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Inhibition of 17beta-hydroxysteroid dehydrogenases by phytoestrogens: Comparison with other steroid metabolizing enzymes
Journal of Steroid Biochemistry & Molecular Biology 93 (2005) 285–292
Inhibition of 17beta-hydroxysteroid dehydrogenases by phytoestrogens: Comparison with other steroid metabolizing enzymes D. Deluca a , A. Krazeisen a , R. Breitling b , C. Prehn a , G. M¨oller a , J. Adamski a, ∗ a
GSF-National Research Center for Environment and Health, Institute for Experimental Genetics, Ingolst¨adter Landstraße 1, 85764 Neuherberg, Germany b University of Glasgow, Molecular Plant Sciences Group and Bioinformatics Research Centre, Institute of Biomedical and Life Sciences, Glasgow G12 8QQ, UK
Abstract Effects of phytoestrogens on human health have been reported for decades. These include not only beneficial action in cancer prevention but also endocrine disruption in males. Since then many molecular mechanisms underlying these effects have been identified. Targets of phytoestrogens comprise steroid receptors, steroid metabolising enzymes, elements of signal transduction and apoptosis pathways, and even the DNA processing machinery. Understanding the specific versus pleiotropic effects of selected phytoestrogens will be crucial for their biomedical application. This review will concentrate on the influence of phytoestrogens on 17beta-hydroxysteroid dehydrogenases from a comparative perspective with other steroid metabolizing enzymes. © 2004 Elsevier Ltd. All rights reserved. Keywords: 17beta-Hydroxysteroid dehydrogenase; Phytoestrogens; Flavonoids
1. Role of phytoestrogens in human health Epidemiological studies comparing disease incidence in countries with high-quality health care have shown striking geographic differences in the occurrence of not only hormone-related types of cancer such as breast and prostate cancers [1–3] but also cancers of the digestive tract [4], and hormone-dependent cardiovascular diseases [5], and also in the progression of post-menopausal-related diseases [6–12]. The incidence of these diseases is significantly lower in Asia compared to Northern Europe and America [13–15]. These studies suggest that environmental factors, especially dietary components, have a major impact on the development and progression of several cancer types and other hormonerelated diseases. One class of substances are suggested to be responsible for these protective effects. These are the phytoestrogens which are particularly abundant in soy products, a major component of the Asian diet. They are functionally
Abbreviations: 17-HSD, 17beta-hydroxysteroid dehydrogenase; P450, cytochrome P450 ∗ Corresponding author. Tel.: +49 89 3187 3155; fax: +49 89 3187 3225. E-mail address: [email protected] (J. Adamski). 0960-0760/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2004.12.035
defined as substances found in plants that promote estrogenic actions in animals [16].
2. Role of phytoestrogens in plants Phytoestrogens are a chemically diverse set of compounds and not all their biological roles in plants are fully understood. However, some of them are thought to act as natural fungicides, UV-protectants, anti-oxidants and flower pigments, and also to participate in pollen germination and stress signalling [17–23].
3. Sources of phytoestrogens Phytoestrogens not only can be found in any plant or plantderived product, but are also detectable in water as a result of plant decomposition. They can be divided into three main classes: flavonoids, coumestans and lignans. Most of them are diphenolic substances usually with structural similarity in their three-dimensional conformation to the natural human steroid hormones. The group of flavonoids comprise flavones,
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Table 1 Phytoestrogens reveal pleiotropic effects on single targets and regulatory pathways Substance
Target
Effect
Reference
Lignans, isoflavones Genistein, naringenin Flavonoids, lignans Quercetin Formononetin, genistein, biochanin A, daidzein, and equol Flavonoids Daidzein, genistein, coumestrol Isoflavones
alpha-Fetoprotein Sex hormone-binding globulin (SHGB) Steroid receptors Guanylate cyclase MAP kinase
Hormone displacement Hormone displacement Activation Activation Inhibition
[58] [59] [39,60–62] [63] [64]
Protein kinase B Prostaglandin synthase Vitamin D synthesis (CYP24 and CYP27B1 transcriptional inhibition) Sulfatase Sulfotransferase 3beta-Hydroxysteroid dehydrogenase delta5/delta4 isomerase Aromatase P450 (CYP19A1) 11beta-Hydroxysteroid dehydrogenase 1 17beta-Hydroxysteroid dehydrogenases 5alpha-Reductase Caspase-3 and -8 Topoisomerase II
Inhibition Activation Activation
[65,66] [67] [36,68,69]
Inhibition Inhibition Inhibition
[70] [70,71] [46,72]
Inhibition Inhibition Inhibition Inhibition Cleavage promotion Inhibition
[46,73,74] [56,75] [46,47,49–51] [38,76] [37] [77]
Daidzein Quercetin, genistein Isoflavones, flavonoids Flavonoids Flavanone Flavonoids Isoflavones Daidzein Coumestrol, genistein
flavanones and isoflavones. Flavonoids are found in almost all plant families in leaves, stems, roots, flowers and seeds [22,24]. The main dietary source for isoflavones is the soybean, containing the glycoside of genistein among others. Flavones and flavanones are widely distributed in all plant families and are found in fruits, vegetables, berries, herbs, beans, and green tea. Most of the flavonoids found in plants are present as glycosides, but only the aglycones exert estrogen-like activities after their release by hydrolytic cleavage during digestion. High levels of coumestrol are found in alfalfa and various beans [8]. The plant lignans are instead found in fibre-rich food, such as unrefined grain products, and are transformed by intestinal bacteria into mammalian lignans such as enterolactone and enterodiol [25]. Additional dietary estrogenic compounds are the resorcyclic acid lactones, e.g. zearalenone which occur as secondary metabolites in mold-infected food [26].
