CHAPTER DISCUSSION Development of sensitive in-vitro estrogen-responsive bioassays as clinical tools to understand biological effects of estrogenic compounds, alone and in combination 187 Ultra-sensitive bioassays expressing ERα and ERβ can be used to detect ER-isoform selective activity and study of structure– function relationships 187 4.1.2 MCF-7 breast cancer cell proliferation assay for quantification of estrogenic activity 191 4.1.3 Estrogenic activities of Epimedium compounds 193 4.1.4 Estrogenic activities of binary mixtures 195 4.1.5 Application of in-vitro ER-responsive bioassays as clinical tools to understand estrogenic effects of compounds in serum 4.1 197 Pharmacokinetics compounds Epimedium 200 4.2.1 Pharmacokinetics of unconjugated prenylflavonoid aglycones from aqueous Epimedium decoction in humans 200 4.2.2 Pharmacokinetics and pharmacodynamics of unconjugated prenylflavonoids after ingestion of an enriched Epimedium preparation by rats 204 4.2.3 Pharmacokinetics and pharmacodynamics of conjugated prenylflavonoids after ingestion of an enriched Epimedium preparation by rats 206 4.2.4 Accumulation of conjugated Epimedium compounds in-vivo 208 4.1.1 4.2 and pharmacodynamics of 185 4.3 Gene expression profiling reveals partially overlapping but distinct genomic actions of different estrogenic compounds in human breast cancer cells 209 Convergence of global gene expression profiles MCF-7 breast cancer cells treated with estrogenic Epimedium prenylflavonoids, genistein and estradiol 209 4.3.2 Discovery of up-regulation of CYP1A1 transcription by Epimedium compounds via global gene expression profiling 211 4.3.3 Epimedium prenylflavonoids are dual activators of the AhR and ER 212 4.3.1 186 4.1 Development of sensitive in-vitro estrogen-responsive bioassays as clinical tools to understand biological effects of estrogenic compounds, alone and in combination 4.1.1 Ultra-sensitive bioassays expressing ERα and ERβ can be used to detect ERisoform selective activity and study of structure–function relationships A panel of stable human cell lines derived from HeLa human cervical adenocarcinoma cells has been developed that specifically respond to estrogenic compounds that interact with human ERα and ERβ proteins thereby allowing the efficient screening of estrogenic activity of chemicals, alone or in combination, such as in a complex mixture encountered in botanical extracts and sera A yeast-based reporter gene assay was not used as such it does not discriminate estrogenic and anti-estrogenic substances (Andersen et al., 1999; Jørgensen et al., 1998) and may not accurately represent human systems, as the permeability of compounds through the yeast cell wall might be different relative to mammalian cell membranes (Scrimshaw & Lester, 2004) HeLa cells were chosen, as in the untransfected form, they endogeneously express undetectable amounts of ERα and ERβ proteins (Escande et al., 2006) The pERE4Luchygro reporter gene was first stably incorporated into HeLa cells consists of tandem copies of consensus vitellogenin ERE cDNA cloned into a pGL3-basic plasmid upstream of the luciferase reporter gene Subsequently, cells with pERE4-Luchygro in their genome were stably transfected with coding sequences for either ERα or ERβ Two highly inducible clones that stably expressed ERα and ERβ proteins respectively were selected for the development of ERα and ERβ cell-based bioassays The production of stable cell lines eliminates the necessity of constantly producing DNA and variability associated with transient transfection procedures (Paris et al., 2002) 187 For the validated ERα bioassay, the intra- and inter-assay variations near the EC50 concentration of the standard estrogen, estradiol, were about 6% and 14% respectively For ERβ, the intra- and inter-assay variations were determined to be 8.1% and 16% respectively Detection limits for estradiol in ERα and ERβ bioassays were 8.4 and 13.1 pM, respectively This set of assays is simple and rapid to perform and offers highthroughput, as cells require only 24 h of incubation with test samples The estrogen agonist properties of estradiol and its metabolites, which include estrone and estriol, were first examined The rank order in terms of decreasing estrogenic potency for this series of compounds in both ERα and ERβ bioassays is estradiol > estriol > estrone This result is in good agreement with those reported by Paris et al., (2002) for their corresponding ERα bioassay Both assays are highly specific to estrogens and they displayed minimal cross reactivity with most steroidal hormones such as the androgens, progestogens and corticosteroids However, when testing samples that contain testosterone, such as sera from males, where testosterone levels can be high, an aromatase inhibitor, such as DL-aminoglutethimide, would have to be added These two assays are also able to distinguish full agonists from partial agonists Estriol and estrone are found to be full agonists like estradiol Interestingly, some compounds elicited superagonist effects