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School of Biosciences and Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
Address all correspondence and requests for reprints to: C. J. Kirk, School of Biosciences and Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. E-mail: c.j.kirk{at}bham.ac.uk.
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1 µM) than those of Finnish vegetarians (5). Soy isoflavones exist mainly as glycoside conjugates, but the free aglycones are absorbed after deconjugation in the gut (6, 7). After absorption, isoflavones are reconjugated with glucuronic acid or sulfate either in the liver, which is also the major site for the conjugation of steroid hormones, or the gut epithelium (8, 9). The relative levels of the various conjugation products of isoflavones show marked intra and interracial variations (10), possibly reflecting polymorphisms among the enzymes involved (11, 12). Isoflavonoids and flavonoids may interact with estrogen receptors, but they also have a diverse range of other biological effects including antiviral and antioxidant properties, interactions with enzymes and pathways associated with control of the cell cycle, and effects on xenobiosis (13). We have previously suggested that these compounds may also disrupt the metabolism of endogenous estrogens, thereby influencing their availability in target cells (14).
In postmenopausal women, who are the group most susceptible to breast cancer, estrogens are generated from dehydroepiandrosterone and androstenedione synthesized in the adrenal cortex via the action of aromatase mainly located in adipose tissue (15, 16). The circulating levels of free estrogens are low, and these hormones are mostly present as inactive estrone sulfate (E1S) synthesized by sulfotransferase enzymes in liver and other tissues. It is now clear that circulating E1S is a major source of free estrogens in breast tumor tissue after local steroid sulfatase action and further metabolism (Fig. 1
) (17, 18, 19). Conversely, free estrogens may be reconverted to sulfoconjugates by the action of sulfotransferase enzymes. This process is mainly catalyzed by estrogen sulfotransferase (SULT1E1), but the thermostable phenolsulfotransferase (SULT1A1) may also be involved (20, 21). This may be especially important if the former is unavailable or only poorly expressed, as appears to be the case in breast tumor tissue (22, 23). A delicate balance between the relative activities of tissue sulfatases and sulfotransferases therefore controls the concentration of active estrogens (Fig. 1).
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Materials and Methods |
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Flavonoids (
97% pure) were purchased as follows: flavone, chrysin (5,7-dihydroxyflavone), 7,8-dihydroxyflavone, apigenin (4',5,7-trihydroxyflavone), narigenin (4',5,7-trihydroxyflavanone), kaempferol (3,4',5,7-tetrahydroxyflavone), luteolin (3',4',5,7-tetrahydroxyflavone), hesperetin (4'-methoxy-3',5,7-trihydroxyflavone), quercetin (3,3',4',5,7-pentahydroxyflavone), rutin (3',4',5,7-tetrahydroxyflavone-3-rutinoside), (+) catechin [(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol], (–) catechin [(2S,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol], (+) epicatechin [(2S,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol], (–) epicatechin [(2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol], daidzein (4',7-dihydroxyisoflavone), genistein (4',5,7-trihydroxyisoflavone], (Sigma, Poole, Dorset, UK); 3-hydroxyflavone, primuletin (5-hydroxyflavone), 3',4'-dihydroxyflavone, galangin (3,5,7-trihydroxyflavone), 3',4',7,8-tetrahydroxyflavone, morin (2',3, 4',5,7-pentahydroxyflavone), 3',4',7-trihydroxyisoflavone, (Lancaster Synthesis, Morecambe, UK); 6-hydroxyflavone, 6-hydroxyflavanone, 7-hydroxyflavone, 3,6-dihydroxyflavone, 3,7-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), fisetin (3,3',4',7-tetrahydroxyflavone) (Aldrich, Gillingham, UK); formononetin (4'-methoxy-7-dihydroxyisoflavone), equol [(S)-3,4-dihydro3-(4-hydroxyphenyl)-2H-1-benzopyran-7-ol] (Fluka, Gillingham, UK), and myricetin (3,3',4',5,5',7-hexahydroxyflavone) (Apin Chemicals, Abingdon, Oxfordshire, UK). Flavonoid sulfates were a generous gift from Dr. Nigel Botting (University of St. Andrews, Fife, UK). Unlabeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS), 3-hydroxytyramine (dopamine), 4-nitrophenol, 4-nitrocatechol sulfate, ß-estradiol, tetrabutylammonium dihydrogen phosphate, and protease inhibitor cocktail were bought from Sigma. Radiolabeled [35S]-PAPS, [2,4,6,7-3H(N)]-estradiol and [6,7-3H(N)]-estrone sulfate ([3H]-E1S) were obtained from NEN Life Science Products (Boston, MA). Other reagents were purchased from standard laboratory suppliers to the highest grade available.