4. Concentrations of phytoestrogens in humans Beans, especially soybeans, are a major nutritional component in the Asian diet. A calculation of the approximate intake of isoflavones by rural Japanese men gave a mean value of about 6 mg daidzein and 10 mg genistein per day. These men have very high concentrations of isoflavones in their plasma [27–30]. The highest individual value exceeded 2 M. Compared to these high levels, the concentration of these flavonoids in urine and plasma in men living in Europe is very low. The mean plasma value in Finnish subjects for daidzein and genistein is about 5 nM each [31]. After consumption of 20 g roasted beans, the following peak concentrations were observed: 0.2 M in human milk, 2 M in plasma and 20 M in urine [23]. Local tissue concentrations
are expected to be two- to three-fold higher. These differences in the concentrations are around three orders of magnitude, and the high concentrations found in the Asian population are in a range that might influence estrogen signalling. These data should attract attention to these substances and to their possible application for treating disorders associated to estrogens.
5. Targets of phytoestrogens The effects of phytoestrogens were reported controversially: Potential activities as endocrine disruptors in males were described in several studies [32–34]. On the other hand, beneficial influence could be observed as well: beneficial role in the modulation of progression of breast and prostate cancers [3,35], enhancement of vitamin D-mediated inhibition of tumour progression [36], alternative to hormone replacement therapies [6], down-regulation of osteoclast differentiation [37], and improvement of neuronal protection and memory [35,38]. Several in vitro and in vivo studies identified various modes of action for phytoestrogens. They modulate signal transduction by the estrogen receptors (alpha and beta), the aryl hydrocarbon receptor, the pregnane X-receptor, the constitutive androstane receptor and the peroxisome proliferatoractivated receptors (alpha and gamma) [3,39], and influence transport proteins, kinases and further enzymes, as shown in Table 1.
6. Function of 17beta-hydroxysteroid dehydrogenases Pre-receptor regulation of steroid hormone action is controlled by hydroxysteroid dehydrogenases and reductases
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converting steroids at positions 3, 5, 11, 17 and 20 of the steroid backbone [40–43]. The subject of this review is the influence of phytoestrogens on 17beta-hydroxysteroid dehydrogenases (17-HSD) in comparison to other steroid metabolising enzymes. By conversion at position 17 of the steroid molecule the 17-HSDs modulate the biological potency of estrogens and androgens: the keto-forms are inactive whereas the hydroxy-forms are active and access the receptors (Fig. 1). 17-Hydroxysteroid dehydrogenases belong to two protein superfamilies: SDR (short-chain dehydrogenase/reductase) and AKR (aldo-ketoreductase) [41,44]. Ten types of human 17-HSDs have been described to date varying in substrate specificity and tissue distribution [45]. 17-HSD 5 is unique among this group of enzymes as it belongs to the AKR family, while the rest of the 17-HSDs belongs to the SDR superfamily [45].