Flavonoids like apigenin and genistein exhibited superagonist effects in both assays Kaempferol and luteolin are superagonists in the ERα bioassay but are partial agonists in the ERβ bioassay The development of stable cell lines that separately express only one ER isoform enables the differentiation of ERα and ERβ bioactivities We validated this aspect of our bioassays using well-known ER-isoform selective ligands, such as genistein, PPT and DPN Similar to the results reported by Escande et al., (2006), PPT, an ERα-selective 188 ligand, strongly activated our ERα bioassay in a dose-dependent manner but did not induce detectable luciferase activity in the ERβ bioassay in the range of concentrations tested Both genistein and DPN, ERβ-selective ligands, displayed ERβ selectivity when tested in both systems The effect of hydroxylation on the estrogenic activities of four common flavonoids - apigenin, kaempferol, luteolin, and quercetin was also studied Previous studies indicated that the two hydroxyls in position of ring A and 4’ of ring B represent the minimal hydroxylation pattern for estrogenic activity (Vaya & Tamir, 2004) and these four phytoestrogens have these two hydroxyl groups Above this number, an inverse relationship between the number of hydroxyl groups and ERα bioactivity was observed Increasing the number of hydroxyl groups decreased the molecule’s hydrophobicity, which reduces binding to the hydrophobic ligand-binding pocket Addition of a hydroxyl group in the 3’ position in benzene ring B, which converts the flavone apigenin to luteolin, reduced estrogenic activity, reflected by higher EC50 values and lower relative peak activities This structure–bioactivity relationship was also observed among the flavonols—the presence of a hydroxyl in the same position changed kaempferol to the less estrogenic quercetin, suggesting a role for the 3’ position of ring B in ERα activity Another active position was the hydroxyl in position of ring C of the chromone backbone: its presence also reduced estrogenic activity (apigenin vs kaempferol; luteolin vs quercetin) This observation was consistent with previous reports that hydroxyls at positions and decrease reporter gene activities (Le Bail et al., 1998) Interestingly, the hydroxyl substitutions in position of ring C, which define flavones and flavonols, suggest that the division of phytoestrogens into these categories also reflect intrinsic ERα activity A similar observation was also seen for ERβ, with the 189 exception of kaempferol, which suggests that the mechanisms of ERβ activation by kaempferol may differ from the rest of the phytoestrogens Positions 3’ of ring B and of ring C may have important roles for estrogenic activity, and more detailed investigations on these two positions may be warranted The molecular basis of these structure–function differences is unlikely to be completely due to differential receptor affinity Cell culture media used in this study had dextran-coated charcoal stripped serum added to it and ex-vivo samples from clinical trials and animal studies use sera samples from volunteers and test animals In the absence of serum, binding of quercetin to recombinant ERα was comparable to that of genistein and kaempferol (Maggiolini et al., 2004; Leung et al., 2004) The presence of serum reduced ERα binding of quercetin in MCF-7 cells, with relative binding affinities for genistein, kaempferol, and quercetin being 0.1, 0.012, and 0.001, respectively (Zava & Duwe, 1997) This apparently low ERα affinity of quercetin in serum is not surprising, considering that quercetin binds extensively to serum proteins (Boulton et al., 1999) Reflecting these changes in relative affinity, transactivation effects of quercetin were comparatively higher in serum-free media compared to experiments done with serum (Maggiolini et al., 2004; Harris et al., 2005) 190 4.1.2 MCF-7 breast cancer cell proliferation assay for quantification of estrogenic activity Unlike HeLa cells, MCF-7 cells are breast cancer cells which express ERα and ERβ to no relevant extent They require estrogenic stimulation for transformation from dormant into proliferating cells MCF-7 cells have been used widely as a highly sensitive tool for the detection of even small agonistic effects at ERα An in-vitro assay based on the proliferation of MCF-7 cells commonly called the ‘E-screen’ is performed by incubating cells with test samples over a period of six days The number of cells is quantified and comparisons can be made between cells treated with test samples with those incubated with the vehicle control Although the assay procedure is more laborious than ERα and ERβ stable cells, the MCF-7 cell proliferation assay is more sensitive towards estrogens as it has a lower detection limit (ERα: 8.45 pM; ERβ: 13.1 pM & MCF-7: 0.112 pM) The rank order in terms of estrogenicity of estradiol, estrone and estriol were similar to that obtained via the stable ERα and ERβ HeLa cell lines where estradiol is the most potent, followed by estriol and estrone To develop the MCF-7 cell