Sources of enzyme preparations
Platelet cytosol, prepared from outdated apheresis packs of platelet concentrates (National Blood Service, Birmingham, UK), was used as a source of SULT1A1 and SULT1A3. Platelets were collected by centrifugation at 8000 x g for 5 min at 4 C and then washed three times in 10 mM PBS (pH 7.0) containing 4 mM EDTA. The platelets were resuspended in 20 ml 10 mM phosphate buffer (pH 7.0) containing 0.25 M sucrose and 2 mM 2-mercaptoethanol, disrupted by ultrasonication, and centrifuged at 100,000 x g for 60 min at 4 C. The supernatant was stored in 1-ml portions at –20 C before use.
SULT1E1 activity was measured in liver cytosol prepared from snap-frozen human donor liver, surplus to surgical requirements. Ethical approval for the use of this tissue for research purposes was granted by South Birmingham Health Authority Local Research Ethics Committee (reference CA/5192). The liver was thawed in 5 volumes of 10 mM triethanolamine buffer containing 0.25 M sucrose and 5 mM 2-mercaptoethanol (pH 7.4 at 4 C). The tissue was finely chopped with scissors and homogenized using a Potter-Elvehjem homogenizer followed by ultrasonication (5 x 10-sec bursts at 4 C). The homogenate was centrifuged at 100,000 x g for 60 min, and portions of the supernatant were stored at –70 C before use.
Membranes obtained from MCF7 cells were used as a source of steroid sulfatase. Confluent cells were harvested by scraping with a rubber policeman and disrupted by osmotic shock and ultrasonication in 8 volumes of homogenization buffer [20 mM Tris, 10 mM EDTA, 10 mM EGTA (pH 6.5)] containing 1% vol/vol protease inhibitor cocktail. The homogenate was centrifuged at 100,000 x g for 60 min, the pellet resuspended in 1 volume homogenization buffer, and stored at –70 C.
Arylsulfatase A was obtained from human urine. Samples were collected before breakfast and stored at –20 C before dialysis. As required, the samples were thawed and 50 ml urine were dialyzed (m.w. cut off 12–14 kDa) against three changes of deionized water at 4 C for 18 h. The dialyzed samples were pooled and reduced to half their original volume using an ultrafiltration cell (model 8050, Amicon Corp., Beverly, MA). The concentrate was stored in 5-ml portions at –20 C before use.
Enzyme assays
All enzyme assays were performed at substrate concentrations close to the Michaelis constant for the particular enzyme activity (26, 27, 28) and were shown to be linear with respect to time and protein concentration. The sulfation of 3 µM 4-nitrophenol and 10 µM dopamine (selective for SULT1A1 and SULT1A3, respectively) were measured using a method based on that of Foldes and Meek (29) as modified by Anderson and Weinshilboum (30). Briefly, diluted cytosol (20 µl) was incubated with 6.7 µM PAPS (containing about 10–20 nCi [35S]PAPS) in a final volume of 150 µl 20 mM phosphate buffer (pH 7.0) containing 1% dimethylsulfoxide (DMSO). Dopamine, 4-nitrophenol, the selective SULT1A1 inhibitor dichloronitrophenol (31), and flavonoids (dissolved in DMSO, maximum final concentration 1% vol/vol) were added as appropriate. The tubes were incubated at 37 C for 40 min and the reactions quenched by the addition of ice-cold 0.1 M barium acetate (200 µl). Any protein, unreacted PAPS, and free sulfate were precipitated by two additions of 0.1 M barium hydroxide (200 µl) followed by 0.1 M zinc sulfate (200 µl). The tubes were centrifuged at 11,500 x g for 3 min after each addition and the radioactivity measured in 500 µl of the final supernatant.
Flavonoid sulfation was measured using a method based on that devised by Varin et al. (32). Incubations were set up as described above. After 40 min at 37 C, the reactions were quenched by the sequential addition (all ice cold) of 15 µl 2.5% aqueous acetic acid, 30 µl freshly made 0.1 M tetrabutylammonium dihydrogen phosphate, and 750 µl ethyl acetate. The tubes were vortexed vigorously for 30 sec and the phases separated by centrifugation at 11,500 x g for 5 min. Radioactivity was determined in the upper ethyl acetate layer (containing the [35S]-labeled product) and in the remaining aqueous phase (containing unreacted [35S]PAPS).