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genases versus other enzymes of steroid metabolism. Given the pleiotropic and sometimes adverse effects of phytoestrogens, understanding the differences in specificity and their molecular basis will be crucial for the development of efficient biomedical applications. 7.1. 3beta-HSD/delta5/delta4 isomerase versus aromatase and 17β-HSD 1 In a comparative study in human placental microsomes Le Bail et al. [46] tested phytoestrogens for inhibition of P450 aromatase, 3-hydroxysteroid dehydrogenase-delta5/delta4 isomerase and 17-HSD 1 (Fig. 1). Isoflavonoids and compounds which presented the phenolic B ring at position 3 of the pyran ring preferentially inhibited 3betaHSD/delta5/delta4 isomerase and/or 17-HSD activities over aromatase activity [46]. However, 7-hydroxy-flavone and apigenin (for structures see Fig. 2) were the most effective aromatase and 17-HSD 1 inhibitors, respectively [47].
7. Comparison of inhibition of hydroxysteroid dehydrogenases by phytoestrogens with other steroid metabolizing enzymes
7.2. Aromatase and 17β-HSD 1
In the following we will present a comparative overview of the effects of phytoestrogens on hydroxysteroid dehydro-
Selected phytoestrogens were checked for aromatase and 17-HSD 1 activity modulation in primary cultures of hu-
Fig. 1. This simplified section of steroid conversions involving 17beta-, 3beta-, 3alfa-HSDs and aromatase activities. Simplified part of steroid metabolism is annotated with enzymes catalyzing reactions inhibited by phytoestrogens. Steroid numbering: dehydroepiandrosterone (1), androstenedione (2), estrone (3), estradiol (4), testosterone (5), dihydrotestosterone (6), androstanediol (7), androsterone (8).
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Fig. 2. Chemical formulas of the most potent inhibitors of steroid metabolizing enzymes. Chemical structures (A) are given together with 3D-representations (B). All molecules are shown in the same orientation as the natural substrates. Numbering: estradiol (4), testosterone (5), 7-hydroxy-flavone (9), flavanone (10), apigenin (11), kaempferol (12), coumestrol (13), quercetin (14), abietic acid (15), zearalenone (16). 3D-coordinates were prepared in ChemDraw Pro and graphics rendered in iMOL. Corresponding PDB files are available from the authors upon request.
man granulosa-luteal cells by Whitehead et al. [48]. Apigenin and zearalenone significantly inhibited the aromatase activity, while biochanin A and quercetin had no effect. None of the phytoestrogens inhibited FSH-induced 17-HSD 1 activity, and only quercetin significantly inhibited progesterone production. 7.3. 17β-HSD 1 and 17β-HSD 2 Makela et al. [49,50] studied the effects of several flavonoids and isoflavonoids on 17beta-oxidoreduction of estrogens by purified 17-HSD 1 and in cell lines expressing 17-HSD 1 (T-47D breast cancer cells) or 17-HSD 2 (PC-3 prostate cancer cells). Several conclusions could be drawn in light of the reported results about these phytoestrogens. Flavones, flavanones, and isoflavones hydroxylated at both the double ring (positions 5 and 7) and ring B (position 4 ) were the most potent inhibitors of estrone reduction (17HSD 1 activity) in T-47D cells, the best being apigenin and coumestrol. The same results were found with purified 17HSD 1. Flavones hydroxylated at positions 3, 5 and 7 of rings A and C, with or without a hydroxyl group in ring B, were ca-
pable of inhibiting estradiol oxidation (17-HSD 2 activity) in PC-3 cells. Changes to the flavanone structure, or hydroxylation at position 3 of ring C of flavones, or methylation of the hydroxyl group at position 4 of ring B of flavones and isoflavones reduced or abolished their inhibitory activity on estrone reduction in T-47D cells. In contrast, the hydroxyl group at position 3 of flavones markedly increased the inhibition of estradiol oxidation in PC-3 cells, kaempferol being in this case the best inhibitor. Thus, changes in the number and location of hydroxyl groups may discriminate between inhibition of estrone reduction and estradiol oxidation. Some of the differences may be due to differences in the pharmacokinetics of these compounds in T-47D and PC-3 cells. Regarding prevention or treatment of estrogenrelated diseases, the substances apigenin, coumestrol, and genistein raise special interest considering the results so far. 7.4. 17β-HSD 5 The influence of phytoestrogens (flavonoids, coumarins, coumestans) on the reductive (androstenedione to testos-
