Department of Physiology, St Georges Hospital Medical School, London, UK
1 To whom correspondence should be addressed at: Department of Physiology, St Georges Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. e-mail: s.whitehead{at}sghms.ac.uk
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Abstract |
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Key words: aromatase/human granulosa luteal cells/4 hydroxy-androstenedione/17 hydroxysteroid dehydrogenase/phytoestrogens
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Introduction |
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Whilst relatively little is known about the absorption, metabolism and bioavailability of various phytoestrogens, on a high soy diet or a vegetarian diet, mean plasma concentrations of the various phytoestrogens that have been measured can range from 100 nmol/l to 1 µmol/l (Mazur and Aldercreutz, 2000). These are concentrations at which phytoestrogens have been shown to have estrogenic effects in vitro (Zava and Duwe, 1997
; Maggiolini et al., 2001
). In this respect, soy is being heavily promoted as a natural alternative to hormone replacement therapy.
There is, however, a paradox. Both epidemiological studies (Aldercreutz, 1998; Barnes, 1998
) and laboratory investigations (Barnes, 1997
) have suggested that dietary compounds may have a major protective effect against post-menopausal breast cancer. Thus, the markedly reduced incidence of breast cancer seen amongst many Asian populations compared with women in Northern Europe and America has been attributed to their diets that are rich in soy products (Aldercreutz, 1998
). These contain high concentrations of conjugated isoflavones that are thought to have a protective role in breast cancer by acting as weak estrogens or anti-estrogens by competing with endogenous estrogens for binding to the ER.
There is, however, increasing evidence that phytoestrogens may bind to aromatase and/or 17-hydroxysteroid dehydrogenase (HSD) and thereby reduce the availability of these enzymes for the production of estrogen from androgen precursors and/or the production of estradiol from weak estrogens (Kao et al., 1998
; Mäkelä et al., 1998
; Le Bail et al., 2000
). In breast tissue of post-menopausal women these enzymes are responsible for the local production of estrogens from circulating adrenal androgens (Simpson et al., 2000
).
Previous studies investigating the effects of phytoestrogens on steroidogenic enzyme activity have typically been carried out on purified or recombinant enzyme preparations, placental microsomes or breast cancer cell lines (Mäkelä et al., 1998; Le Bail et al., 2000
; Krazeisen et al., 2001
). These studies showed that the concentrations inducing a 50% inhibition of enzyme activity (IC50) of the most potent phytoestrogens investigated were generally between 1 and 20 µmol/l. Thus, the purpose of the present study was to establish an experimental protocol that would allow an investigation of the effects of phytoestrogens on aromatase and 17
-HSD type 1 in primary cultures of human cells. The phytoestrogens investigated were two flavones (apigenin and quercetin), an isoflavone (biochanin A) and the mycotoxin, zearalenone. These compounds have been shown to have relatively potent effects on aromatase and/or 17
-HSD compared with other phytoestrogens (Le Bail et al., 1998
; Jeong et al., 1999
; Krazeisen et al., 2001
), although their reported potency varies according to the experimental model employed.
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Materials and methods |
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For the chronic experiments, the protocol was identical except that the cells were exposed to phytoestrogens for 24 h prior to their 4 h exposure to steroid substrates with or without test compounds. For the time-course studies, 25 µl of medium was taken at 1 and 2 h and the volume replaced with fresh medium. The dilution factor was taken into account in the analysis of the data.
At the end of the experiment, media samples were taken and stored at 20°C and viability of cells was routinely tested either with the Trypan blue exclusion test or with Almar blue (Serotec, Oxford, UK) according to the manufacturers instructions. This is a non-toxic REDOX indicator that provides a measure of mitochondrial activity and has been found to be more sensitive than the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, particularly when relatively small numbers of cells are being assayed.
Drugs
The following drugs were used in these experiments: apigenin (4',5,7-trihydroxyflavone), biochanin A (4'methoxy-5,7-dihydroxyisoflavone), quercetin (3,3', 4',5,7, pentahydroxyflavone), zearalenone, androstenedione, 4-hydroxyandrostene-3,17-dione (4OH-A), estrone, pregnenolone, testosterone, human FSH (hFSH) and recombinant hCG (rhCG). hFSH and rhCG were kindly supplied by The National Hormone and Pituitary Agency, Torrance, CA, USA; all other drugs were supplied by Sigma UK. All steroids were initially dissolved in ethanol and diluted appropriately with culture medium before being stored as stock solutions. Stock solutions were then further diluted with medium for each experiment and added to cultures in 10 µl volumes to give the desired final concentration. Controls were performed to ensure that the maximum volume of ethanol diluent (1 µl) did not affect cellular responses.
