ToxSci Advance Access originally published online on August 19, 2004
Toxicological Sciences 2004 82(1):70-79; doi:10.1093/toxsci/kfh257
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Toxicological Sciences vol. 82 no. 1 © Society of Toxicology 2004; all rights reserved.
Induction and Inhibition of Aromatase (CYP19) Activity by Natural and Synthetic Flavonoid Compounds in H295R Human Adrenocortical Carcinoma Cells
* Institute for Risk Assessment Sciences (IRAS), Utrecht University, 3508 TD, Utrecht, The Netherlands; Department of Environmental Toxicology, and Department of Chemistry, University of California, Davis, California 95616
Received June 2, 2004; accepted August 9, 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Flavonoids and related structures (e.g., flavones, isoflavones, flavanones, catechins) exert various biological effects, including anticarcinogenic, antioxidant and (anti-)estrogenic effects, and modulation of sex hormone homeostasis. A key enzyme in the synthesis of estrogens from androgens is aromatase (cytochrome P450 19; CYP19). We investigated the effects of various natural and synthetic flavonoids on the catalytic activity and promoter-specific expression of aromatase in H295R human adrenocortical carcinoma cells. Natural flavones were consistently more potent inhibitors than flavanones. IC50 values for 7-hydroxyflavone, chrysin, and apigenin were 4, 7, and 20 µM, respectively; for the flavanones 7-hydroxyflavanone and naringenin the IC50 values were 65 and 85 µM, respectively. The steroidal aromatase inhibitor (positive control) 4-hydroxyandrostenedione had an IC50 of 20 nM. The inhibition by apigenin and naringenin coincided with some degree of cytotoxicity at 100 µM. The natural flavonoid derivative rotenone (IC50 0.3 µM) was the most potent aromatase inhibitor tested. Several synthetic flavonoid and structurally related quinolin-4-one analogs inhibited aromatase activity. The most potent inhibitor was 4'-tert-butyl-quinolin-4-one (IC50 2 µM), followed by two 2-pyridinyl-substituted alpha-naphthoflavones (IC50s 5 and >30 µM). The two 2-pyridinyl-substituted gamma-naphthoflavones consistently produced biphasic concentration-response curves, causing about 1.5-fold aromatase induction at concentrations below 1 µM and inhibition above that level (IC50s 7 and >30 µM). The natural flavone quercetin and isoflavone genistein induced aromatase activity 4- and 2.5-fold induction, respectively, at 10 µM. This coincided with increased intracellular cAMP concentrations and increased levels of the cAMP-dependent pII and to a lesser extent 1.3 promoter-specific aromatase transcripts. These results shed light on the structure–activity relationships for aromatase inhibition as well as mechanisms of induction in human H295R cells.
Key Words: H295R; flavonoids; quinolin-4-ones; catechins; aromatase; endocrine disruption; induction; inhibition; transcription.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Flavonoids are a group of polyphenolic phytochemicals that include flavones, isoflavones, (iso)flavanones, catechins, and chalcones, among other chemicals. They occur in relatively high concentrations in fruits, vegetables, nuts and grains, and in various herbs and spices. Flavonoids are known to have widely diverse beneficial biological effects, such as anti-inflammatory (Middleton, 1998
), antioxidant (Pietta, 2000), antiviral (Jassim and Naji, 2003), and anticancer effects (Adlercreutz, 2002; Frei and Higdon, 2003; Rietveld and Wiseman, 2003). They also modulate the function of sex hormones and their receptors. Certain flavonoids, such as the isoflavone genistein, are estrogenic (Wang et al., 1996; Zand et al., 2000), whereas others, such as chrysin, can interfere with steroid synthesis and metabolism. Although flavonoids are often called phytoestrogens, only a limited number are in fact estrogen receptor agonists. In contrast, many flavonoids are known to interfere to a greater or lesser extent with various cytochrome P450 enzymes, including those involved in steroidogenesis.
Several studies have addressed the ability of flavonoids to interfere with the catalytic activity or expression of aromatase (cytochrome P450 19; CYP19), the enzyme responsible for the conversion of androgens to estrogens (Ibrahim and Abul-Hajj, 1990; Kellis and Vickery, 1984; Le Bail et al., 1998, 2000; Pelissero et al., 1996; Whitehead and Lacey, 2003). Most of these studies were performed in human placental microsomes, and they reveal significant differences in the relative inhibition potencies of flavonoids. Although the use of microsomes for the study of enzyme inhibitors has numerous advantages, it also has several drawbacks. One aspect is that factors such as cellular uptake and metabolism of flavonoids and their effects on enzyme regulation are generally not taken into consideration. In addition, flavonoid test concentrations used in some studies exceed solubility or are in a range that would normally be toxic to cells or the organism. The use of cell lines may address some of these limitations, and it obviates the need to resort to in vivo experiments. In the present study the human adrenocortical (H295R) cell line was selected as a model system to examine the activity of a variety of natural and synthetic flavonoids on the catalytic activity and gene expression of aromatase. The H295R cell line expresses numerous steroidogenic enzymes, including aromatase, and is a useful bioassay to screen for interferences with steroidogenesis (Rainey et al., 1994; Sanderson et al., 2000; Staels et al., 1993). This cell line has been used to determine inhibitory potencies of compounds toward several steroidogenic enzymes (Johansson et al., 2002; Ohno et al., 2002; Sanderson et al., 2002) and to study the effects of chemicals on regulation of expression of these enzymes (Harvey and Everett, 2003; Li et al., 2004; Sanderson et al., 2001). Here we use the H295R cell line to determine inhibition and induction potencies of a large number of natural and synthetic flavonoids to delineate structure–activity relationships for modulation of aromatase expression and catalytic activity.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chemicals. Natural flavonoid compounds were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands) and were greater than 96% pure. Synthetic flavonoid derivatives were synthesized at the Department of Chemistry, University of California, Davis, CA). These compounds were purified by recrystallization, or by column chromatography when recrystallization was inefficient. Compound integrity and purity were confirmed by thin layer chromatography (TLC) and nuclear magnetic resonance (NMR), and all compounds included in the study were at least 95% pure. All compounds were dissolved in dimethyl sulfoxide (DMSO cell culture tested; Sigma-Aldrich) as 1000-fold concentrated stock solutions.