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terone) and oxidative (androstanediol to androsterone) 17-activity of the recombinant purified human 17-HSD 5 was tested by Krazeisen et al. [51]. The best inhibitors for the reductive activity were zearalenone, coumestrol, quercetin and biochanin A (IC50 4 M, 4 M, 9 M and 14 M, respectively). The oxidative activity was inhibited most strongly by zearalenone, quercetin, 7-hydroxy-flavone and biochanin A (IC50 2 M, 5 M, 7 M and 8 M, respectively). Among the group of flavones the inhibitor potency is growing with increasing number of hydroxylations in ring A, the most effective being a hydroxylation at position 7. For flavonoids the issue is more complex due to the presence or absence of a double bond in ring C, the position of ring B as well as the hydroxylation pattern. Under reductive conditions a double bond in ring C increases the inhibitory potency (e.g. naringenin versus apigenin). In contrast, the flavanones (lacking the double bond) have stronger influence on the oxidative activity of 17-HSD 5. For both 17-hHSD 5 activities, inhibitor potency decreases when ring B is found at position 3 as it is in isoflavones. Flavones carrying a 5methoxy group at ring A are more potent than those with a hydroxy group at this position. However, within the group of flavones methylation of position 5 increases inhibition of the reductive pathway but decreases activity in oxidative conversion. Within the group of isoflavones methylation of the hydroxy group at position 4 correlates with a high inhibitor activity. On the other hand, substances like the synthetic antiestrogens tamoxifen, diethylstilbestrol, daidzein and coumarin had no influence on both types of reaction. An interesting inhibition pattern is observed for 18-glycyrrhetinic acid with 17-HSD 5, as in this case this compound has no influence on the oxidative, but only on the reductive reaction of the enzyme. This result could be explained with a deprotonation of the His 117 in the active center at higher pH values, at which the oxidation tests were performed in vitro. The deprotonated side chain of His 117 is then unable to bind the substrate 18-glycyrrethinic acid. This indicates that this substrate binds at the active center of the enzyme in a pH- and cofactor-dependent way [51]. The proposed model can now be further analysed in light of
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the recently determined crystal structure of 17-HSD 5 [52]. The dietary substances described above shown to be potent inhibitors of 17-HSD 5 seem to play an important role in delaying the development of hormone-dependent cancers such as that of prostate carcinoma. 7.5. 11β-HSD and 17β-HSD and 17,20-lyase The inhibitory capacity of 18-glycyrrhetinic acid, contained in licorice, has been firstly observed toward 11-HSD (Fig. 3) [53] and later toward other enzymes as well, as it is reported above with 17-HSD5. Previously it has been reported that the serum testosterone level in men consuming about 7 g of a commercial preparation of licorice (containing 0.5 g of 18-glycyrrhetinic acid) a day is significantly reduced [54]. The inhibitory effect of 18-glycyrrhetinic acid may be the reason for that, since it has been demonstrated that licorice consumption inhibits 11-HSD [55], 17-HSD and 17,20-lyase activity (Fig. 4) [54]. Decreased testosterone levels result in reduced libido or other sexual dysfunctions but might have beneficial effects in cases of abnormal prostate growth. 7.6. 11β-HSD 1 and 11β-HSD 2 Abietic acid inhibited both 11-HSD 1 (IC50 27 M for reduction and 2.8 M for oxidation) and 11-HSD 2 (IC50 12 M). Flavanone and 2 -hydroxyflavanone efficiently (IC50 18 and 10 M, respectively) inhibited reductive but not oxidative activity of 11-HSD 1 as shown by Schweizer et al. [56]. These assays were performed with lysates of cells stably transfected with 11-HSD 1 and 11-HSD 2. This observation reveals that a variety of environmental compounds exert distinct inhibitory effects on 11-HSD 1 and 11HSD 2, opening the possibility for selectively modulating local cortisone/cortisol availability in vivo. Amide derivatives of the 18-glycyrrethinic acid at position 30 were studied as possible inhibitors of 11-HSD 1 and 11-HSD 2. The study opens the way to a new class of substances and a specific inhibitor for 11-HSD 2 has already been identified [57].
Fig. 3. Steroid conversions at position 11beta. Steroid numbering: cortisol (17), cortisone (18).
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References
Fig. 4. Activity of 17-hydroxylase, 17,20-lyase. Steroid numbering: pregnenolone (19), 17-hydroxy-pregnenolone (20), dehydroepiandrosterone (1).
8. Conclusion Although phytoestrogens can inhibit quite a broad spectrum of steroid metabolizing enzymes, there is always an enzyme-specific preference. For example, 18glycyrrethinic acid is a potent inhibitor of 11-HSDs but not a very effective modulator of 17-HSDs. Future work should be able to exploit our improved understanding of these specific effects for the development of targeted inhibitors of steroid metabolism.