Steroid assays
Progesterone and estradiol samples were measured in duplicate using a direct radioimmunoassay (RIA) kit (ICN Pharmaceuticals Ltd, Basingstoke, UK) according to the manufacturers instructions. All drugs used in these experiments were tested for their possible cross-reactivity with the anti-sera used, but none was detected. The cross-reactivity of the progesterone anti-serum with 20-dihydroprogesterone, 17
-hydroxyprogesterone and pregnenolone was 5.4, 0.6 and 0.4% respectively, and that with the estradiol anti-serum with estrone, estriol, progesterone and testosterone and androstenedione was 20, 1.5, <0.01, <0.01 and <0.01% respectively. Inter- and intra-assay coefficients of variation were respectively 6.2 and 3.0% for the progesterone assay, and 9.1 and 5.2% for the estrogen assay. For each experiment, the cross-reactivity of estrone with the estradiol antibody was monitored by incubating the appropriate concentration of estrone in culture medium without GL cells for 4 h. The mean estradiol concentration measured in the blank wells was subtracted from the appropriate experimental measurements. When 10 µmol/l estrone was added to the cultures the cross-reactivity was >20% and thus these results were excluded.
Statistical analysis
Data shown represent mean ± SEM of triplicate cultures obtained from at least three independent experiments, and n = the total number of individual observations. When comparing several groups of data, statistical differences were obtained with an analysis of variance, followed by Gabriels test. A Students t-test was used to test significance between two groups of data.
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Results |
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The time course of the conversion of the various substrates showed that between 1 and 2 h there was no marked increase in either estradiol or progesterone production, but between 2 and 4 h there was a marked and significant increase in steroid production (Figure 3a and b). It was thus established that a 24-h exposure to FSH followed by a 4-h exposure to steroid substrates at a dose of 100 nmol/l was a suitable protocol for investigating estradiol production.
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Discussion |
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Several previous studies have shown that GL cells obtained from natural cycles are sensitive to FSH, but that those obtained from stimulated cycles are insensitive (Bergh et al., 1991; Mason et al., 1993
; Lambert et al., 1995
; Foldesi et al., 1998
). It has been reported that a long pre-incubation period of 72 h followed by exposure to FSH for 46 days increased basal estradiol production in GL cells in the absence of any androgenic substrate, but in the presence of testosterone FSH had no effect on estradiol production. More recently, it has been reported that GL cells exposed to FSH for 24 h dose-dependently increased estradiol synthesis from testosterone over a 2 h period (Lambert et al., 2000
). In the present study, using a protocol similar to that used by Lambert and colleagues, no increase in aromatase activity (as assessed by the conversion of testosterone to estradiol) was observed. In contrast, however, FSH priming significantly increased the production of estradiol from both estrone and androstenedione. The former requires 17
-HSD type 1 activity and the latter aromatase and 17
-HSD type 1 if the conversion is via estrone, or 17
-HSD type 5 and aromatase if the conversion is via testosterone. Both type 1 and type 5 17
-HSD are present in the human ovary (Labrie et al., 2000
).
Others (Ghersevich et al., 1994a) have reported that in rat granulosa cells, rFSH stimulated expression and activity of 17
-HSD type 1, and subsequently it was shown that FSH induction of this enzyme was through a protein kinase A (PKA)-dependent pathway (Kaminiski et al., 1997
). In cultured human granulosa-luteal cells, gonadotrophins were shown to have no effect on 17
-HSD, although aromatase expression and activity was increased (Ghersevich et al., 1994b
). Interestingly, there is evidence of an ovarian deficiency of the enzyme(s) in polycystic ovary syndrome (Toscano et al., 1990
).
It has generally been assumed that the action of aromatase on the conversion of testosterone to estradiol has been the major pathway through which estrogens are produced by granulosa cells. Recent studies have shown, however, that human type 5 17-HSD, which converts androstenedione to testosterone, is expressed exclusively in thecal cells (Luu-The et al., 2001
). It was suggested that the production of estradiol in ovarian granulosa cells is preferentially via aromatization of 4-androstenedione to estrone and a subsequent conversion of estrone to estradiol by type 1 17
-HSD. This would be in agreement with the higher affinity of aromatase for androstenedione compared with testosterone (Luu-The et al., 2001
).
The present studies certainly agree with this hypothesis, as the production of estradiol from precursors that involved the activity of type 1 17-HSD generally produced higher concentrations of estradiol than testosterone alone (Figure 1). They also show that 17
-HSD type 1 in human GL cells can be modulated by FSH rather than aromatase, since the mean increase in estradiol production after FSH priming compared with control values was 16, 56 and 68% for testosterone, estrone and androstenedione respectively. This is consistent with the observations that FSH induction of aromatase is not generally observed in GL cells (see Lambert et al., 2000
). Whether or not type 5 17
-HSD is also modulated by FSH can neither be supported nor negated by this study.
