHE1) and estriol (E3), are estrogen agonists {5}.

16OHE1 has several unique properties: it is capable of binding covalently to the estrogen receptor {6}, to nuclear histone proteins {7,8}, and to DNA. Because of this covalent linkage to the receptor, 16OHE1 shows persistent biological responses {9}. The formation of 16OHE1 is elevated in women with breast cancer {10,11}, women at high risk for breast cancer {12}, and in strains of mice with a high incidence of spontaneous mammary tumors {13}. Estradiol and estrone 16a-hydroxylation has been shown to take place in murine and human breast epithelial cell lines, both normal and transformed {14,15,16}, and has also been demonstrated in terminal duct lobular units (TDLU), the functional units of the breast.

In addition to the persistence in estrogenic activity described above, it was recently established that 16OHE1, unlike E2 or E3, possesses both initiator and promoter activities in normal (non-transformed) mammary epithelial cells {17}. In proliferation assays 16OHE1 had activity comparable to that observed for dimethlybenzanthracene (DMBA), unlike E2 and E3, both of which showed only minimal responses. In a mutagenic assay measuring unscheduled DNA repair, 16OHE1 was likewise considerably more potent than estrone (E1), E2 or E3. Measurements of anchorage-independent colony formation of mammary epithelial cells grown in soft agar showed that 16OHE1 was far more potent than E1, E2, or E3 at increasing growth.

16OHE1 is the only estrogen that has been shown to be mutagenic in the Ames test, causing his+ revertants in 2 of 5 cell lines tested [18]. Because the 16a-hydroxylated compounds are very potent estrogens, these significant changes may have important hyper-estrogenic consequences that could have a bearing upon the etiology of gynecologic cancers, especially breast cancer. With this association proven, the ratio 2OHE1/16OHE1 could serve as an innovative intermediate biomarker for breast cancer risk, and provide an analytical framework for the development of new pharmaceutical and dietary intervention strategies.