Cell culture conditions. H295R cells were obtained from the American Type Culture Collection (ATCC #CRL-2128) and grown in 1:1 (v/v) Dulbecco's modified Eagle medium/Ham's F-12 nutrient mix (DMEM/F12) containing 365 mg/ml L-glutamine and 15 mM HEPES (GibcoBRL). The medium was supplemented with 10 mg/l insulin, 6.7 µg/l sodium selenite, and 5.5 mg/l transferrin (ITS-G, GibcoBRL), 1.25 mg/l bovine serum albumin (Sigma-Aldrich, Zwijndrecht, The Netherlands), 100 U/l penicilline/100 µg/l streptomycin (GibcoBRL), and 2% steroid-free replacement serum Ultroser SF (Soprachem, France). For the aromatase experiments, cells were treated as described previously (Sanderson et al., 2000). In brief, cells (between 1 x 105 and 2 x 105 cells/well) in 24-well culture plates containing 1 ml medium per well were exposed to various concentrations of the natural (Sigma-Aldrich) and synthetic flavonoid compounds dissolved in 1 µl of DMSO. Negative control cells received 1 µl of DMSO. As positive control for aromatase inhibition, cells were exposed to 1 µM of the steroidal aromatase inhibitor 4-hydroxyandrostenedione in DMSO; as positive control for induction, 300 µM of 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) or 20 µM of forskolin dissolved in medium containing 0.1% (v/v) DMSO was used. Unexposed cells were included as further controls. All treatments were tested in quadruplicate, unless stated otherwise; each concentration-response experiment was performed three times. For the mRNA isolation experiments, cells in 6-well plates were exposed to 4 µl DMSO or the test chemicals in DMSO; a positive control (300 µM 8-Br-cAMP) was included on each plate. Each treatment was tested in triplicate; each experiment was performed three times. DMSO at 0.1% had no effect on CYP19 expression or catalytic activity relative to unexposed cells. Protein concentrations were determined by the method of Lowry (Lowry et al., 1951), with bovine serum albumin (Sigma-Aldrich) as standard. All exposures were for 24 h, unless stated otherwise.
Isolation and amplification of RNA. RNA was isolated using the RNA Insta-Pure System (Eurogentec, Belgium) according to the instructions of the supplier and stored at –70°C. The purity of the RNA preparations was verified by denaturing agarose gel electrophoresis. Reverse-transcriptase polymerase chain reactions (RT-PCR) were performed using the Access RT-PCR System (Promega, Madison, WI). The primer pairs and experimental conditions used for promoter-specific amplification of CYP19 mRNA were reported recently (Heneweer et al., 2004). The sequences of the pII- and 1.3-promoter-specific mRNAs were constructed from National Center for Biology Information (NCBI) accession numbers S52794 and D21241, respectively and reported by Heneweer et al. (2004). For both primers, amplification was performed using an annealing temperature of 61°C, 1 min extension, and 35 cycles. As reference, RT-PCR was performed on ß-actin mRNA as described previously (Sanderson et al., 2002). Beta-actin mRNA expression was not affected by treatment with DMSO, 30 µM quercetin, or 300 µM 8Br-cAMP; it was increased slightly but not statistically significantly (<15%) by 30 µM of genistein). Under these conditions ß-actin could be used as a reliable reference amplification response. Amplification products were detected with agarose gel electrophoresis and ethidium bromide staining. Intensities of the ethidium bromide stains were quantified with a FluorImager (Molecular Dynamics, Sunnyvale, CA).
Aromatase assay. The catalytic activity of aromatase was determined based on the method of Lephart and Simpson (1991) with minor modifications. Cells were exposed to 54 nM 1ß-3H-androstenedione (New England Nuclear Research Products [PerkinElmer] Wellesley, MA) dissolved in serum-free culture medium and incubated for 1.5 h at 37°C in an atmosphere of 5% CO2 and 95% air. All further steps were as reported previously (Sanderson et al., 2000). Aromatase activity was expressed in picomoles of androstenedione converted per hour per milligram cellular protein. The specificity of the aromatase assay based on the release of tritiated water was verified by measuring the production of estrone, using a 125I-labeled double-antibody radioimmunoassay kit (DSL-8700; Diagnostic Systems Inc, Plantation, FL), and by using the irreversible aromatase inhibitor 4-hydroxyandrostenedione (Brodie et al., 1977) to block the formation of tritiated water. Under these conditions, unexposed or DMSO-exposed cells had a basal aromatase activity of about 2.4 ± 0.2 pmole/h/mg protein.
