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Many women consider botanical dietary supplements (BDSs) as safe alternatives to hormone therapy for menopausal symptoms. However, the effect of BDSs on breast cancer risk is largely unknown. In the estrogen chemical carcinogenesis pathway, P450 1B1 metabolizes estrogens to 4-hydroxylated catechols, which are oxidized to genotoxic quinones that initiate and promote breast cancer. In contrast, P450 1A1 catalyzed 2-hydroxylation represents a detoxification pathway. The current study evaluated the effects of red clover, a popular BDS used for women's health, and its isoflavones, biochanin A (BA), formononetin (FN), genistein (GN), and daidzein (DZ), on estrogen metabolism. The methoxy estrogen metabolites (2-MeOE1, 4-MeOE1) were measured by LC-MS/MS, and CYP1A1 and CYP1B1 gene expression was analyzed by qPCR. Nonmalignant ER-negative breast epithelial cells (MCF-10A) and ER-positive breast cancer cells (MCF-7) were derived from normal breast epithelial tissue and ER+ breast cancer tissue. Red clover extract (RCE, 10 μg/mL) and isoflavones had no effect on estrogen metabolism in MCF-10A cells. However, in MCF-7 cells, RCE treatments downregulated CYP1A1 expression and enhanced genotoxic metabolism (4-MeOE1/CYP1B1 > 2-MeOE1/CYP1A1). Experiments with the isoflavones showed that the AhR agonists (BA, FN) preferentially induced CYP1B1 expression as well as 4-MeOE1. In contrast, the ER agonists (GN, DZ) downregulated CYP1A1 expression likely through an epigenetic mechanism. Finally, the ER antagonist ICI 182,780 potentiated isoflavone-induced XRE-luciferase reporter activity and reversed GN and DZ induced downregulation of CYP1A1 expression. Overall, these studies show that red clover and its isoflavones have differential effects on estrogen metabolism in "normal" vs breast cancer cells. In breast cancer cells, the AhR agonists stimulate genotoxic metabolism, and the ER agonists downregulate the detoxification pathway. These data may suggest that especially breast cancer patients should avoid red clover and isoflavone based BDSs when making choices for menopausal symptom relief.

Since the Women’s Health Initiative reported that hormone therapy correlated with increased risk of breast cancer, many women have turned to botanical dietary supplements to reduce menopausal symptoms. For example, soy bean (SBE) and red clover (RCE) extracts and their contained isoflavones are consumed by many women for the alleviation of hot flashes. Little is known about isoflavones’ potential ability to affect estrogen chemical carcinogenesis, and whether the presence of estradiol/estrone (E2/E1) in breast tissue influences this modulation. The estrogenic genotoxic pathway involves 4-hydroxylation of E2/E1 by P450 1B1, whereas the detoxification pathway involves 2-hydroxylation by P450 1A1. Both pathways are classically regulated by the aryl hydrocarbon receptor (AhR), and the detoxification pathway is epigenetically downregulated by estrogen receptor alpha (ERalpha). Isoflavone containing extracts were analyzed in a breast cancer cell model (MCF-7) with high, low (1 nM, 1 pM) and no E2 pretreatment and compared to their respective controls. A novel spheroid EROD assay developed for this investigation was applied in conjunction with computational modeling to evaluate extracts/compounds’ activity. 3D-EROD measured the activity of P450 1A/1B1, qPCR determined CYP1A1/1B1 gene expression, and a LC-MS/MS assay measured downstream estrogen metabolites. With no E2 present, RCE, containing both ERalpha and AhR agonists, preferentially increased the genotoxic P450 1B1 pathway. The estrogenic constituents genistein (GN) and daidzein (DZ) and the primarily estrogenic SBE selectively decreased P450 1A activity relative to the negative control. However, in the presence of 1 nM E2 and, to a lesser extent, 1 pM E2, the selective decrease in P450 1A activity reverses. An LC-MS/MS assay indicated that with 1 microM pre- and cotreatment, off target effects of the phytoestrogens, such as the COMT downregulation by GN, are observed. These data indicate that the phytoestrogens likely do not have an adverse effect on the detoxification pathway of estrogens in women, as pre-and postmenopausal breast tissue contains E2/E1. These results emphasize the importance of investigating the effects of botanical dietary supplements and their compounds in different model systems to better understand their efficacy and safety. Supported by NIH Grant P50AT000155.