Estradiol (E2) sulfation was measured using a method based on that of Qian et al. (33). Diluted cytosol (20 µl) was incubated with 100 µM PAPS in a final volume of 200 µl 100 mM Tris buffer with 10 mM magnesium acetate and 0.1 mM EDTA (pH 7.9 at 37 C). Reactions were started by the addition of [2,4,6,7-3H(N)]-estradiol, incubated at 37 C for 40 min and quenched by the sequential addition (both ice cold) of deionized water (200 µl) and dichloromethane (800 µl). The tubes were vortexed vigorously for 30 sec, the phases separated by centrifugation at 11,500 x g for 5 min, and radioactivity determined in the upper aqueous phase (containing [3H]-E1S).
Steroid sulfatase activity was measured as described previously (27). Briefly, diluted membrane suspension (20 µl) was incubated with 40 µM [3H]-E1S (250 nCi) in a final volume of 100 µl 20 mM Tris buffer (pH 6.5). Flavonoids and isoflavonoids (dissolved in DMSO, maximal final concentration 1% vol/vol) were added as required, and the tubes were incubated for 60 min at 37 C. The reactions were quenched by the sequential addition of 300 µl ice-cold aqueous sodium bicarbonate (0.1 M) followed by 600 µl water. The reaction products were extracted into 3 ml toluene and radioactivity was determined in the organic and aqueous layers, containing free E1 and E1S, respectively.
Arylsulfatase A activity was measured by the method of Baum et al. (34). Dialyzed urine (0.25 ml) was mixed with an equal volume of 4-nitrocatechol reagent [10 mM 4-nitrocatecholsulfate, 0.5 mM sodium pyrophosphate, 10% wt/vol sodium chloride in 0.5 M sodium acetate buffer (pH 5.0)] and incubated for 60 min at 37 C. The reaction was quenched by the addition of 1 M sodium hydroxide (0.75 ml) and the liberated 4-nitrocatechol determined by measuring the absorbance at 515 nm.
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Preliminary experiments revealed that the ratios of SULT1A1/SULT1A3 activities in platelet and liver preparations were 1:2 and 10:1, respectively (results not shown). In platelets, most of the flavonoids with two or more hydroxyl groups were sulfated at detectable rates, and only one of them (baicalein: 5,6,7-trihydroxyflavone) showed substrate inhibition at high concentrations (Table 1). The two simplest flavonoids to be appreciably sulfated were 7-hydroxyflavone and 3',4'-dihydroxyflavone showing that hydroxyl groups on both rings can be sulfated by platelet enzymes.
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suggests three general rules that describe the sulfation of most of the flavonoids tested in platelets: 1) Flavon-3-ols are usually poorer substrates than the parent flavone.
2) If the B ring is hydroxylated, the addition of an hydroxyl group in the 5 position makes the compound a better substrate.
3) Flavon-3-ols are better substrates than flavan-3-ols. However, trans-substituted flavan-3-ols (catechins) appear to be better substrates than their cis-substituted counterparts (epicatechins).
Kinetic analysis, by Eadie-Hofstee transformation, of the sulfation of five flavonoids by platelet cytosol at concentrations of between 0.1 and 25 µM (Fig. 2 and Table 2) demonstrated that it was biphasic, indicating that the sulfation of these compounds is under the control of two enzymes. One enzyme appeared to be mainly active at sub- to low micromolar concentrations, whereas the other predominated at concentrations above the low micromolar. The low-affinity enzyme was susceptible to substrate inhibition by 7-hydroxyflavone and baicalein at concentrations in excess of 5 and 1 µM, respectively. The two major isoforms of sulfotransferases present in platelet cytosol are SULT1A1 and SULT1A3 (11). We examined the involvement of these isoforms in flavonoid sulfation in more detail using chrysin (5,7-dihydroxyflavone) as a substrate. Chrysin was incubated in the presence of dichloronitrophenol (which selectively inhibits SULT1A1) (31, 35) or using cytosol that had been heated to 41 C for 15 min (which preferentially denatures SULT1A3) (35). The results (Table 3) indicate that at very low concentrations (0.1 µM) chrysin was mainly sulfated by SULT1A1, whereas at higher concentrations (>2.5 µM) SULT1A3 was almost exclusively involved.