MTT reduction assay. Mitochondrial function, as an indicator of cytotoxicity, was assessed by measuring the capacity of H295R cells to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan (Denizot and Lang, 1986). MTT is reduced to the blue-colored formazan by the mitochondrial enzyme succinate dehydrogenase, which is considered a reliable and sensitive measure of mitochondrial function. The cells in each well on the 24-well plate were incubated for 30 min at 37°C, with 0.5 ml of MTT (1 mg/ml) dissolved in serum-free medium. Then, the MTT solution was removed, after which the cells were washed twice with phosphate-buffered saline (PBS). The formazan formed in the cells was extracted by adding 1 ml of isopropanol and incubating for 10 min at room temperature. The isopropanol was added directly to a plastic cuvette for spectrophotometric analysis (Shimadzu UV-160A, Shimadzu Benelux, Belgium) at an absorbance wavelength of 560 nm. MTT reduction was linear with time for about 45 min and was not affected by DMSO treatment.
Cyclic AMP measurements. Intracellular cAMP concentrations were determined using a commercial enzyme-linked immunoassay kit (DE0450, R&D systems, Abingdon, UK) according to the instructions provided. Briefly, H295R cells were exposed to the test compounds in 12-well plates for up to 6 h, after which the medium was removed and the cells were washed with 50 mM Tris buffer containing 0.9% NaCl (PBS was not used because phosphate interferes with the immunoassay). The cells were lysed for 20 min in 300 µl of 0.1 N HCl; then the lysate was transferred to a 1.5 ml plastic vial, vortexed, and centrifuged at 12,000 x g for 10 min. The supernatant was diluted 1 in 5 with the assay buffer provided by the kit and underwent all other steps, including an acetylation step according to the instructions of the supplier. Each assay was accompanied by a cAMP standard curve.
Data analysis. All responses are presented as means of triplicate or quadruplicate determinations with standard deviations. Each full concentration-response curve was produced three times. Statistically significant differences were determined by one-way analysis of variance (ANOVA) with a significance level of 0.05. IC50 values were determined using Prism 3.00 (GraphPad Software, Inc, San Diego, CA).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aromatase Inhibition by Natural Flavonoids in H295R Cells
Various classes of naturally occurring flavonoids, including flavones, flavanones, isoflavones, and catechins (structures shown in Table 1) were tested for effects on aromatase activity and cell viability in H295R human adrenocortical carcinoma cells. Rotenone (a flavonoid derivative) was by far the most potent aromatase inhibitor, with an IC50 value of about 0.3 µM (Fig. 1; Table 1). This was well below the first statistically significant sign of cytotoxicity at a concentration of 3 µM (15% decrease in mitochondrial function; Table 1). In comparison, our positive control 4-hydroxyandrostendione had an IC50 of 20 nM (data not shown). Among the flavones, 7-hydroxyflavone, chrysin, and to lesser extent apigenin inhibited aromatase activity concentration dependently in H295R cells after a 24 h exposure (Fig. 1). 7-Hydroxyflavone, chrysin, and apigenin had IC50 concentrations of about 4, 7 and 20 µM, respectively, values that were well below concentrations that caused the first signs of cytotoxicity (Table 1). The first statistically significant decreases in cell viability (based on MTT reduction) of about 20% were observed at 30 µM for chrysin, and at 100 µM for 7-OH-flavone and apigenin. 7-Methoxyflavone had no statistically significant effect on aromatase activity at concentrations up to 100 µM (Fig. 1), a concentration at which the first statistically significant decrease in cell viability of about 30% was observed (Table 1). The flavanones were considerably less potent inhibitors. 7-Hydroxyflavanone and naringenin had IC50 values close to the highest tested concentration of 100 µM (Fig. 2), a concentration at which both compounds caused a 20% decrease in cell viability (Table 1). 7-Methoxyflavanone had no effect on aromatase activity and caused a 15% decrease in cell viability at 100 µM.
|
|
|
Aromatase Induction by Natural Flavonoids and Effects on Promoter-Specific CYP19 Transcript Levels in H295R Cells
Several (iso)flavone structures were inducers of aromatase activity in H295R cells after a 24 h exposure. Flavone and quercetin, as well as the isoflavone genistein, induced aromatase activity between 2-fold and 4.5-fold, with an apparent maximum attained at concentrations between 10 and 30 µM for quercetin and around 30 µM for genistein and flavone (Fig. 3). The sharp decline in quercetin-induced aromatase activity at 100 µM could be explained by cell toxicity, as indicated by the 40% decrease in cell viability based on MTT reduction activity (Table 1). Positive controls for aromatase induction, such as 8Br-cAMP (300 µM) and forskolin (20 µM) induced aromatase activity about 4-fold to 5-fold in H295R cells.
|
To further investigate the mechanism of induction of aromatase in H295R cells by the flavonoid quercetin and isoflavone genistein, their effect on intracellular cAMP production and the promoter-specific expression of mRNA coding for CYP19 was examined. Quercetin and genistein increased intracellular cAMP levels about 1.7-fold and 1.4-fold, respectively, after a 4 h exposure. In comparison, the positive control forskolin elevated cAMP levels over 5-fold. Initial RT-PCR experiments performed to detect cAMP promoter-specific transcript for CYP19 demonstrated that the cAMP analog 8Br-cAMP significantly increased pII and I.3 transcripts in a RNA concentration range between 0.1 and 100 ng (Fig. 4). The relative expression level of pII appeared to be greater than that of I.3, as did its inducibility by 8Br-cAMP, although we emphasize that the RT-PCR method was semiquantitative. Forskolin (data not shown), 8Br-cAMP, genistein and quercetin (Fig. 5) increased CYP19 mRNA levels that were specific for the aromatase promoters pII and I.3 after a 24 h exposure. Quercetin increased pII and I.3-specific transcript about 2.6-fold and 2-fold, respectively; genistein 2.3-fold and 1.8-fold, respectively (Fig. 5). The positive control 8Br-cAMP increased pII and I.3-specific transcript about 2.7-fold and 2.3-fold, respectively (Fig. 5).