proliferation assay for quantification of estrogenic activity of ex-vivo samples, we incubated large doses of steroids such as dihydrotestosterone, progesterone, and cortisol in the presence of estradiol and found that these not significantly affect the bioassay However, there is a need to include the aromatase inhibitor DL-aminoglutethimide to prevent conversion of testosterone to estradiol by aromatase in MCF-7 cells Like ERα and ERβ cell-based bioassays, the MCF-7 cell proliferation assay also possesses the ability of being able to distinguish full agonists from partial agonists 191 Superagonism in cell proliferation was not observed in the range of compounds and concentrations tested The difference in maximal stimulatory levels in ERα and ERβ bioassays; and MCF-7 cell proliferation assay could be a reflection of differences in endogenous versus transfected genes Superagonistic ER activity exhibited by cells that have been stably transfected with a reporter gene had also been observed for genistein and reservatrol (Legler et al., 1999; Harris et al., 2005; Gehm et al., 2004) One possible mechanism of this superagonist effect may be related to the function of the ER activation function-1 domain, since its removal prevented enhanced reporter gene activity with resveratrol (Gehm et al., 2004) Because the ERα activation function-1 domain is subjected to phosphorylation, superagonist effects could be contributed by the actions of the AP-1 or the mitogen-activated protein kinase-responsive pathway (Frigo et al., 2002) 192 4.1.3 Estrogenic activities of Epimedium compounds The estrogenicities of prenylated flavonoid compounds found in the Traditional Chinese Medicine herb, Epimedium, namely, icariin, icariside I, icariside II, icaritin and desmethylicaritin, were examined using ERα and ERβ; and MCF-7 cell proliferation assays in this study Out of the five Epimedium compounds, icariin was not estrogenic and this is in agreement with results reported in an earlier study by Liu et al., 2005 Icariside I, icariside II, icaritin and desmethylicaritin, were found to be estrogenic in all three assays but were less potent compared to estradiol and the well-known soy isoflavone, genistein In terms of the magnitude of maximal inducible responses, those elicited by genistein, desmethylicaritin and icaritin were similar to that given by estradiol, albeit at varying concentrations, indicating these three phytoestrogens are full estrogen agonists in the MCF-7 cell proliferation assay This contrasts with results obtained from experiments involving stable ERα and ERβ HeLa cell lines where genistein was found to be a superagonist where the maximal luciferase induction of genistein exceeded that achieved by estradiol In both stable ERα and ERβ HeLa cell lines, desmethylicaritin and icaritin were partial agonists where their maximal luciferase inductions were lower than estadiol Icariside I and icarside II were also both found to be partial estrogen agonists and less potent than icaritin and desmethylicaritin in all three assays In a paper involving protein–ligand docking simulations published by Wang et al., (2006), the prototypical estradiol molecule docked in the ERα ligand-binding domain was found to be characterized by three hydrogen bonds formed between amino acid residues Arg-394, Glu-353, and His-524 with estradiol, which are known to be essential for the molecule’s agonistic activity Using the same approach on the two aglycones, namely, 193 icaritin and desmethylicaritin, the former compound’s higher estrogenicity could be attributed to the ability of the hydroxyl group on ring B to interact with Glu-353 and Arg394 and another similar interaction involving the hydroxyl group on ring A with His-524 In contrast, for the case of icaritin, the interaction with Glu-353 and Arg-394 in the ERα ligand-binding domain was blocked due to the presence of the hydrophobic methyl group in ring C The lower estrogenicities of icariside I, icariside II and icariin could be explained by the steric hindrance produced by the presence of bulky sugar groups which prevents the required docking to the receptor site and increases the molecules’ hydrophilicity Prenylation at the 8-position of the flavonoid backbone was observed to lead to an enhancement of estrogenicity Desmethylicaritin is the prenylated version of kaempferol and the estrogenic potency of the desmethylicaritin is ~ 55 times higher than that of kaempferol A similar observation has been reported in studies comparing the estrogenicities of naringenin and its prenylated derivative, 8-prenylnaringenin, which is found in hops and beer (Milligan et al., 2000; Schaefer et al., 2003; Kretzschmar et al., 2010) Icaritin and icariside I were discovered to be ERα-selective ligands in this study This is quite unlike common flavonoids, such as apigenin, kaempferol, luteolin, quercetin and genistein, which are mainly ERβ-selective 8-Prenylnaringenin, the prenylated version of naringenin, was also found to be ERα-selective (Schaefer et al., 2003) 194 The use of poorly