Previous studies investigating the inhibitory effect of phytoestrogens on 17-HSD or aromatase activity in placental microsomes or partially purified or recombinant enzyme preparations show that the most potent phytoestrogens induce 50% inhibition of enzyme activity (IC50) at concentrations in the range of 0.5 to 20 µmol/l. Thus, for these initial studies to screen the effects of phytoestrogens, a relatively high dose of 10 µmol/l was used. This is the same concentration of 4OH-A used to inhibit aromatase activity by competitively binding to the enzyme.
In human GL cells, 10 µmol/l apigenin had only weak inhibitory effects on enzyme activity, although after cells had been exposed to this phytoestrogen for 24 h there was a marked loss of both aromatase and 17-HSD type 1 activity. In contrast, the other flavone, quercetin was without effect on either enzyme. It has been reported (Mäkelä et al., 1995, 1998) that 1.2 µmol/l apigenin inhibited the conversion of tritiated estrone to estradiol by
40% and 80% in a purified 17
-HSD type 1 preparation and in T-47D breast cancer cells respectively, whilst quercetin at the same dose had no effect in either preparation. In human placental microsomes, apigenin was similarly shown to be a relatively potent inhibitor of 17
-HSD type 1 activity, with an IC50 of 0.3 µmol/l (Le Bail et al., 1998
). Others (Krazeisen et al., 2001
) showed that apigenin and quercetin were the most potent flavones inhibiting the reductive activity of recombinant 17
-HSD type 5, with IC50 values in the order of 20 µmol/l. With regard to the effects of flavones on aromatase activity, apigenin has been reported to inhibit this enzyme, with an IC50 of 2.9 µmol/l, in placental microsomes (Le Bail et al., 1998
) and
5 µmol/l in partially purified placental aromatase (Jeong et al., 1999
). Both apigenin and quercetin were found to be potent inhibitors of the aromatase enzyme complex from rainbow trout (Pelissero et al., 1996
), and quercetin was also found to inhibit aromatase activity in colorectal cancer cell lines (Fiorelli et al., 1999
).
Like apigenin, the isoflavone genistein has also been reported to inhibit 17-HSD type 1 activity (Le Bail et al., 1998
; Whitehead et al., 2002
), whilst the effects of genistein and other isoflavones on aromatase inhibition have generally been reported to be either weak or undetectable (Kao et al., 1998
; Le Bail et al., 1998
; Whitehead et al., 2002
).
In the present study, it was found that another isoflavone, biochanin A, had no significant effects on either 17-HSD type 1 or aromatase, although others (Le Bail et al., 2000
) reported that biochanin A inhibited human placental aromatase, 17
-HSD and 3
-HSD activities with IC50s of 49, 4.9 and 10 µmol/l respectively. Current investigations include the examination of doseresponse curves to several phytoestrogens, and indeed someincluding biochanin Ado show a weak dose-related inhibition of aromatase in human GL cells. Compared with controls, however, significance is only reached at a ten times higher dose (100 µmol/l) than those used in the present series of experiments.
The mycotoxin, zearalenone, showed potent effects on inhibiting the production of estradiol from androstenedione and testosterone, but had no effect on 17-HSD type 1 activity converting estrone to estradiol. The latter result agrees with those of others (Mäkelä et al., 1995
) on purified 17
-HSD type 1. Interestingly, it has also been found (Krazeisen et al., 2001
) that the five most potent phytoestrogens inhibiting the reductive activity of 17
-HSD type 5 were zearalenone, coumestrol, quercetin, biochanin A and apigenin, with IC50s of 4, 5, 9, 14 and 20 µmol/l respectively.
The effects of the phytoestrogens investigated in the present study were not always in agreement with studies concerned with their effects on cell-free systems, or indeed with cancer cell lines (Mäkelä et al., 1998; Fiorelli et al., 1999
). One explanation is that the enzyme concentration/activity is very high in primary cultures of GL cells. Indeed, preliminary studies in our laboratory on MCF-7 cells have found that the production of estradiol/75 000 cells from the various substrates is five to ten times lower after a 48 h period compared with that of human GL cells after a 4 h period.
In conclusion, these studies have established an experimental protocol for investigating the effects of phytoestrogens on enzyme activity in primary cultures of GL cells, and have shown that FSH primingin line with other studiesdid not induce aromatase in GL cells but did induce 17-HSD type 1. In contrast with other studies there was no acute effect of phytoestrogens on enzyme activity but, when cells had been exposed to the compounds for 24 h and then exposed to steroid substrates for 4 h, inhibition of aromatase activity was observed. This suggests either that the lipid solubility of phytoestrogens is low and so permeation into the cells is slow, or that the compounds are being metabolized to inactive substances and/or the compounds and their metabolites are altering the expression, and hence concentration, of intracellular steroidogenic enzymes.
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Acknowledgements |
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References |
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Submitted on June 25, 2002; resubmitted on October 3, 2002; accepted on November 26, 2002.