|
|
Effects of Synthetic Flavonoid Derivatives on Aromatase Activity in H295R Cells
Various synthetic flavonoid derivatives (structures shown in Table 2) affected the catalytic activity of aromatase in H295R cells. The most potent inhibitor was 4'-tert-butyl-quinolin-4-one (A4: IC50 = 2 µM), followed by two alpha-naphthoflavone derivatives with the B-ring substituted with 2-pyridinyl-groups (AJ2: IC50 = 5 µM, AJ6: IC50 > 30 µM; Fig 6). The 2-pyridinyl-substituted gamma-naphthoflavones consistently produced biphasic concentration-response curves, causing about 1.5-fold aromatase induction at concentrations below 1 µM and inhibition above that level (BC8: IC50 = 7, BC6: IC50 > 100 µM; Fig. 6). None of the beta-naphthoflavone derivatives were active. Various flavones substituted with a bromine, fluorine, or nitro-group at the 2'-, 3'- or 4'-position of the B-ring (A7, A8, A9, Z8, and Z10) were also inactive, as were two 2-pyridinyl-substituted flavones (AL3 and AL4). BC6 and BC8, the two compounds to show initial aromatase induction, did not statistically significantly increase intracellular cAMP concentrations or levels of the promoter-specific aromatase transcripts pII and I.3 (data not shown).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aromatase Inhibition and Cytotoxicity by Rotenone
To our knowledge this is the first report of the aromatase-inhibiting capacity of the natural pesticide rotenone. Rotenone is found in tropical plants of the genus Lonchocarpus or Derris and is commonly used as a nonselective fish poison. Rotenone exerts its toxic effect by inhibiting mitochondrial function through inhibition of NADH-dependent coenzyme Q oxidoreductase (Singer and Ramsay, 1994
), thus blocking passage of electrons from NADH to coenzyme Q in the oxidative phosphorylation chain. Despite its mitochondrial toxicity in fish, rotenone was a relatively poor inhibitor of mitochondrial MTT reduction in H295R cells. A similarly poor inhibition by rotenone was observed by Berridge and coworker in 32D human bone marrow–derived cells (Berridge and Tan, 1993). MTT is reduced by the enzyme succinate dehydrogenase, which is responsible for the oxidation of succinate to fumarate in the tricarboxylic acid cycle. Succinate oxidation produces electrons that enter the oxidative phosphorylation chain directly via coenzyme Q, thus bypassing the inhibition site of rotenone. As succinate dehydrogenase activity is only weakly impaired by rotenone in the 0.01–10 µM concentration range, the energy status of the H295R cell is likely to be relatively unaffected. Similarly, the study in 32D cells showed no effect of rotenone on MTT reduction at concentrations up to 2 µM (Berridge and Tan, 1993). Rotenone has been found to cause some lipid peroxidation and only a moderate reduction of ATP levels in rat hepatocytes, but this effect was observed at the relatively high concentration of 100 µM (Zhang et al., 2001). We observed no overt signs of cytotoxicity microscopically after a 24 h exposure of H295R cells to a rotenone concentration of 10 µM. This in contrast to SK-N-MC neuroblastoma cells (Sherer et al., 2003), in which a concentration of 1 µM caused significant cell death after 24 h, although lower concentrations did not cause cytotoxicity until after about 36 to 40 h. Thus, it is possible that a 24 h exposure to rotenone is too short to assess its cytotoxic potential. Another possibility is that H295R cells are relatively insensitive to inhibition of NADH-dependent coenzyme Q oxidoreductase. Rotenone may cause aromatase inhibition via several possible mechanisms. It could interact with the heme group via the oxo-group on the 4-position of the C ring, which is common to the flavonoid structures and appears essential (although not necessarily sufficient) for aromatase inhibition. It is also possible that rotenone inhibits CYP enzymes such as CYP19 by interfering with the electron transfer to the heme-iron, by an interaction with either NADPH-cytochrome P450 reductase or NADH-cytochrome b5. The mechanism of action of CYP inhibition by rotenone is of great interest and warrants further investigation.
Aromatase Inhibition by Other Natural Flavonoids
The ability of various natural flavonoid structures to inhibit aromatase activity in vitro has been documented previously in human placental microsomes (Ibrahim and Abul-Hajj, 1990; Kellis and Vickery, 1984; Le Bail et al., 1998), in human preadipocytes (Campbell and Kurzer, 1993), in JEG-3 human placental choriocarcinoma cells (Saarinen et al., 2001), and in rainbow trout ovarian microsomes (Pelissero et al., 1996). However, considerable qualitative and quantitative differences were observed among the different test systems. Comparisons of IC50 values among different test systems is not very useful, because these values are highly dependent on experimental conditions. The observed marked qualitative differences, however, are of greater interest. For example, quercetin was found to inhibit human aromatase activity in placental microsomes (Kellis and Vickery, 1984), had no effect in JEG-3 cells (Saarinen et al., 2001), and caused aromatase induction in H295R cells in the present study. Also, the ranking of relative inhibition potencies differed among test systems, although some general trends are apparent. In JEG-3 cells (Saarinen et al., 2001), apigenin (IC50 values) was more potent (0.18 µM) than 7-hydroxyflavone (0.35 µM), chrysin (0.5 µM), naringenin (1.4 µM), and quercetin (>100 µM). In human placenta (Ibrahim and Abul-Hajj, 1990; Kellis and Vickery, 1984; Le Bail et al., 1998), 7-hydroxyflavone and chrysin (0.2–0.7 µM) were more potent than apigenin (1.2–2.9 µM), naringenin (9.2 µM), and quercetin (12 µM). In the present study with H295R cells, 7-hydroxyflavone (4 µM) was the most potent inhibitor, followed closely by chrysin (7 µM), then apigenin (20 µM) and naringenin (85 µM); quercetin was an inducer. In general these studies show that the flavones hydroxylated on the A ring were more potent aromatase inhibitors than those with additional hydroxyl-groups on the B ring; substitution of the flavone base-structure (7-hydroxyflavanone, apigenin) with a flavanone (7-hydroxyflavanone, naringenin) resulted in considerably weaker aromatase inhibition, a finding that is consistent with previous reports (Le Bail et al., 1998; Saarinen et al., 2001). In strong contrast to our study, where we found no effect of 7-methoxylated flavone and flavanone, LeBail and co-workers (1998) found them to be relatively potent inhibitors in placental microsomes with IC50 values of 3.2 and 2.6 µM, respectively.