defined botanical entities has resulted in controversial results despite many studies of estrogenic flavonoids, including soy products (Williamson & Manach, 2005) Cell-based bioassays developed in the present study were used to examine the bioavailability and biological effectiveness in sera of the decoction when administered orally Ingestion of a defined Epimedium decoction resulted in a 6% increase in ERα AUC effect over baseline in the subjects The increase in Epimedium ERα estrogenicity was contributed to a large extent by the 24 h time period and was more than 10-fold lower than that of observed following ingestion of estradiol valerate A single dose of this traditionally prepared Epimedium pubescens decoction may not to exert physiological effects The development of viable and robust bioassays facilitates the rapid screening of similar herbal extracts with purported estrogenic properties and the measurement of estrogenic activity ex-vivo at close time points for pharmacokinetics and pharmacodynamics studies 199 4.2 Pharmacokinetics and pharmacodynamics of Epimedium compounds 4.2.1 Pharmacokinetics of unconjugated prenylflavonoid aglycones from aqueous Epimedium decoction in humans In this pilot study, only the concentrations of icaritin and desmethylicaritin were measured as they are prenylflavonoids unique to Epimedium In addition, icaritin and desmethylicaritin are both aglycones that exert stronger estrogenic activities than their glycosides and flavonoids such as apigenin, kaempferol, luteolin and quercetin (Table 4) Human sera, obtained from healthy male volunteers over a 48 h period following ingestion of a traditional Epimedium decoction, were analyzed for their icaritin and desmethylicaritin content Icaritin was first detected at h where the mean value was 0.24 nM, which suggests that very little of the aglycone was directly absorbed In contrast, genistein or diosmin, which were shown to be directly absorbed exhibited high concentrations in blood within h (Kano et al., 2006) This was also consistent with the low levels of the aglycone in Epimedium decoction (119.9 μg/g) The level of icaritin was also observed to rise rapidly after h, reaching a peak at h (1.51±1.6 nM) and returning to almost baseline (0.25±0.17 nM) after 48 h The estrogenicity of these trace amounts of unconjugated icaritin was well below the detection limits of the ERα and ERβ, as well as, MCF-7 cell-based bioassays The low amounts of the compound were likely to be largely bound to serum proteins, particularly, human serum albumin but not sex hormone-binding globulin, which would influence its bioavailability A large proportion of proteins found in serum are made up of albumin, which binds and transports acidic and neutral bioactive molecules and food nutrients (Xiao et al., 2011b) Studies that examined the bioavailability of flavonoids in human serum albumin reported that flavonoid bioavailability is often poor due to interaction with 200 this protein (Bolli et al., 2010) Walle et al (2001) demonstrated that binding of the flavonoid chrysin can be as high as > 99% Via fluorescence spectroscopy, Dufour & Dangles (2004) reported that flavonoids display moderate affinities for albumins and flavones and flavonols being most tightly bound Methylation of hydroxyl groups improved the affinities for human serum albumin by to 16-fold (Xiao et al., 2011a) whereas glycosidation and sulfation could lower the affinity to albumin by one order of magnitude depending on the conjugation site (Dufour & Dangles, 2004) Globulins can also be found in serum and are used in the transport of ions, hormones and lipids responsible for immune function (Xiao et al., 2011b) The sex hormone-binding globulin transports and regulates the access of sex steroids such as testosterone and estradiol to their target tissues The number of steroid-binding sites in the sex hormone-binding globulin far exceeds the molar concentrations of sex steroids and can accommodate ligands such as phytoestrogens and fatty acids (Hodgert Jury et al., 2000) In a study by Hodgert Jury et al (2000), the relative binding affinities (RBAs) of various compounds in comparison with estradiol (RBA= 100) to sex hormone-binding globulin determined using competitive displacement assays Some phytoestrogens bound with RBAs of 0.12 (coumestrol) to 0.04 (naringenin) Many compounds did not bind to sex hormone-binding globulin with sufficient affinity to allow RBA measurements and these include several phytoestrogens, such as genistein and kaempferol An earlier study by Déchaud et al (1999) reported binding affinity constants of a range of xenoestrogens measured in equilibrium conditions via solid phase binding assay, also had relatively similar findings Flavonoids such as genistein and naringenin were showed weak binding affinities that were at least three orders of magnitude lower than testosterone and/or estradiol Flavonoid glucoside derivatives, genistin and naringin not bind to the sex 201 hormone-binding globulin Hence, it is not likely that icaritin will bind