Acknowledgement This work was supported in part by a DFG grant to Jerzy Adamski.
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Inhibition of 17beta-hydroxysteroid dehydrogenases by phytoestrogens: Comparison with other steroid metabolizing enzymes D. Deluca a , A. Krazeisen a , R. Breitling b , C. Prehn a , G. M¨oller a , J. Adamski a, ∗ a
GSF-National Research Center for Environment and Health, Institute for Experimental Genetics, Ingolst¨adter Landstraße 1, 85764 Neuherberg, Germany b University of Glasgow, Molecular Plant Sciences Group and Bioinformatics Research Centre, Institute of Biomedical and Life Sciences, Glasgow G12 8QQ, UK
Abstract Effects of phytoestrogens on human health have been reported for decades. These include not only beneficial action in cancer prevention but also endocrine disruption in males. Since then many molecular mechanisms underlying these effects have been identified. Targets of phytoestrogens comprise steroid receptors, steroid metabolising enzymes, elements of signal transduction and apoptosis pathways, and even the DNA processing machinery. Understanding the specific versus pleiotropic effects of selected phytoestrogens will be crucial for their biomedical application. This review will concentrate on the influence of phytoestrogens on 17beta-hydroxysteroid dehydrogenases from a comparative perspective with other steroid metabolizing enzymes. © 2004 Elsevier Ltd. All rights reserved. Keywords: 17beta-Hydroxysteroid dehydrogenase; Phytoestrogens; Flavonoids
1. Role of phytoestrogens in human health Epidemiological studies comparing disease incidence in countries with high-quality health care have shown striking geographic differences in the occurrence of not only hormone-related types of cancer such as breast and prostate cancers [1–3] but also cancers of the digestive tract [4], and hormone-dependent cardiovascular diseases [5], and also in the progression of post-menopausal-related diseases [6–12]. The incidence of these diseases is significantly lower in Asia compared to Northern Europe and America [13–15]. These studies suggest that environmental factors, especially dietary components, have a major impact on the development and progression of several cancer types and other hormonerelated diseases. One class of substances are suggested to be responsible for these protective effects. These are the phytoestrogens which are particularly abundant in soy products, a major component of the Asian diet. They are functionally
Abbreviations: 17-HSD, 17beta-hydroxysteroid dehydrogenase; P450, cytochrome P450 ∗ Corresponding author. Tel.: +49 89 3187 3155; fax: +49 89 3187 3225. E-mail address: [email protected] (J. Adamski). 0960-0760/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2004.12.035
defined as substances found in plants that promote estrogenic actions in animals [16].
2. Role of phytoestrogens in plants Phytoestrogens are a chemically diverse set of compounds and not all their biological roles in plants are fully understood. However, some of them are thought to act as natural fungicides, UV-protectants, anti-oxidants and flower pigments, and also to participate in pollen germination and stress signalling [17–23].
3. Sources of phytoestrogens Phytoestrogens not only can be found in any plant or plantderived product, but are also detectable in water as a result of plant decomposition. They can be divided into three main classes: flavonoids, coumestans and lignans. Most of them are diphenolic substances usually with structural similarity in their three-dimensional conformation to the natural human steroid hormones. The group of flavonoids comprise flavones,
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Table 1 Phytoestrogens reveal pleiotropic effects on single targets and regulatory pathways Substance
Target
Effect
Reference
Lignans, isoflavones Genistein, naringenin Flavonoids, lignans Quercetin Formononetin, genistein, biochanin A, daidzein, and equol Flavonoids Daidzein, genistein, coumestrol Isoflavones
alpha-Fetoprotein Sex hormone-binding globulin (SHGB) Steroid receptors Guanylate cyclase MAP kinase
Hormone displacement Hormone displacement Activation Activation Inhibition
[58] [59] [39,60–62] [63] [64]
Protein kinase B Prostaglandin synthase Vitamin D synthesis (CYP24 and CYP27B1 transcriptional inhibition) Sulfatase Sulfotransferase 3beta-Hydroxysteroid dehydrogenase delta5/delta4 isomerase Aromatase P450 (CYP19A1) 11beta-Hydroxysteroid dehydrogenase 1 17beta-Hydroxysteroid dehydrogenases 5alpha-Reductase Caspase-3 and -8 Topoisomerase II
Inhibition Activation Activation
[65,66] [67] [36,68,69]
Inhibition Inhibition Inhibition
[70] [70,71] [46,72]
Inhibition Inhibition Inhibition Inhibition Cleavage promotion Inhibition
[46,73,74] [56,75] [46,47,49–51] [38,76] [37] [77]
Daidzein Quercetin, genistein Isoflavones, flavonoids Flavonoids Flavanone Flavonoids Isoflavones Daidzein Coumestrol, genistein
flavanones and isoflavones. Flavonoids are found in almost all plant families in leaves, stems, roots, flowers and seeds [22,24]. The main dietary source for isoflavones is the soybean, containing the glycoside of genistein among others. Flavones and flavanones are widely distributed in all plant families and are found in fruits, vegetables, berries, herbs, beans, and green tea. Most of the flavonoids found in plants are present as glycosides, but only the aglycones exert estrogen-like activities after their release by hydrolytic cleavage during digestion. High levels of coumestrol are found in alfalfa and various beans [8]. The plant lignans are instead found in fibre-rich food, such as unrefined grain products, and are transformed by intestinal bacteria into mammalian lignans such as enterolactone and enterodiol [25]. Additional dietary estrogenic compounds are the resorcyclic acid lactones, e.g. zearalenone which occur as secondary metabolites in mold-infected food [26].