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). All the monohydroxy flavonoids were sulfated to a degree that cannot be explained by the higher proportion of SULT1A1 present in the liver, strongly suggesting that at least one other cytosolic sulfotransferase, perhaps SULT1E1 or SULT2A1, is involved. In addition, the sulfation of many of the flavonoids showed a pronounced substrate inhibition in liver that was absent in platelets. This, together with the low proportion of SULT1A3 present in liver cytosol, also suggests that SULT1A3 is responsible for sulfating flavonoids at higher concentrations in platelets. Flavone, which has no free hydroxyl group, was also shown to be sulfated by cytosol from liver and, to a lesser extent, platelets. Whereas there are cytosolic keto-reductases that could reduce the 4-keto group to a hydroxyl, they are unlikely to be active due to a lack of NADH/NADPH in our assay mixture. A likely explanation for this result could be the presence of either an hydroxy contaminant or a flavanone that could undergo tautomerism producing a transient aromatic hydroxyl group at the 4 position.
In contrast to the flavonoids, isoflavonoids are generally rather poor substrates for platelet preparations. Of the three compounds examined in detail, all showed marked substrate inhibition. The daidzein metabolite equol (which is generated by the intestinal flora of 30% of the population) was by far the best substrate with maximum sulfation achieved between 2 and 4 µM (Fig. 3A). In contrast, formononetin (4'-methoxy-daidzein) was only weakly sulfated, suggesting either that it is the 4'-hydroxyl group that is sulfated in equol or that the 4'-methoxy group is an excellent inhibitor of platelet sulfotransferases.
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). The concentration-dependence for the sulfation of these substrates was similar to that observed with platelet cytosol, suggesting that SULT1A1 predominated. This is in contrast to the sulfation of 3',4',7-trihydroxyisoflavone, which was one of the best substrates tested in platelets but was only poorly sulfated by comparison with genistein in liver, suggesting that it is mainly a substrate for SULT1A3, even at fairly low concentrations (Table 1). Flavonoids inhibit sulfotransferases but not sulfatases
SULT1A1.
Some flavonoids have previously been shown to be potent inhibitors of SULT1A1 (14, 24), an enzyme that may have a role in sulfating estrogens in breast tumor cells (20, 21, 22, 23). These experiments used an assay involving barium hydroxide/zinc sulfate precipitation of protein and unreacted PAPS. In preliminary experiments, we found that this also causes polyhydroxyflavonoids and their sulfates to be precipitated (results not shown) (32). Here, we used the ion-pairing/ethyl acetate extraction method developed by Varin et al. (32) and were able to demonstrate substantial sulfation of flavonoids at sub- and low micromolar concentrations. However, many of these same compounds are also able to inhibit the sulfation of 3 µM 4-nitrophenol by SULT1A1 at concentrations at which their own sulfation is negligible, compared with that of the probe substrate (Table 4). The most potent of these was 3',4'-dihydroxyflavone with an IC50 of less than 1 nM against 3 µM 4-nitrophenol. Of the common, naturally occurring flavonoids, quercetin was the most potent inhibitor (IC50, 60 nM), but its glycosylated derivative rutin was almost without effect (IC50 >>25 µM). The catechins and epicatechins, which have the same pattern of hydroxylation as quercetin, were rather poor inhibitors with the most potent, (–) catechin, having an IC50 of 4 µM. The major soy isoflavones, genistein and daidzein, inhibited SULT1A1 with IC50 values of 500 and 600 nM, respectively. The most potent isoflavone was 3',4',7-trihydroxyisoflavone, with an IC50 of 20 nM. The sulfates of two of the more interesting compounds, 7-hydroxyflavone and equol, were not precipitated, and therefore their ability to inhibit the sulfation of the probe substrates could not be measured by these assays.
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suggest that the following rules apply to the action of flavonoids as inhibitors of SULT1A1 in platelets: 1) All planar aglycone molecules with a 3',4'-dihydroxy motif are potent inhibitors.
2) Flavon-3-ols are more potent inhibitors than the parent flavonoid, provided that the B ring is not substituted.
3) A 5-hydroxyl group increases the IC50.
4) Flavones are more potent inhibitors than the corresponding flavanone.
SULT1E1.