to the sex hormone-binding globulin protein found in serum The late appearance of icaritin in sera of human volunteers was hypothesized to be due to the metabolism of icariin to icaritin which occurs mainly in the intestine This view is supported by animal data showing that icariin is stable in gastric juice, and that hydrolysis of icariin to icaritin occurs in the intestine (Qiu et al., 1999) The aglycone is formed through successive hydrolysis of the diglycoside icariin and triglycosides by intestinal enzymes Significant differences in peak concentrations between subjects were observed ranging from 5.11 nM in subject E to 0.61 nM in subject G These may reflect differences in enzymatic activity contributed by inter-individual variability in bacteria and enzyme activities (Walle et al., 2005) Polymorphisms in uridine diphosphate glucuronosyltransferases which attach a glucuronide residue to flavonoids may contribute to variability in flavonoid bioavailability but the effects of these polymorphisms have not been widely studied The selectivity of conjugation of the flavonoids by UDP-glucuronosyltransferases depends on structure of the flavonoid and the enzyme Studies on UDP-glucuronosyltransferase polymorphisms, to date, mainly focus on the glucuronidation of drugs and various xenobiotics and similar effects may be seen with ingested flavonoids (Lampe & Chang, 2007) A minute fraction of ingested flavonoids are conjugated with a sulfate residue by sulfatases found in the liver and gastrointestinal tract Single-nucleotide polymorphisms in sulfatases 1A1 and 2A1 have been reported and were found to be associated with altered response to therapeutic agents and sex steroid concentrations, respectively and this could influence the disposition of phytochemicals metabolized by sulfatases (Nowell & Falany, 2006) 202 Flavonoids are also extensively metabolized by intestinal bacteria that reside in the gut Notable examples in literature include the conversions of isoflavone daidzein to equol, lignans, such as, secoisolariciresinol diglucoside and matairesinol to enterolignans which include enterodiol and enterolactone, and hop-derived prenylchalcone xanthohumol to 8-prenylnaringenin by intestinal bacteria The reasons for such interindividual differences still remain unknown and diet and host genetics have been thought to be the contributory factors (Lampe & Chang, 2007) Desmethylicaritin was not detected in any one of the human serum samples One contributory reason may be because the amount of desmethyicaritin (0.19 mg) in the water decoction was only a quarter that of icaritin (0.75 mg) The low concentrations of unconjugated icaritin and desmethylicaritin could also be brought about by extensive conjugation to form sulfates, glucuronides and/or methylated conjugates via the respective action of sulfatases, uridine diphosphate glucuronosyltransferases and catechol-O-methyltransferases found in the small intestines and liver 203 4.2.2 Pharmacokinetics and pharmacodynamics of unconjugated prenylflavonoids after ingestion of an enriched Epimedium preparation by rats In view of the low bioactivity measured in a traditionally prepared, aqueous Epimedium pubescens extract, a prenylflavonoid-enriched extract based on Epimedium brevicornu was subsequently formulated and fed to female ovariectomized Sprague– Dawley rats The concentrations of icariin, as well as two other estrogenic glycosides, namely, icariside I and icariside II, together with the two aglycones, icaritin and desmethylicaritin were measured in rat sera The quantification of prenylflavonoid glycosides, such as, icariin, icariside I and icariside II, will allow the understanding on whether the lack of estrogenic activity was due to conjugation of prenylflavonoids In this second study, an early phase during which unconjugated icariin and icariside II reached peak levels in 0.5 to h and a late phase wherein unconjugated icariside I, icaritin and desmethylicaritin peaked at h Treatment with Epimedium extract resulted in non-linear increases in AUC and delayed peaks Icariin was the first prenylflavonoid to be detected at tmax 0.5 h which increased in a non-linear dose-dependent manner after oral administration of the extract This was similar to that observed when icariin was administered as a pure compound (Liu & Lou, 2004) and consistent with direct absorption (Xu et al., 2007) Although icariin was the main flavonoid glycoside in the extract (14% by weight), peak concentrations of this glycoside in serum were in the low nanomolar range, suggesting poor bioavailability of the intact molecule On the other hand, icariside II was detected at several fold higher concentrations than icariin despite of its lower concentration in the extract (icariin:icariside II, 7.8:1.0;w:w) Compared to icariin, icariside II lacks a glucose moiety at position on ring 204 A and this may make the latter more bioavailable There is evidence from the rat intestinal model that apical to basolateral permeability of monoglycosides can be more than 2-fold greater than