4. Concentrations of phytoestrogens in humans Beans, especially soybeans, are a major nutritional component in the Asian diet. A calculation of the approximate intake of isoflavones by rural Japanese men gave a mean value of about 6 mg daidzein and 10 mg genistein per day. These men have very high concentrations of isoflavones in their plasma [27–30]. The highest individual value exceeded 2 M. Compared to these high levels, the concentration of these flavonoids in urine and plasma in men living in Europe is very low. The mean plasma value in Finnish subjects for daidzein and genistein is about 5 nM each [31]. After consumption of 20 g roasted beans, the following peak concentrations were observed: 0.2 M in human milk, 2 M in plasma and 20 M in urine [23]. Local tissue concentrations
are expected to be two- to three-fold higher. These differences in the concentrations are around three orders of magnitude, and the high concentrations found in the Asian population are in a range that might influence estrogen signalling. These data should attract attention to these substances and to their possible application for treating disorders associated to estrogens.
5. Targets of phytoestrogens The effects of phytoestrogens were reported controversially: Potential activities as endocrine disruptors in males were described in several studies [32–34]. On the other hand, beneficial influence could be observed as well: beneficial role in the modulation of progression of breast and prostate cancers [3,35], enhancement of vitamin D-mediated inhibition of tumour progression [36], alternative to hormone replacement therapies [6], down-regulation of osteoclast differentiation [37], and improvement of neuronal protection and memory [35,38]. Several in vitro and in vivo studies identified various modes of action for phytoestrogens. They modulate signal transduction by the estrogen receptors (alpha and beta), the aryl hydrocarbon receptor, the pregnane X-receptor, the constitutive androstane receptor and the peroxisome proliferatoractivated receptors (alpha and gamma) [3,39], and influence transport proteins, kinases and further enzymes, as shown in Table 1.
6. Function of 17beta-hydroxysteroid dehydrogenases Pre-receptor regulation of steroid hormone action is controlled by hydroxysteroid dehydrogenases and reductases
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converting steroids at positions 3, 5, 11, 17 and 20 of the steroid backbone [40–43]. The subject of this review is the influence of phytoestrogens on 17beta-hydroxysteroid dehydrogenases (17-HSD) in comparison to other steroid metabolising enzymes. By conversion at position 17 of the steroid molecule the 17-HSDs modulate the biological potency of estrogens and androgens: the keto-forms are inactive whereas the hydroxy-forms are active and access the receptors (Fig. 1). 17-Hydroxysteroid dehydrogenases belong to two protein superfamilies: SDR (short-chain dehydrogenase/reductase) and AKR (aldo-ketoreductase) [41,44]. Ten types of human 17-HSDs have been described to date varying in substrate specificity and tissue distribution [45]. 17-HSD 5 is unique among this group of enzymes as it belongs to the AKR family, while the rest of the 17-HSDs belongs to the SDR superfamily [45].
