6-Prenylnaringenin from Hops Disrupts ERα-Mediated Downregulation of CYP1A1 to Facilitate Estrogen Detoxification.

Botanical dietary supplements (BDS) containing hops are sold as women's health supplements due to the potent hop phytoestrogen, 8-prenylnaringenin (8-PN), and the cytoprotective chalcone, xanthohumol. Previous studies have shown a standardized hop extract to beneficially influence chemical estrogen carcinogenesis in vitro by fostering detoxified 2-hydroxylation over genotoxic 4-hydroxylation estrogen metabolism. In this study, hop extract and its bioactive compounds were investigated for its mechanism of action within the chemical estrogen carcinogenesis pathway, which is mainly mediated through the 4-hydroxylation pathway catalyzed by CYP1B1 that can form gentoxic quinones. Aryl hydrocarbon receptor (AhR) agonists induce CYP1A1 and CYP1B1, while estrogen receptor alpha (ERα) inhibits transcription of CYP1A1, the enzyme responsible for 2-hydroxylated estrogens and the estrogen detoxification pathway. An In-Cell Western MCF-7 cell assay revealed hop extract and 6-prenylnaringenin (6-PN) degraded ERα via an AhR-dependent mechanism. Reverse transcription PCR and xenobiotic response element luciferase assays showed hop extract and 6-PN-mediated activation of AhR and induction of CYP1A1. A reduction in estrogen-mediated DNA (cytosine-5)-methyltransferase 1 (DNMT1) downregulation of CYP1A1 accompanied this activity in a chromatin immunoprecipitation assay. Ultimately, hop extract and 6-PN induced preferential metabolism of estrogens to their detoxified form in vitro. These results suggest that the standardized hop extract and 6-PN activate AhR to attenuate epigenetic inhibition of CYP1A1 through degradation of ERα, ultimately increasing 2-hydroxylated estrogens. A new mechanism of action rationalizes the positive influence of hop BDS and 6-PN on oxidative estrogen metabolism in vitro and, thus, potentially on chemical estrogen carcinogenesis. The findings underscore the importance of elucidating various biological mechanisms of action and standardizing BDS to multiple phytoconstituents for optimal resilience promoting properties.

The Multiple Biological Targets of Hops and Bioactive Compounds.

Botanical dietary supplements for women's health are increasingly popular. Older women tend to take botanical supplements such as hops as natural alternatives to traditional hormone therapy to relieve menopausal symptoms. Especially extracts from spent hops, the plant material remaining after beer brewing, are enriched in bioactive prenylated flavonoids that correlate with the health benefits of the plant. The chalcone xanthohumol (XH) is the major prenylated flavonoid in spent hops. Other less abundant but important bioactive prenylated flavonoids are isoxanthohumol (IX), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN). Pharmacokinetic studies revealed that these flavonoids are conjugated rapidly with glucuronic acid. XH also undergoes phase I metabolism in vivo to form IX, 8-PN, and 6-PN. Several hop constituents are responsible for distinct effects linked to multiple biological targets, including hormonal, metabolic, inflammatory, and epigenetic pathways. 8-PN is one of the most potent phytoestrogens and is responsible for hops' estrogenic activities. Hops also inhibit aromatase activity, which is linked to 8-PN. The weak electrophile, XH, can activate the Keap1-Nrf2 pathway and turn on the synthesis of detoxification enzymes such as NAD(P)H-quinone oxidoreductase 1 and glutathione S-transferase. XH also alkylates IKK and NF-κB, resulting in anti-inflammatory activity. Antiobesity activities have been described for XH and XH-rich hop extracts likely through activation of AMP-activated protein kinase signaling pathways. Hop extracts modulate the estrogen chemical carcinogenesis pathway by enhancing P450 1A1 detoxification. The mechanism appears to involve activation of the aryl hydrocarbon receptor (AhR) by the AhR agonist, 6-PN, leading to degradation of the estrogen receptor. Finally, prenylated phenols from hops are known inhibitors of P450 1A1/2; P450 1B1; and P450 2C8, 2C9, and 2C19. Understanding the biological targets of hop dietary supplements and their phytoconstituents will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties.