prenylflavonoids with or more sugar moieties due to higher absorptive permeability and carrier-mediated transport in the intestine (Chen et al., 2008) When icariin was administered alone, icariside II can be rapidly derived from icariin by firstpass deglycosylation and displays an early tmax ~ h (Xu et al., 2007), similar to that observed in our study Thus, the early peak of icariside II with tmax h is likely to be the sum of absorption of icariside II in the extract and metabolism of icariin At the highest Epimedium dose, a secondary delayed icariside II peak with tmax of h was observed This delayed peak of icariside II was not seen when it was derived from icariin administered alone (Xu et al., 2007) but was reminiscent of the delayed re-entry peak observed when biochanin A was administered as a mixture with quercetin and (−)epigallocatechin-3-gallate (Moon & Morris, 2007) Remarkably, icariside I, icaritin and desmethylicaritin also exhibited a major peak at tmax h At tmax h, the most abundant flavonoid was icariside I with an AUC of 538 nMh−1 at the highest drug dose In comparison, AUC of the aglycones icaritin and desmethylicaritin were an order of magnitude lower Rat sera were examined ex-vivo for ERα bioactivity Rats administered with low doses of Epimedium did not display any significant ERα activity At the highest Epimedium dose (600 mg/kg), ER bioactivity was observed at h This peak was comparable in size to that of the minor peak after estradiol benzoate administration Thus, estrogenic activity in sera after ingestion of Epimedium lagged by several hours compared to estradiol benzoate, and corresponded to the appearance of the bioactive metabolites desmethylicaritin, icaritin and icariside I at h 205 4.2.3 Pharmacokinetics and pharmacodynamics of conjugated prenylflavonoids after ingestion of an enriched Epimedium preparation by rats To measure the amounts of conjugated flavonoids in test sera, analyses were repeated after digestion by β-glucuronidase and sulfatase The concentrations of three compounds, namely, icariside II, icaritin and desmethylicaritin, could be studied in rat serum samples after enzyme hydrolysis as under these conditions, icariin and icariside I were deglycosylated completely to give icariside II and icaritin respectively, and could not be detected in enzyme-treated sera A 0.5 h, a peak for icariside II was detected which was consistent with rapid absorption and conjugation (Xu et al., 2007) The AUC value of icariside II after digestion was observed to be several orders of magnitude higher than non-digested samples and this increase reflected the sum of deconjugated icariside II and deglycosylated icariin On the other hand, both desmethylicaritin and icaritin exhibited two peaks, a minor one at 0.5 h and a higher one at h where the Cmax values of icaritin and desmethylicaritin reached ~ and ~ 0.25 µM, respectively The higher concentrations of both compounds after treatment with β-glucuronidase and sulfatase that were two to three orders of magnitude higher than before exposure to enzyme treatment, suggested that the greater proportion of these two compounds and their precursors existed in conjugated forms This was consistent with data with the flavanone, naringenin, whereby > 95% of the compound exists in the conjugated form (Ma et al., 2006) Non-linear dose-dependent AUC increases were observed and saturation effects at higher Epimedium doses were most evident for icaritin, possibly due to either reduced absorption or increased metabolism The isoflavone, genistein, also displayed non-linear 206 pharmacokinetics, attributed to reduced absorption at high doses (Zhou et al., 2008) Remarkably, conjugated icaritin can be detected at micromolar levels up to 72 h after administration of the high dose Epimedium extract This has not been observed when genistein alone was administered, where conjugated genistein reached baseline levels after 36 h (Zhou et al., 2008) Sera from rats fed Epimedium exhibited strong ERα bioactivity throughout most of the 72 h study period Dose-dependent increases in ERα activity were observed with AUC of 374, 543 and 771 pM E2 h−1 with increasing doses of Epimedium The peaks of ERα activity for Epimedium were biphasic with a small first peak a 0.5 to h and larger second peak at h This biphasic pattern reflected the appearance of icaritin, desmethylicaritin, and icariside II at 0.5 and h Concentrations of desmethylicaritin and icaritin reached 0.2 and 0.4 µM respectively at 0.5 h, corresponding values at the h time-point being 0.6 and 1.5 µM Since the EC50 of desmethylicaritin and icaritin was 0.07 and 0.83 µM respectively, it is plausible that estrogenic activity of Epimedium is contributed by desmethylicaritin and icaritin 207 4.2.4 Accumulation of conjugated Epimedium compounds in-vivo A unique feature of Epimedium is its prolonged effect, with strong estrogenic activity (40% of peak activity) being detected up to 72 h This prolonged estrogenic activity at higher Epimedium doses correlated with the persistence of micromolar