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genases versus other enzymes of steroid metabolism. Given the pleiotropic and sometimes adverse effects of phytoestrogens, understanding the differences in specificity and their molecular basis will be crucial for the development of efficient biomedical applications. 7.1. 3beta-HSD/delta5/delta4 isomerase versus aromatase and 17β-HSD 1 In a comparative study in human placental microsomes Le Bail et al. [46] tested phytoestrogens for inhibition of P450 aromatase, 3-hydroxysteroid dehydrogenase-delta5/delta4 isomerase and 17-HSD 1 (Fig. 1). Isoflavonoids and compounds which presented the phenolic B ring at position 3 of the pyran ring preferentially inhibited 3betaHSD/delta5/delta4 isomerase and/or 17-HSD activities over aromatase activity [46]. However, 7-hydroxy-flavone and apigenin (for structures see Fig. 2) were the most effective aromatase and 17-HSD 1 inhibitors, respectively [47].
7. Comparison of inhibition of hydroxysteroid dehydrogenases by phytoestrogens with other steroid metabolizing enzymes
7.2. Aromatase and 17β-HSD 1
In the following we will present a comparative overview of the effects of phytoestrogens on hydroxysteroid dehydro-
Selected phytoestrogens were checked for aromatase and 17-HSD 1 activity modulation in primary cultures of hu-
Fig. 1. This simplified section of steroid conversions involving 17beta-, 3beta-, 3alfa-HSDs and aromatase activities. Simplified part of steroid metabolism is annotated with enzymes catalyzing reactions inhibited by phytoestrogens. Steroid numbering: dehydroepiandrosterone (1), androstenedione (2), estrone (3), estradiol (4), testosterone (5), dihydrotestosterone (6), androstanediol (7), androsterone (8).
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Fig. 2. Chemical formulas of the most potent inhibitors of steroid metabolizing enzymes. Chemical structures (A) are given together with 3D-representations (B). All molecules are shown in the same orientation as the natural substrates. Numbering: estradiol (4), testosterone (5), 7-hydroxy-flavone (9), flavanone (10), apigenin (11), kaempferol (12), coumestrol (13), quercetin (14), abietic acid (15), zearalenone (16). 3D-coordinates were prepared in ChemDraw Pro and graphics rendered in iMOL. Corresponding PDB files are available from the authors upon request.
man granulosa-luteal cells by Whitehead et al. [48]. Apigenin and zearalenone significantly inhibited the aromatase activity, while biochanin A and quercetin had no effect. None of the phytoestrogens inhibited FSH-induced 17-HSD 1 activity, and only quercetin significantly inhibited progesterone production. 7.3. 17β-HSD 1 and 17β-HSD 2 Makela et al. [49,50] studied the effects of several flavonoids and isoflavonoids on 17beta-oxidoreduction of estrogens by purified 17-HSD 1 and in cell lines expressing 17-HSD 1 (T-47D breast cancer cells) or 17-HSD 2 (PC-3 prostate cancer cells). Several conclusions could be drawn in light of the reported results about these phytoestrogens. Flavones, flavanones, and isoflavones hydroxylated at both the double ring (positions 5 and 7) and ring B (position 4 ) were the most potent inhibitors of estrone reduction (17HSD 1 activity) in T-47D cells, the best being apigenin and coumestrol. The same results were found with purified 17HSD 1. Flavones hydroxylated at positions 3, 5 and 7 of rings A and C, with or without a hydroxyl group in ring B, were ca-
pable of inhibiting estradiol oxidation (17-HSD 2 activity) in PC-3 cells. Changes to the flavanone structure, or hydroxylation at position 3 of ring C of flavones, or methylation of the hydroxyl group at position 4 of ring B of flavones and isoflavones reduced or abolished their inhibitory activity on estrone reduction in T-47D cells. In contrast, the hydroxyl group at position 3 of flavones markedly increased the inhibition of estradiol oxidation in PC-3 cells, kaempferol being in this case the best inhibitor. Thus, changes in the number and location of hydroxyl groups may discriminate between inhibition of estrone reduction and estradiol oxidation. Some of the differences may be due to differences in the pharmacokinetics of these compounds in T-47D and PC-3 cells. Regarding prevention or treatment of estrogenrelated diseases, the substances apigenin, coumestrol, and genistein raise special interest considering the results so far. 7.4. 17β-HSD 5 The influence of phytoestrogens (flavonoids, coumarins, coumestans) on the reductive (androstenedione to testos-