The sulfation of E2 by SULT1E1 was less sensitive to inhibition by flavonoids and isoflavonoids than was that of 4-nitrophenol by SULT1A1 (Table 4). The two most potent inhibitors were found to be 6-hydroxyflavone and equol, both of which have hydroxyl groups that can potentially superimpose with the 3-hydroxyl group of E2. However, daidzein, which is structurally the most closely related isoflavonoid to equol, was a much less potent inhibitor than the more distantly related genistein. The 4'-hydroxyl group appears to play an important role in the inhibitory mechanism because formononetin, which lacks this group, was the least potent of all the compounds tested. Kinetic analysis of flavonoid inhibition was too complex to allow full interpretation, but a competitive element was identified that appeared to account for most of the inhibition (results not shown). Kinetic analysis of isoflavonoid inhibition proved to be more straightforward and demonstrated that the compounds were mainly competitive inhibitors, although there was some interaction at an allosteric site (Fig. 4 and Table 5). Of the major dietary phytoestrogens, equol and genistein were the most potent, exhibiting inhibitory constants at the active site (Ki values) of 0.4 and 0.5 µM, and at an allosteric site (K'i values) of 2 and 5 µM, respectively. None of the isoflavone sulfates tested inhibited SULT1E1 at concentrations that are likely to occur in vivo. The most potent was daidzein 4,7-bisulfate, which had an IC50 of 10 µM.
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). Because most flavonoids appear to be substrates for this enzyme at high concentrations, at which they are sulfated at similar rates to dopamine, it is likely that the latter binds far more strongly to the active site. The most potent inhibitor of SULT1A3 was baicalein (IC50, 500 nM), which also showed signs of substrate inhibition in platelets. It is difficult to ascribe structural rules to the inhibition of SULT1A3 by flavonoids because the IC50 of most compounds exceeds the concentration at which they can be reliably tested. Of the isoflavonoids only 3',4',7-trihydroxyisoflavone, which is also a substrate of SULT1A3 (Table 1), inhibited the enzyme (IC50 2 µM) at the concentrations tested here. Arylsulfatase A and E1 sulfatase
None of the compounds tested were found to have any effect on the activity of either sulfatase at concentrations likely to be achieved from the diet. Only the isoflavone sulfates had IC50s less than 25 µM for either enzyme. The most potent of these was daidzein 4,7-bisulfate, which exhibited an IC50 for steroid sulfatase (vs. 1 µM E1S) of 0.8 µM (data not shown). In parallel experiments, daidzein 4-sulfate, daidzein 7-sulfate, and genistein 4-sulfate inhibited steroid sulfatase activity with IC50s of 14, 15, and more than 20 µM, respectively (data not shown). The ability of the isoflavone sulfates to inhibit arylsulfatase activity was not investigated.
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). Intracellular concentrations of flavonoids have not been reported in the literature, but if they reflect those found in the plasma and if these compounds are able to interact with sulfotransferases as we have observed in vitro, the activity of both SULT1A1 and SULT1E1 would be greatly reduced by their presence. Moreover, by acting as substrates for SULT1A3 and other cytosolic sulfotransferases, these high concentrations of flavonoids might rapidly exhaust the available supplies of PAPS, thereby effectively inhibiting all sulfotransferase activity. It has previously been reported that dietary flavonoids and/or their sulfates can inhibit a number of key enzymes of steroid metabolism including aromatase (41, 42, 43), 17-ß-hydroxysteroid dehydrogenase type 5 (43, 44), 3'-ß-hydroxysteroid dehydrogenase (43, 45), and steroid sulfatase (28, 46). However, our studies show that some sulfotransferases are at least 10 times more sensitive to inhibition by flavonoids than any of the enzymes listed above and that these enzymes may be inhibited by concentrations of flavonoids that can occur in vivo. We have also confirmed that steroid sulfatase activity was unaffected by physiological concentrations of flavonoids or flavonoid sulfates. All unconjugated flavonoids exhibited IC50s for steroid sulfatase inhibition of more than 25 µM in our experiments, slightly higher than the estimates of approximately 10 µM for the inhibition of steroid sulfatase by quercetin, naringenin, and kaempferol previously reported in human liver (28). The IC50s for inhibition of steroid sulfatase by daidzein 4,7-bisulfate and daidzein 4-sulfate (0.8 and 14 µM, respectively) are quite similar to values previously reported by Wong and Keung for inhibition of dehydroepiandrosterone sulfation in hamster liver extracts (46). The isoflavone sulfates studied in our experiments included the major sulfate metabolites of soy isoflavones. However, less than 0.1% of the conjugation products of isoflavones in humans are bisulfates (47). Hence, concentrations of daidzein 4,7-bisulfate, even in those consuming high soy/isoflavone-supplemented diets, would not be sufficient to influence steroid sulfatase activity in vivo.