levels of icaritin at the 48 to 72 h sampling period It is relevant to note that pharmacological doses of flavonoids were administered in this study At the level of the enterocyte, cycling of flavonoid conjugates back to the intestinal lumen at high doses may be markedly decreased, reflecting a saturation of the intestinal conjugation pathways (Silberberg et al., 2006) The long elimination time for the highest dose of icaritin may also be due to differences in the type of Phase II enzymes that are activated The possibility exists that sulfatases, rather than glucuronosyltransferases, may be preferentially recruited at higher doses, resulting in the production of very hydrophilic metabolites which may be less efficiently eliminated by the kidney (Liu & Hu, 2007), thereby contributing to the prolonged presence of icaritin in sera even 72 h after dosing Since intestinal and hepatic metabolism of flavonoids at high doses may differ from more physiological doses, our results may not be directly applicable to all doses of flavonoids Nevertheless, similar depot effects have been observed in ERE-luc transgenic mice, whereby a single physiological dose of the isoflavone was able to induce significant ER/ERE-driven activity in diverse target organs such as liver, cerebral cortex and testis for over 24 h (Montani et al., 2008) Such delayed effects may persist for at least weeks, were unmasked by fasting and are thought to be due to bioaccumulation in the intestines, liver and reproductive tissues (Penza et al., 2007) 208 4.3 Gene expression profiling reveals partially overlapping but distinct genomic actions of different estrogenic compounds in human breast cancer cells 4.3.1 Convergence of global gene expression profiles MCF-7 breast cancer cells treated with estrogenic Epimedium prenylflavonoids, genistein and estradiol Illumina's bead-based DNA microarray platform was used in this study to understand the biological effects and their underlying mechanisms in MCF-7 breast cancer cells that have been treated with a standardised, prenylflavonoid-enriched Epimedium extract and a series of Epimedium-derived compounds Each gene on the microarray acts as a reporter gene and gene expression profiling was used to identify specific genes in a biological pathway that are involved in the mode of action To date, this study is the first to obtain gene expression profiles of a standardized Epimedium extract and its compounds The expression profiles obtained for the range of treatments were analyzed via two ways - responses of genes by hierarchial clustering and correlation analysis of gene expression profiles Hierarchial clustering grouped the range of treatments into two main categories One group consisted of well-known estrogenic compounds such as estradiol and genistein, together with icariside I, icaritin, desmethylicaritin and the standardized Epimedium extract The other group comprised ER-antagonist, 4-hydroxytamoxifen, the vehicle control, together with icariin and icariside II, which were non-estrogenic at the dose tested Correlation analyses of expression profiles was performed to compare the gene expression profiles of estradiol, an established estrogen, with that of genistein, as well as, the various estrogenic treatments encountered in this study The overall gene expression profiles of estradiol and soy isoflavone, genistein, were found to be largely similar due to the high correlation coefficient value that was obtained and this result was similar to those 209 reported in other studies (Ise et al., 2005; Dip et al., 2008) Estrogenic Epimediumderived compounds such as icariside I, icaritin and desmethylicaritin showed gene regulation that were largely similar with that of estradiol, due to their significant high correlation coefficients when their respective gene expression profiles were compared Further analysis was performed based on the functions of the 99-gene set obtained in this study Each gene was categorized into one of the six functional groups, namely, enzymes, signaling, proliferation, transcription, transport and other functions Correlation analysis was then used to compare the similarity between functional groups of estradiol with that of genistein, the standardized Epimedium extract, icariside I, icaritin and desmethylicaritin Genistein exhibited excellent correlation with estradiol in all functional categories Treatments involving the standardized Epimedium extract, icariside I, icaritin and desmethylicaritin showed significant correlations with estradiol for the genes in all functional groups except those classified under enzymes A striking outlier that contributed variation to the correlation plots of each of these treatments against estradiol was found to be CYP1A1 210 4.3.2 Discovery of up-regulation of CYP1A1 transcription by Epimedium compounds via global gene expression profiling In this study, Epimedium extract, icariside I, icariside II, icaritin and desmethylicaritin were found to upregulate the CYP1A1 gene In contrast, cells treated with estradiol and genistein did