Aromatase Induction by Natural Flavonoids
The human aromatase gene is known to be under the differential control of several tissue-specific promoters (Bulun et al., 2003; Harada et al., 1993; Simpson et al., 1993). For example, aromatase in human gonads is regulated mainly through the proximal promoter pII, and to a lesser extent through promoter I.3, both of which are stimulated by the cAMP-dependent protein kinase A (PKA) second messenger pathway. Healthy breast adipose stromal tissue utilizes promoter 1.4, which is stimulated by the glucocorticoid signaling pathway. However, in malignant breast tumors a promoter-switch appears to occur, resulting in strongly increased pII and 1.3 promoter activity (Agarwal et al., 1996; Kamat et al., 2002). In H295R cells, it would appear that several promoters are more or less actively involved in the induction of aromatase activity. The pII-specific and I.3-specific aromatase transcripts have been detected in unstimulated and forskolin/cAMP-stimulated H295R cells in two recent studies (Heneweer et al., 2004; Watanabe and Nakajin, 2004). Both studies could not detect 1.4 promoter activity. Clearly, further characterization of the promoter-specific regulation aromatase in H295R cells is required and of great interest. Nevertheless, we can use the cell line to evaluate the effects of various compounds on the pII-specific and I.3-specific regulation of aromatase expression, providing us with information on two promoters that are important in aromatase regulation in gonads and malignant breast tissue.
In the present study, quercetin was found to be a relatively potent and efficacious inducer of aromatase activity in H295R cells, followed by genistein, as also shown previously (Sanderson et al., 2002), and flavone. Both quercetin and genistein increased intracellular cAMP concentrations, and elevated cAMP-mediated pII and 1.3 promoter-specific mRNA levels in H295R cells, indicating that these (iso)flavones act through the PKA second messenger pathway. As quercetin, genistein, and to a lesser extent flavone are known phosphodiesterase inhibitors in several tissues (Kuppusamy and Das, 1992; Nichols and Morimoto, 2000), it is likely that the flavonoids cause aromatase induction in H295R cells via this mechanism. The observed potency (in decreasing order) of quercetin, genistein, and flavone to inhibit phosphodiesterase activity corresponded in rank-order with their capacity to stimulate cAMP-mediated lipolysis in rat adipocytes (Kuppusamy and Das, 1992), as well as with their ability to induce aromatase activity in H295R cells in the present study.
Implications for Human Health
Concentrations of (iso)flavonoids in human blood fall within the nM to lower µM range (Manach et al., 2004). Blood plasma concentrations of quercetin and genistein, for example, generally range from less than 15 nM to about 1.5 µM, although in soy-rich diets peak concentrations of over 4 µM may be reached shortly after ingestion (Manach et al., 2004). Blood plasma concentrations do not necessarily reflect target tissue concentrations, which may well be higher in intestine and liver than in other tissues. We observed the initial signs of aromatase induction by genistein and quercetin at concentrations above 1 µM in H295R cells. However, in vivo induction of aromatase activity by these flavonoids in experimental animals has not been established so far. It is also unknown whether pII-specific and 1.3-specific aromatase expression can be influenced by xenobiotics in humans. Thus, at present it is not possible to say whether, and to what extent, aromatase expression in vivo would be affected by high dietary intakes of genistein and quercetin. The biological significance and toxicological implications of the observed aromatase induction in vitro requires further in vivo studies.
Little information is available about human blood plasma concentrations of flavonoids such as apigenin, chrysin, and naringenin. The first inhibitory effects on aromatase activity in vitro in H295R cells were observed at concentrations above 1 µM for chrysin and apigenin, and above 10 µM for naringenin. Blood levels of naringenin, for example, are generally around 100 to 150 nM (Erlund et al., 2002), but they may reach peak levels of over 2 µM after consumption of orange or grapefruit juice (Manach et al., 2004). As a human diet contains several flavonoids with aromatase inhibitory properties in various concentrations, it may be possible for combined tissue concentrations to be reached that result in a certain degree of aromatase inhibition. However, under normal dietary conditions, flavones occur in complex mixtures of which the individual components have various (inhibitory or inductive) effects on enzymes such as aromatase, making the prediction of a net effect difficult. Also, the ultimate beneficial effects usually assigned to flavonoid exposure are the product of a balanced set of interactions; these include not only the effects of these compounds on aromatase activity but also their effects on other enzyme systems and their (often more potent) antioxidant (Bravo, 1998; Duthie and Crozier, 2000), anti-inflammatory, and anti-proliferative effects (Kuntz et al., 1999). Therefore, the effects of flavonoids on aromatase activity cannot be seen in isolation. However, the situation may be different for exposures to single flavonoids (e.g., chrysin) in the form of food supplements. It is likely that consumption of high quantities of such food supplements, often containing more than 100 times the normal dietary intake, results in tissue concentrations of a single flavonoid that are sufficiently high to inhibit aromatase activity. Strong decreases of aromatase activity in females may result in a number of reproductive disturbances, such as disruption of the menstrual cycle (Brodie et al., 1989), and loss of bone density (Turner et al., 1994). In men, decreased estrogen synthesis may also result in deleterious effects on bone homeostasis (Vanderschueren et al., 1998) and disruption of spermatogenesis (Carreau et al., 2003).