The bioavailability of estrogens in postmenopausal women is regulated by the release of free estrogens from the circulating pool of E1S by steroid sulfatase and their reconjugation by SULT1E1. The importance of this balance to the development of hormone-dependent tumors is well illustrated by a recent report showing that low SULT1E1 and/or high steroid sulfatase expression in breast tumor tissue correlates with a poor prognosis for disease progression (23). The results we report here show that concentrations of isoflavonoids that may be achieved in individuals consuming a high-soy diet may inhibit SULT1E1 activity while exerting no effect on steroid sulfatase. The most potent isoflavonoids were genistein, daidzein, and its metabolic product equol. Indeed, equol was the most potent inhibitor of SULT1E1 that we tested, so the effects of a high-soy diet could be particularly important in the approximately 30% of the population whose gut flora produces significant amounts of this compound (48).
SULT1A1 also sulfates estrogens at physiologically relevant concentrations and may be the major route for estrogen sulfation in some mammary tumor cells and cell lines that lack SULT1E1 (22, 23). Furthermore, when cDNA for SULT1E1 was transfected into hormone-dependent MCF-7 breast cancer cells that lack this enzyme, their proliferative response to estrogens was significantly reduced, suggesting that an inability to inactivate estrogens via SULT1E1 is partially responsible for the transformed phenotype (33, 49). The sulfation of estrogens by SULT1A1 may represent an important back-up pathway in these tissues (20, 21, 22, 23), so our observation that SULT1A1 is also inhibited by flavonoids, even at relatively modest concentrations, may have a particular significance in breast tumor tissue.
The physiological consequences of this selective inhibition of sulfotransferase activity in vivo may depend on the location of the enzymes most affected by dietary phytoestrogens. If the effect of these compounds was exerted primarily on those enzymes responsible for the generation of the circulating E1S pool, then diets rich in phytoestrogens might diminish the availability of active estrogens to peripheral tissues. However, those few studies that exist suggest that flavonoid consumption has little or no effect on circulating estrogen levels (50, 51, 52). One study reported elevated E2 concentrations in a group of white Americans consuming a dietary supplement of soy protein isolate (53). If the effects of isoflavonoids were exerted in breast tissue itself, then reduced sulfotransferase activity would increase the local concentrations of free E1 and E2, thus driving proliferation of hormone-dependent tumor cells. This could be of particular significance if the tissues affected already expressed diminished sulfotransferase activity as appears to be the case in breast cancer cell lines and some breast tumors (22, 23). In such cases, phytoestrogen ingestion might contribute to the further worsening of an already unfavorable ratio of estrogen/estrogen sulfate in tissues that are subject to hormone-stimulated proliferation.
The literature concerning dietary phytoestrogen intake and breast cancer risk has recently been reviewed by Adlercreutz (3). Although some studies have shown a weak inverse correlation of breast cancer risk with soy consumption in Asian cultures (54), Adlercreutz (3) concluded that there is no direct evidence indicating that isoflavonoid intake during adult life is protective against the disease in Western populations. Despite this uncertainty, dietary supplements containing phytoestrogens, especially isoflavonoids, are widely advertised as being protective against breast cancer. It is likely that individuals who perceive themselves to be at risk from the disease may be particularly susceptible to such advertising.
The results of our in vitro studies suggest that large doses of dietary isoflavonoids may have a profound effect upon the metabolism of steroid hormones in humans, possibly leading to elevated levels of active estrogens in target tissues including mammary gland. These changes are unlikely to offer any protection against breast cancer and could be deleterious for those in the early stages of the disease or at particular risk of developing it. Hence, there are good reasons to question whether the widespread use of these compounds in dietary supplements is wise, and there is an urgent need for further work to clarify this issue. Dietary trials with human populations could investigate the effects of phytoestrogens on the balance between circulating E1S and active estrogens and the activity and expression of these key sulfotransferases in accessible target tissues such as blood cells.
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Acknowledgments |
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Footnotes |
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Abbreviations: DMSO, Dimethylsulfoxide; E1, estrone; E1S, estrone sulfate; [3H]-E1S, [6,7-3H(N)]-estrone sulfate; E2, estradiol; Ki, inhibitory constant (active site); Ki, inhibitory constant (allosteric site); PAPS, adenosine 3'-phosphate 5'-phosphosulfate; SULT1A1, phenolsulfotransferase; SULT1A3, monoamine phenolsulfotransferase; SULT1E1, estrogen sulfotransferase.
Received September 18, 2003.
Accepted December 23, 2003.
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References |
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,25-dihydroxyvitamin D3. Biochem J 355:361–371[CrossRef][Medline]
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), genistein (
), and equol (
).