16 research outputs found
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 womenâs
health, and its isoflavones, biochanin A (BA), formononetin (FN),
genistein (GN), and daidzein (DZ), on estrogen metabolism. The methoxy
estrogen metabolites (2-MeOE<sub>1</sub>, 4-MeOE<sub>1</sub>) 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 ÎŒg/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-MeOE<sub>1</sub>/CYP1B1 > 2-MeOE<sub>1</sub>/CYP1A1). Experiments
with the isoflavones showed that the AhR agonists (BA, FN) preferentially
induced CYP1B1 expression as well as 4-MeOE<sub>1</sub>. 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 ânormalâ 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
Differential Effects of <i>Glycyrrhiza</i> Species on Genotoxic Estrogen Metabolism: Licochalcone A Downregulates P450 1B1, whereas Isoliquiritigenin Stimulates It
Estrogen chemical carcinogenesis
involves 4-hydroxylation of estrone/estradiol
(E<sub>1</sub>/E<sub>2</sub>) 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 â« GG > GU and LigC â
LicA â« 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-MeOE<sub>1</sub> as nontoxic and
4-MeOE<sub>1</sub> as genotoxic biomarkers in the nontumorigenic human
mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased
4-MeOE<sub>1</sub>, whereas GI and LicA inhibited 2- and 4-MeOE<sub>1</sub> levels. GG, GU (5 ÎŒg/mL), and LigC (1 ÎŒM) also
enhanced P450 1B1 expression and activities, which was further increased
by inflammatory cytokines (TNF-α and IFN-γ). LicA (1,
10 ÎŒM) decreased cytokine- and TCDD-induced P450 1B1 gene expression
and TCDD-induced xenobiotic response element luciferase reporter (IC<sub>50</sub> = 12.3 ÎŒM), suggesting an antagonistic effect on the
aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI
(5 ÎŒg/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 womenâs health. Additionally,
the differential effects of the <i>Glycyrrhiza</i> species
on estrogen metabolism emphasize the importance of standardization
of botanical supplements to species-specific bioactive compounds
Raloxifene and Desmethylarzoxifene Block Estrogen- Induced Malignant Transformation of Human Breast Epithelial Cells
There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to
contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A
human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen
components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and
histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of
CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs
correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE
adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of
estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation
induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention
Dynamic Residual Complexity of the IsoliquiritigeninâLiquiritigenin Interconversion During Bioassay
Bioactive
components in food plants can undergo dynamic processes
that involve multiple chemical species. For example, 2âČ-hydroxychalcones
can readily isomerize into flavanones. Although chemically well documented,
this reaction has barely been explored in the context of cell-based
assays. The present time-resolved study fills this gap by investigating
the isomerization of isoliquiritigenin (a 2âČ-hydroxychalcone)
and liquiritigenin (a flavanone) in two culture media (Dulbeccoâs
modified eagle medium and Roswell Park Memorial Institute medium)
with and without MCF-7 cells, using high-performance liquid chromatographyâdiode
array detectorâelectrospray ionization/atmospheric pressure
chemical ionizationâmass spectrometry for analysis. Both compounds
were isomerized and epimerized under all investigated biological conditions,
leading to mixtures of isoliquiritigenin and <i>R</i>/<i>S</i>-liquiritigenin, with 19.6% <i>R</i> enantiomeric
excess. Consequently, all three species can potentially modulate the
biological responses. This exemplifies dynamic residual complexity
and demonstrates how both nonchiral reactions and enantiomeric discrimination
can occur in bioassay media, with or without cells. The findings highlight
the importance of controlling in situ chemical reactivity, influenced
by biological systems when evaluating the mode of action of bioactives
DESIGNER Extracts as Tools to Balance Estrogenic and Chemopreventive Activities of Botanicals for Womenâs Health
Botanical dietary supplements contain
multiple bioactive compounds
that target numerous biological pathways. The lack of uniform standardization
requirements is one reason that inconsistent clinical effects are
reported frequently. The multifaceted biological interactions of active
principles can be disentangled by a coupled pharmacological/phytochemical
approach using specialized (âknock-outâ) extracts. This
is demonstrated for hops, a botanical for menopausal symptom management.
Employing targeted, adsorbent-free countercurrent separation, <i>Humulus lupulus</i> extracts were designed for pre- and postmenopausal
women by containing various amounts of the phytoestrogen 8-prenylnaringenin
(8-PN) and the chemopreventive constituent xanthohumol (XH). Analysis
of their estrogenic (alkaline phosphatase), chemopreventive (NADÂ(P)ÂH-quinone
oxidoreductase 1 [NQO1]), and cytotoxic bioactivities revealed that
the estrogenicity of hops is a function of 8-PN, whereas their NQO1
induction and cytotoxic properties depend on XH levels. Antagonization
of the estrogenicity of 8-PN by elevated XH concentrations provided
evidence for the interdependence of the biological effects. A designed
postmenopausal hop extract was prepared to balance 8-PN and XH levels
for both estrogenic and chemopreventive properties. An extract designed
for premenopausal women contains reduced 8-PN levels and high XH concentrations
to minimize estrogenic while retaining chemopreventive properties.
This study demonstrates the feasibility of modulating the concentrations
of bioactive compounds in botanical extracts for potentially improved
efficacy and safety
Hop (<i>Humulus lupulus</i> L.) Extract and 6âPrenylnaringenin Induce P450 1A1 Catalyzed Estrogen 2âHydroxylation
<i>Humulus lupulus</i> 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-MeOE<sub>1</sub>) 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 <i>O</i>-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-<i>p</i>-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
Different <i>Glycyrrhiza</i> species and their bioactive compounds induce ER dependent estrogenic response in MCF-7 cells.
<p>ERE-luciferase induction in ERα (+) MCF-7 cells by A) licorice and hops extracts and B) their respective compounds. Cells were cotransfected with pERE and pRL-TK 24 h before being treated with either extracts (2 ”g/mL, open bars and 10 ”g/mL, closed bars) or pure compounds (0.1 ”M, open bars and 1 ”M, closed bars). 17ÎČ-Estradiol (1 nM) was used as positive control. Since hops extract showed a considerable estrogenic activity at 2 ÎŒg/mL, higher concentrations were not tested. Chemiluminescence analysis was performed after 24 h. Results were normalized for transfection efficiency, and they are shown as a fold induction relative to the level observed in cells treated with vehicle only. Results are the means of three independent determinations in duplicates ± SD.</p
Estrogenic compounds in licorice and hops are formed from chalcones.
<p>Metabolism of bioactive compounds from <b>A</b>) hops and <b>B</b>) licorice.</p
<i>Glycyrrhiza</i> species and their bioactive compounds induce the estrogenic marker,<i>Tff1</i> mRNA, in T47D cells.
<p>Estrogen responsive gene (<i>Tff1</i>) induction in T47D cells by A) licorice and hops extracts (10 ”g/mL) and B) the related compounds 8-PN (100 nM), LigF (5 ”M), and Lig C (5 ”M). 17ÎČ-Estradiol (100 nM) was used as positive control. Results are the means of four independent determinations in duplicates ± SD.</p
Liquiritigenin selectively binds to ERÎČ.
<p>Competitive ER binding using human recombinant A) ERα and B) ERÎČ.</p