Structure–Activity Relationships for Aromatase Inhibition by Flavonoids and Their Derivatives in H295R Cells
An essential contributor to the aromatase inhibitory effect of flavonoids is the 4-oxo group on the C ring of the flavone base structure (Kao et al., 1998). Hydroxylation of the 7-position on the A ring enhances the inhibitory potency considerably, whereas methoxylation diminishes this effect. As for the effect of additional hydroxylation at the 5-position of the A ring (chrysin), it has been suggested that this reduces aromatase inhibition potency after formation of a hydrogen bond with the 4-oxo group (Kao et al., 1998). However, we did not observe a dramatic decrease in potency when comparing 7-hydroxyflavone with chrysin. It is also clear that the presence of a 7-hydroxy group on the A ring does not appear to be essential, as various alpha-naphthoflavones (7,8-benzoflavones) are also potent aromatase inhibitors.
Flavones (7-hydroxyflavone, apigenin) were significantly more potent aromatase inhibitors than flavanones (7-hydroxyflavanone and naringenin). It is plausible that the lack of 2,3-double bond in the flavanones results in reduced electronegativity of the 4-oxo group and, subsequently, a weaker interaction of this group with the heme prosthetic group of the aromatase enzyme. Obliteration of aromatase inhibition capacity is seen when flavonoid structures are substituted on the 3-position of the C ring, as was observed for quercetin and genistein. This is consistent with the generally very weak or nonexistent inhibitory effects of these compounds found in other studies (Campbell and Kurzer, 1993; Kao et al., 1998; Pelissero et al., 1996; Saarinen et al., 2001).
Substitutions on the B ring of the flavone base structure generally decrease aromatase inhibitory potency, as can be seen by comparing the natural flavone chrysin to apigenin or 7-OH-flavone to the synthetic flavone Z10. One notable exception is the relatively potent inhibitory effect of 4'-tert-butyl-quinolin-4-one (A4), indicating that a t-butyl substitution on the B ring is an important contributing factor. Whether the influence of the t-butyl group is steric, electronic (by donating electrons), or a consequence of increased lipophilicity is presently unknown. Fluorination of the B ring obliterates aromatase inhibition by alpha-naphthoflavone derivatives (AJ2, AJ6, AJ8, and AJ9) or quinolin-4-one derivatives (A2 and A3). Effects of substitutions on the B ring of the synthetic flavones A7, A8, and A9 could not be discerned in our test system because flavone itself was not an inhibitor.
In summary, the 4-oxo group on the C ring appears to be essential for a flavonoid compound to be able to interact with the heme iron of CYP19 and cause aromatase inhibition. This finding rules out any significant capacity for related compounds that lack the 4-oxo group such as catechins or anthocyanidins, but not the chalcones, to inhibit aromatase activity. Inhibition potency is improved by the presence of a 7-hydroxy or 7,8-benzo group on the A ring, although their presence is not essential. Inhibition potency is also improved by the presence of electron donating groups on the B ring, although again this presence is not essential, whereas an electron withdrawing group results in decreased potency. Decreased aromatase inhibition potency is also caused by groups that interfere (either electronically or sterically) with the 4-oxo group, such as substitutions on the 3 position of the C ring, and large substitutions on the 5- or 6-position of the A ring.
|
NOTES |
|---|
1 To whom correspondence should be addressed at Institute for Risk Assessment Sciences (IRAS), University of Utrecht, P.O. Box 80176, 3508 TD Utrecht, The Netherlands. Fax: +31-30–253–5077. E-mail: t.sanderson{at}iras.uu.nl.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adlercreutz, H. (2002). Phyto-oestrogens and cancer. Lancet Oncol. 3, 364–373.[CrossRef][Web of Science][Medline]
Agarwal, V. R., Bulun, S. E., Leitch, M., Rohrich, R., and Simpson, E. R. (1996). Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J. Clin. Endocrinol. Metab. 81, 3843–3849.
Berridge, M. V., and Tan, A. S. (1993). Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch. Biochem. Biophys. 303, 474–482.[CrossRef][Web of Science][Medline]
Bravo, L. (1998). Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev. 56, 317–333.[Web of Science][Medline]
Brodie, A. M., Hammond, J. O., Ghosh, M., Meyer, K., and Albrecht, E. D. (1989). Effect of treatment with aromatase inhibitor 4-hydroxandrostenedione on the nonhuman primate menstrual cycle. Cancer Res. 49, 4780–4784.
Brodie, A. M., Schwarzel, W. C., Shaikh, A. A., and Brodie, H. J. (1977). The effect of an aromatase inhibitor, 4-hydroxy-4-androstene-3,17-dione, on estrogen-dependent processes in reproduction and breast cancer. Endocrinology 100, 1684–1695.