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terone) and oxidative (androstanediol to androsterone) 17-activity of the recombinant purified human 17-HSD 5 was tested by Krazeisen et al. [51]. The best inhibitors for the reductive activity were zearalenone, coumestrol, quercetin and biochanin A (IC50 4 M, 4 M, 9 M and 14 M, respectively). The oxidative activity was inhibited most strongly by zearalenone, quercetin, 7-hydroxy-flavone and biochanin A (IC50 2 M, 5 M, 7 M and 8 M, respectively). Among the group of flavones the inhibitor potency is growing with increasing number of hydroxylations in ring A, the most effective being a hydroxylation at position 7. For flavonoids the issue is more complex due to the presence or absence of a double bond in ring C, the position of ring B as well as the hydroxylation pattern. Under reductive conditions a double bond in ring C increases the inhibitory potency (e.g. naringenin versus apigenin). In contrast, the flavanones (lacking the double bond) have stronger influence on the oxidative activity of 17-HSD 5. For both 17-hHSD 5 activities, inhibitor potency decreases when ring B is found at position 3 as it is in isoflavones. Flavones carrying a 5methoxy group at ring A are more potent than those with a hydroxy group at this position. However, within the group of flavones methylation of position 5 increases inhibition of the reductive pathway but decreases activity in oxidative conversion. Within the group of isoflavones methylation of the hydroxy group at position 4 correlates with a high inhibitor activity. On the other hand, substances like the synthetic antiestrogens tamoxifen, diethylstilbestrol, daidzein and coumarin had no influence on both types of reaction. An interesting inhibition pattern is observed for 18-glycyrrhetinic acid with 17-HSD 5, as in this case this compound has no influence on the oxidative, but only on the reductive reaction of the enzyme. This result could be explained with a deprotonation of the His 117 in the active center at higher pH values, at which the oxidation tests were performed in vitro. The deprotonated side chain of His 117 is then unable to bind the substrate 18-glycyrrethinic acid. This indicates that this substrate binds at the active center of the enzyme in a pH- and cofactor-dependent way [51]. The proposed model can now be further analysed in light of
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the recently determined crystal structure of 17-HSD 5 [52]. The dietary substances described above shown to be potent inhibitors of 17-HSD 5 seem to play an important role in delaying the development of hormone-dependent cancers such as that of prostate carcinoma. 7.5. 11β-HSD and 17β-HSD and 17,20-lyase The inhibitory capacity of 18-glycyrrhetinic acid, contained in licorice, has been firstly observed toward 11-HSD (Fig. 3) [53] and later toward other enzymes as well, as it is reported above with 17-HSD5. Previously it has been reported that the serum testosterone level in men consuming about 7 g of a commercial preparation of licorice (containing 0.5 g of 18-glycyrrhetinic acid) a day is significantly reduced [54]. The inhibitory effect of 18-glycyrrhetinic acid may be the reason for that, since it has been demonstrated that licorice consumption inhibits 11-HSD [55], 17-HSD and 17,20-lyase activity (Fig. 4) [54]. Decreased testosterone levels result in reduced libido or other sexual dysfunctions but might have beneficial effects in cases of abnormal prostate growth. 7.6. 11β-HSD 1 and 11β-HSD 2 Abietic acid inhibited both 11-HSD 1 (IC50 27 M for reduction and 2.8 M for oxidation) and 11-HSD 2 (IC50 12 M). Flavanone and 2 -hydroxyflavanone efficiently (IC50 18 and 10 M, respectively) inhibited reductive but not oxidative activity of 11-HSD 1 as shown by Schweizer et al. [56]. These assays were performed with lysates of cells stably transfected with 11-HSD 1 and 11-HSD 2. This observation reveals that a variety of environmental compounds exert distinct inhibitory effects on 11-HSD 1 and 11HSD 2, opening the possibility for selectively modulating local cortisone/cortisol availability in vivo. Amide derivatives of the 18-glycyrrethinic acid at position 30 were studied as possible inhibitors of 11-HSD 1 and 11-HSD 2. The study opens the way to a new class of substances and a specific inhibitor for 11-HSD 2 has already been identified [57].
Fig. 3. Steroid conversions at position 11beta. Steroid numbering: cortisol (17), cortisone (18).
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References
Fig. 4. Activity of 17-hydroxylase, 17,20-lyase. Steroid numbering: pregnenolone (19), 17-hydroxy-pregnenolone (20), dehydroepiandrosterone (1).
8. Conclusion Although phytoestrogens can inhibit quite a broad spectrum of steroid metabolizing enzymes, there is always an enzyme-specific preference. For example, 18glycyrrethinic acid is a potent inhibitor of 11-HSDs but not a very effective modulator of 17-HSDs. Future work should be able to exploit our improved understanding of these specific effects for the development of targeted inhibitors of steroid metabolism.
Acknowledgement This work was supported in part by a DFG grant to Jerzy Adamski.
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