not show a similar upregulation in these CYP1 genes CYP1A1 is part of the cytochrome P450 superfamily of enzymes that are involved in phase I xenobiotic and drug metabolism The expression of the CYP1 gene is mediated by the AhR, which responds to a number of coal tar carcinogens, such as benzo[α]pyrene and potent tumor promoters such as TCDD (Denison & Nagy, 2003) where it metabolizes these pro-carcinogens into ultimate carcinogens (Androutsopoulos et al., 2009b) Besides Epimedium prenylflavonoids, a number of flavonoids have been reported to induce CYP1A1 transcription via the interaction with AhR in MCF-7 breast cancer cells Naturally occurring flavonoids are generally less active than the prototypic AhR ligand TCDD, and previous studies have demonstrated that their CYP1-inducing potential in MCF7 cells lies between 0.5 and 10 μM (Ciolino et al., 1998; Ciolino & Yeh, 1999) Diosmetin, galangin and quercetin have been reported to increase the transcription of the CYP1A1 gene in MCF-7 cells (Ciolino et al., 1998, 1999; Ciolino & Yeh, 1999) Interestingly, kaempferol, which lacked one hydroxyl group on the same ring as quercetin, was completely inactive (Ciolino et al., 1999) Androutsopoulos et al., (2009a) reported eupatorin and cirsiliol found in Lantana montevidensis induced CYP1 enzyme expression in MCF-7 breast cancer cells 211 4.3.3 Epimedium prenylflavonoids are dual activators of the AhR and ER Epimedium compounds, namely, icariside I, II, icaritin and desmethylicaritin are estrogenic compounds In this study, they were also found to bring about the upregulation of CYP1A1 and the expression of this enzyme is mediated by the AhR In a recent study by Tiong (2010), icaritin was confirmed to be an agonist of the AhR via an independent quantitative RT-PCR experiment involving CYP1A1 and competitive ligand-binding assay with radioactive TCDD Icaritin also induced dose-dependent luciferase activation of an AhR-driven xenobiotic response element reporter gene assay based on MCF-7 breast cancer cells AhR has been regarded as an inducible transcription factor that controls the expression of enzymes that metabolize potentially dangerous xenobiotic chemicals (Nebert et al., 2004) Recent studies, however, have shown more diverse physiological functions of the AhR which include a possible role in liver development (Nguyen & Bradfield, 2008) and activation of expression of genes that metabolize estrogen (Yager & Davidson, 2006; Jefcoate et al., 2000) into non-carcinogenic and chemopreventive metabolites In breast cells, AhR has also been reported to be able to initiate the degradation of the ER and suppress estrogen signaling (Wormke et al., 2003; Ohtake, et al., 2007) It also promoted the proteolysis of ERα through assembly of the ERα-specific ubiquitin ligase complex, CUL4-BAHR (Ohtake et al., 2007; Ohtake et al., 2009) By mediating with these pathways, AhR can influence breast cancer initiation and progression One of the most important etiologies for breast cancer cell proliferation is the hormonal control of its growth via ERα MCF-7 cells with stable knockdown of ERα exhibited suppression in proliferation and enhanced apoptosis (Fu et al., 2006) MCF-7 212 cells treated with icaritin showed reduced ERα protein levels in a dose-dependent manner and combination of estradiol and icaritin inhibited ERα protein levels more than either estradiol or icaritin alone (Tiong, 2010) Icaritin’s modulation of estradiol-stimulated MCF-7 cell proliferation was subsequently found to be due to AhR-mediated proteasomal degradation of ERα in-vitro via the activation of AhR-E3 ubiquitin ligase pathway (Tiong, 2010) This observation was supported via knockdown experiments of AhR using siRNA, which blocked the suppressive effects of icaritin on estradiol-stimulated breast cancer cell proliferation and profound degradation of ERα induced by estradiol/icaritin combination The knockdown of AhR gene did not restore estradiol-mediated degradation of ERα consistent with the fact that estradiol is not a ligand for AhR Based on the results obtained from this preliminary study, icaritin and related Epimedium compounds should be further examined for development of a drug that can be co-administered during hormone replacement therapy to reduce breast cancer risk caused by hormone replacement therapy Ligands, such as icaritin, which are dual activators for ERα and AhR mechanistically represent a new class of SERMs 213 ... that of genistein and kaempferol (Maggiolini et al., 20 04; Leung et al., 20 04) The presence of serum reduced ERα binding of quercetin in MCF-7 cells, with relative binding affinities for genistein,... effectiveness in sera of the decoction when administered orally Ingestion of a defined Epimedium decoction resulted in a 6% increase in ERα AUC effect over baseline in the subjects The increase in Epimedium. .. group on ring B to interact with Glu-353 and Arg3 94 and another similar interaction involving the hydroxyl group on ring A with His-5 24 In contrast, for the case of icaritin, the interaction with