Evidence for Chemopreventive and Resilience Activity of Licorice: Glycyrrhiza Glabra and G. Inflata Extracts Modulate Estrogen Metabolism in ACI Rats.

Women are increasingly using botanical dietary supplements (BDS) to reduce menopausal hot flashes. Although licorice (Glycyrrhiza sp.) is one of the frequently used ingredients in BDS, the exact plant species is often not identified. We previously showed that in breast epithelial cells (MCF-10A), Glycyrrhiza glabra (GG) and G. inflata (GI), and their compounds differentially modulated P450 1A1 and P450 1B1 gene expression, which are responsible for estrogen detoxification and genotoxicity, respectively. GG and isoliquiritigenin (LigC) increased CYP1A1, whereas GI and its marker compound, licochalcone A (LicA), decreased CYP1A1 and CYP1B1 The objective of this study was to determine the distribution of the bioactive licorice compounds, the metabolism of LicA, and whether GG, GI, and/or pure LicA modulate NAD(P)H quinone oxidoreductase (NQO1) in an ACI rat model. In addition, the effect of licorice extracts and compounds on biomarkers of estrogen chemoprevention (CYP1A1) as well as carcinogenesis (CYP1B1) was studied. LicA was extensively glucuronidated and formed GSH adducts; however, free LicA as well as LigC were bioavailable in target tissues after oral intake of licorice extracts. GG, GI, and LicA caused induction of NQO1 activity in the liver. In mammary tissue, GI increased CYP1A1 and decreased CYP1B1, whereas GG only increased CYP1A1 LigC may have contributed to the upregulation of CYP1A1 after GG and GI administration. In contrast, LicA was responsible for GI-mediated downregulation of CYP1B1 These studies highlight the polypharmacologic nature of botanicals and the importance of standardization of licorice BDS to specific Glycyrrhiza species and to multiple constituents.

Formation and biological targets of botanical o-quinones.

The formation of o-quinones from direct 2-electron oxidation of catechols and/or two successive one electron oxidations could explain the cytotoxic/genotoxic and/or chemopreventive effects of several phenolic botanical extracts. For example, poison ivy contains urushiol, an oily mixture, which is oxidized to various o-quinones likely resulting in skin toxicity through oxidative stress and alkylation mechanisms resulting in immune responses. Green tea contains catechins which are directly oxidized to o-quinones by various oxidative enzymes. Alternatively, phenolic botanicals could be o-hydroxylated by P450 to form catechols in vivo which are oxidized to o-quinones. Examples include, resveratrol which is oxidized to piceatannol and further oxidized to the o-quinone. Finally, botanical o-quinones can be formed by O-dealkylation of O-alkoxy groups or methylenedioxy rings resulting in catechols which are further oxidized to o-quinones. Examples include safrole, eugenol, podophyllotoxin and etoposide, as well as methysticin. Once formed these o-quinones have a variety of biological targets in vivo resulting in various biological effects ranging from chemoprevention -> no effect -> toxicity. This U-shaped biological effect curve has been described for a number of reactive intermediates including o-quinones. The current review summarizes the latest data on the formation and biological targets of botanical o-quinones.

Red Clover Aryl Hydrocarbon Receptor (AhR) and Estrogen Receptor (ER) Agonists Enhance Genotoxic Estrogen Metabolism.

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.

Botanicals and Their Bioactive Phytochemicals for Women's Health.