Bulun, S. E., Sebastian, S., Takayama, K., Suzuki, T., Sasano, H., and Shozu, M. (2003). The human CYP19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters. J. Steroid Biochem. Mol. Biol. 86, 219–224.[CrossRef][Web of Science][Medline]
Campbell, D. R., and Kurzer, M. S. (1993). Flavonoid inhibition of aromatase enzyme activity in human preadipocytes. J. Steroid Biochem. Mol. Biol. 46, 381–388.[CrossRef][Web of Science][Medline]
Carreau, S., Lambard, S., Delalande, C., Denis-Galeraud, I., Bilinska, B., and Bourguiba, S. (2003). Aromatase expression and role of estrogens in male gonad : a review. Reprod. Biol. Endocrinol. 1, 35.[CrossRef][Medline]
Denizot, F., and Lang, R. (1986). Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89, 271–277.[CrossRef][Web of Science][Medline]
Duthie, G., and Crozier, A. (2000). Plant-derived phenolic antioxidants. Curr. Opin. Clin. Nutr. Metab. Care 3, 447–451.[CrossRef][Medline]
Erlund, I., Silaste, M. L., Alfthan, G., Rantala, M., Kesaniemi, Y. A., and Aro, A. (2002). Plasma concentrations of the flavonoids hesperetin, naringenin and quercetin in human subjects following their habitual diets, and diets high or low in fruit and vegetables. Eur. J. Clin. Nutr. 56, 891–898.[CrossRef][Web of Science][Medline]
Frei, B., and Higdon, J. V. (2003). Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J. Nutr. 133, 3275S–3284S.
Harada, N., Utsumi, T., and Takagi, Y. (1993). Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis. Proc. Natl. Acad. Sci. U. S. A. 90, 11312–11316.
Harvey, P. W., and Everett, D. J. (2003). The adrenal cortex and steroidogenesis as cellular and molecular targets for toxicity: critical omissions from regulatory endocrine disrupter screening strategies for human health? J. Appl. Toxicol. 23, 81–87.[CrossRef][Web of Science][Medline]
Heneweer, M., van den Berg, M., and Sanderson, J. (2004). A comparison of human H295R and rat R2C cell lines as in vitro screening tools for effects on aromatase. Toxicol. Lett. 146, 183–194.[CrossRef][Web of Science][Medline]
Ibrahim, A. R., and Abul-Hajj, Y. J. (1990). Aromatase inhibition by flavonoids. J. Steroid Biochem. Mol. Biol. 37, 257–260[CrossRef][Web of Science][Medline]
Jassim, S. A., and Naji, M. A. (2003). Novel antiviral agents: a medicinal plant perspective. J. Appl. Microbiol. 95, 412–427.[CrossRef][Medline]
Johansson, M. K., Sanderson, J. T., and Lund, B. O. (2002). Effects of 3-MeSO2-DDE and some CYP inhibitors on glucocorticoid steroidogenesis in the H295Rhuman adrenocortical carcinoma cell line. Toxicol. In Vitro 16, 113–121.[CrossRef][Web of Science][Medline]
Kamat, A., Hinshelwood, M. M., Murry, B. A., and Mendelson, C. R. (2002). Mechanisms in tissue-specific regulation of estrogen biosynthesis in humans. Trends Endocrinol. Metab. 13, 122–128.[CrossRef][Web of Science][Medline]
Kao, Y. C., Zhou, C., Sherman, M., Laughton, C. A., and Chen, S. (1998). Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: A site-directed mutagenesis study. Environ. Health Perspect. 106, 85–92.[Web of Science][Medline]
Kellis, J. T., Jr., and Vickery, L. E. (1984). Inhibition of human estrogen synthetase (aromatase) by flavones. Science 225, 1032–1034.
Kuntz, S., Wenzel, U., and Daniel, H. (1999). Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines. Eur. J. Nutr. 38, 133–142.[CrossRef][Web of Science][Medline]
Kuppusamy, U. R., and Das, N. P. (1992). Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes. Biochem. Pharmacol. 44, 1307–1315.[CrossRef][Web of Science][Medline]
Le Bail, J. C., Champavier, Y., Chulia, A. J., and Habrioux, G. (2000). Effects of phytoestrogens on aromatase, 3beta and 17beta-hydroxysteroid dehydrogenase activities and human breast cancer cells. Life Sci. 66, 1281–1291.[CrossRef][Web of Science][Medline]
Le Bail, J. C., Laroche, T., Marre-Fournier, F., and Habrioux, G. (1998). Aromatase and 17beta-hydroxysteroid dehydrogenase inhibition by flavonoids. Cancer Lett. 133, 101–106.[CrossRef][Web of Science][Medline]
Lephart, E. D., and Simpson, E. R. (1991). Assay of aromatase activity. Methods Enzymol. 206, 477–483.[Web of Science][Medline]
Li, L. A., Wang, P. W., and Chang, L. W. (2004). Polychlorinated biphenyl 126 stimulates basal and inducible aldosterone biosynthesis of human adrenocortical H295R cells. Toxicol. Appl. Pharmacol. 195, 92–102.[CrossRef][Web of Science][Medline]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Manach, C., Scalbert, A., Morand, C., Remesy, C., and Jimenez, L. (2004). Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 79, 727–747.
Middleton, E., Jr. (1998). Effect of plant flavonoids on immune and inflammatory cell function. Adv. Exp. Med. Biol. 439, 175–182.[Web of Science][Medline]
Nichols, M. R., and Morimoto, B. H. (2000). Differential inhibition of multiple cAMP phosphodiesterase isozymes by isoflavones and tyrphostins. Mol. Pharmacol. 57, 738–745.
