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.

中文翻译：

---

红三叶草芳烃受体（AhR）和雌激素受体（ER）激动剂可增强遗传毒性雌激素代谢

许多妇女认为植物性膳食补充剂（BDSs）是绝经症状激素治疗的安全替代品。但是，BDS对乳腺癌风险的影响在很大程度上尚不清楚。在雌激素化学致癌途径中，P450 1B1将雌激素代谢为4-羟基化的邻苯二酚，后者被氧化为引发并促进乳腺癌的遗传毒性醌。相反，P450 1A1催化的2-羟基化代表解毒途径。目前的研究评估了红三叶草（一种用于女性健康的流行BDS）及其异黄酮，生物素A（BA），单果糖素（FN），金雀异黄素（GN）和黄豆苷原（DZ）对雌激素代谢的影响。甲氧基雌激素代谢物（2-MeOE ₁，4- MeOE ₁通过LC-MS / MS测定），并通过qPCR分析CYP1A1和CYP1B1基因表达。非恶性ER阴性乳腺癌上皮细胞（MCF-10A）和ER阳性乳腺癌细胞（MCF-7）来自正常的乳腺癌上皮组织和ER +乳腺癌组织。红三叶草提取物（RCE，10μg/ mL）和异黄酮对MCF-10A细胞中的雌激素代谢没有影响。然而，在MCF-7细胞中，RCE处理下调了CYP1A1的表达并增强了遗传毒性代谢（4-MeOE ₁ / CYP1B1> 2-MeOE ₁ / CYP1A1）。异黄酮的实验表明，AhR激动剂（BA，FN）优先诱导CYP1B1表达以及4-MeOE ₁。相反，ER激动剂（GN，DZ）可能通过表观遗传机制下调了CYP1A1的表达。最后，ER拮抗剂ICI 182,780增强了异黄酮诱导的XRE-荧光素酶报道分子的活性，并逆转了GN和DZ诱导的CYP1A1表达下调。总体而言，这些研究表明，红三叶草及其异黄酮在“正常”细胞与乳腺癌细胞中对雌激素代谢具有不同的作用。在乳腺癌细胞中，AhR激动剂刺激遗传毒性代谢，而ER激动剂下调解毒途径。这些数据可能表明，特别是乳腺癌患者在选择更年期症状缓解时应避免使用基于红三叶草和异黄酮的BDS。