Botanical dietary supplements are increasingly popular for women's health, particularly for older women. The specific botanicals women take vary as a function of age. Younger women will use botanicals for urinary tract infections, especially Vaccinium macrocarpon (cranberry), where there is evidence for efficacy. Botanical dietary supplements for premenstrual syndrome (PMS) are less commonly used, and rigorous clinical trials have not been done. Some examples include Vitex agnus-castus (chasteberry), Angelica sinensis (dong quai), Viburnum opulus/prunifolium (cramp bark and black haw), and Zingiber officinale (ginger). Pregnant women have also used ginger for relief from nausea. Natural galactagogues for lactating women include Trigonella foenum-graecum (fenugreek) and Silybum marianum (milk thistle); however, rigorous safety and efficacy studies are lacking. Older women suffering menopausal symptoms are increasingly likely to use botanicals, especially since the Women's Health Initiative showed an increased risk for breast cancer associated with traditional hormone therapy. Serotonergic mechanisms similar to antidepressants have been proposed for Actaea/Cimicifuga racemosa (black cohosh) and Valeriana officinalis (valerian). Plant extracts with estrogenic activities for menopausal symptom relief include Glycine max (soy), Trifolium pratense (red clover), Pueraria lobata (kudzu), Humulus lupulus (hops), Glycyrrhiza species (licorice), Rheum rhaponticum (rhubarb), Vitex agnus-castus (chasteberry), Linum usitatissimum (flaxseed), Epimedium species (herba Epimedii, horny goat weed), and Medicago sativa (alfalfa). Some of the estrogenic botanicals have also been shown to have protective effects against osteoporosis. Several of these botanicals could have additional breast cancer preventive effects linked to hormonal, chemical, inflammatory, and/or epigenetic pathways. Finally, although botanicals are perceived as natural safe remedies, it is important for women and their healthcare providers to realize that they have not been rigorously tested for potential toxic effects and/or drug/botanical interactions. Understanding the mechanism of action of these supplements used for women's health will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties.

Hop (Humulus lupulus L.) Extract and 6-Prenylnaringenin Induce P450 1A1 Catalyzed Estrogen 2-Hydroxylation.

Humulus lupulus L. (hops) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief. In addition to its efficacy for menopausal symptoms, hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer. In the present study, an enriched hop extract and the key bioactive compounds [6-prenylnarigenin (6-PN), 8-prenylnarigenin (8-PN), isoxanthohumol (IX), and xanthohumol (XH)] were tested for their effects on estrogen metabolism in breast cells (MCF-10A and MCF-7). The methoxyestrones (2-/4-MeOE1) were analyzed as biomarkers for the nontoxic P450 1A1 catalyzed 2-hydroxylation and the genotoxic P450 1B1 catalyzed 4-hydroxylation pathways, respectively. The results indicated that the hop extract and 6-PN preferentially induced the 2-hydroxylation pathway in both cell lines. 8-PN only showed slight up-regulation of metabolism in MCF-7 cells, whereas IX and XH did not have significant effects in either cell line. To further explore the influence of hops and its bioactive marker compounds on P450 1A1/1B1, mRNA expression and ethoxyresorufin O-dealkylase (EROD) activity were measured. The results correlated with the metabolism data and showed that hop extract and 6-PN preferentially enhanced P450 1A1 mRNA expression and increased P450 1A1/1B1 activity. The aryl hydrocarbon receptor (AhR) activation by the isolated compounds was tested using xenobiotic response element (XRE) luciferase construct transfected cells. 6-PN was found to be an AhR agonist that significantly induced XRE activation and inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced XRE activity. 6-PN mediated induction of EROD activity was also inhibited by the AhR antagonist CH223191. These data show that the hop extract and 6-PN preferentially enhance the nontoxic estrogen 2-hydroxylation pathway through AhR mediated up-regulation of P450 1A1, which further emphasizes the importance of standardization of botanical extracts to multiple chemical markers for both safety and desired bioactivity.

Differential Effects of Glycyrrhiza Species on Genotoxic Estrogen Metabolism: Licochalcone A Downregulates P450 1B1, whereas Isoliquiritigenin Stimulates It.

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 µg/mL), and LigC (1 µM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 µM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 µM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 µg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women's health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.