Ohno, S., Shinoda, S., Toyoshima, S., Nakazawa, H., Makino, T., and Nakajin, S. (2002). Effects of flavonoid phytochemicals on cortisol production and on activities of steroidogenic enzymes in human adrenocortical H295R cells. J. Steroid Biochem. Mol. Biol. 80, 355–363.[CrossRef][Web of Science][Medline]
Pelissero, C., Lenczowski, M. J., Chinzi, D., Davail-Cuisset, B., Sumpter, J. P., and Fostier, A. (1996). Effects of flavonoids on aromatase activity, an in vitro study. J. Steroid Biochem. Mol. Biol. 57, 215–223.[CrossRef][Web of Science][Medline]
Pietta, P. G. (2000). Flavonoids as antioxidants. J. Nat. Prod. 63, 1035–1042.[CrossRef][Medline]
Rainey, W. E., Bird, I. M., and Mason, J. I. (1994). The NCI-H295 cell line: a pluripotent model for human adrenocortical studies. Mol. Cell Endocrinol. 100, 45–50.[CrossRef][Web of Science][Medline]
Rietveld, A., and Wiseman, S. (2003). Antioxidant effects of tea: evidence from human clinical trials. J. Nutr. 133, 3285S–3292S.
Saarinen, N., Joshi, S. C., Ahotupa, M., Li, X., Ammala, J., Makela, S., and Santti, R. (2001). No evidence for the in vivo activity of aromatase-inhibiting flavonoids. J. Steroid Biochem. Mol. Biol. 78, 231–239.[CrossRef][Web of Science][Medline]
Sanderson, J. T., Boerma, J., Lansbergen, G. W., and van den Berg, M. (2002). Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol. Appl. Pharmacol. 182, 44–54.[CrossRef][Web of Science][Medline]
Sanderson, J. T., Seinen, W., Giesy, J. P., and van den Berg, M. (2000). 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? Toxicol. Sci. 54, 121–127.
Sanderson, J. T., Slobbe, L., Lansbergen, G. W., Safe, S., and van den Berg, M. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells. Toxicol. Sci. 61, 40–48.
Sherer, T. B., Betarbet, R., Testa, C. M., Seo, B. B., Richardson, J. R., Kim, J. H., Miller, G. W., Yagi, T., Matsuno-Yagi, A., and Greenamyre, J. T. (2003). Mechanism of toxicity in rotenone models of Parkinson's disease. J. Neurosci. 23, 10756–10764.
Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Corbin, C. J., and Mendelson, C. R. (1993). Tissue-specific promoters regulate aromatase cytochrome P450 expression. Clin. Chem. 39, 317–324.[Abstract]
Singer, T. P., and Ramsay, R. R. (1994). The reaction sites of rotenone and ubiquinone with mitochondrial NADH dehydrogenase. Biochim. Biophys. Acta 1187, 198–202.[Medline]
Staels, B., Hum, D. W., and Miller, W. L. (1993). Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal. Mol. Endocrinol. 7, 423–433.
Turner, R. T., Riggs, B. L., and Spelsberg, T. C. (1994). Skeletal effects of estrogen. Endocrinol. Rev. 15, 275–300.
Vanderschueren, D., Boonen, S., and Bouillon, R. (1998). Action of androgens versus estrogens in male skeletal homeostasis. Bone 23, 391–394.[Medline]
Wang, T. T., Sathyamoorthy, N., and Phang, J. M. (1996). Molecular effects of genistein on estrogen receptor mediated pathways. Carcinogenesis 17, 271–275.
Watanabe, M., and Nakajin, S. (2004). Forskolin up-regulates aromatase (CYP19) activity and gene transcripts in the human adrenocortical carcinoma cell line H295R. J. Endocrinol. 180, 125–133.[Abstract]
Whitehead, S. A., and Lacey, M. (2003). Phytoestrogens inhibit aromatase but not 17beta-hydroxysteroid dehydrogenase (HSD) type 1 in human granulosa-luteal cells: evidence for FSH induction of 17beta-HSD. Hum. Reprod. 18, 487–444.
Zand, R. S., Jenkins, D. J., and Diamandis, E. P. (2000). Steroid hormone activity of flavonoids and related compounds. Breast Cancer Res. Treat. 62, 35–49.[CrossRef][Web of Science][Medline]
Zhang, J. G., Tirmenstein, M. A., Nicholls-Grzemski, F. A., and Fariss, M. W. (2001). Mitochondrial electron transport inhibitors cause lipid peroxidation-dependent and -independent cell death: protective role of antioxidants. Arch. Biochem. Biophys. 393, 87–96.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S.-M. Hsia, Y.-H. Kuo, W. Chiang, and P. S. Wang Effects of adlay hull extracts on uterine contraction and Ca2+ mobilization in the rat Am J Physiol Endocrinol Metab, September 1, 2008; 295(3): E719 - E726. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Rice and S. A Whitehead Phytoestrogens and breast cancer -promoters or protectors? Endocr. Relat. Cancer, December 1, 2006; 13(4): 995 - 1015. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. Sanderson The Steroid Hormone Biosynthesis Pathway as a Target for Endocrine-Disrupting Chemicals Toxicol. Sci., November 1, 2006; 94(1): 3 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Auborn Can Indole-3-Carbinol-Induced Changes in Cervical Intraepithelial Neoplasia Be Extrapolated to Other Food Components? J. Nutr., October 1, 2006; 136(10): 2676S - 2678S. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||