Institut für Toxikologie, Universität Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany
Received February 7, 2001; accepted May 7, 2001
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ABSTRACT |
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Key Words: daidzein; estrogen; isoflavone; disposition.
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INTRODUCTION |
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Daidzein has been found to act as an estrogen receptor agonist and induces receptor-mediated estrogenic responses both in in vivo and in vitro assays for estrogenicity. For example, daidzein induces receptor-mediated increases in cell proliferation in human breast cancer cells (E-screen) and a dose-dependent weight increase of the uterus of rats (disruption of ovarian cyclicity; Kaldas and Hughes, 1989). In humans, high consumption of soy and soy-based food has also been connected with estrogenic responses in humans (Baird et al., 1995; Cassidy et al., 1994
; Hargreaves et al., 1999
; Setchell et al., 1987
), along with other effects such as antioxidative and antiproliferative responses (Setchell, 1998
) and a lower incidence of cardiovascular diseases in Asia (Murkies et al., 1998
). In some animal species, consumption of diets rich in phytoestrogens has been associated with toxic effects on reproduction (Kaldas and Hughes, 1989
).
Humans are also exposed to a variety of industrial compounds that are weak estrogens in different assays used to determine estrogenicity. These chemicals are sometimes called "environmental estrogens" or "endocrine disrupters" (Guillette et al., 1995; Sharpe and Skakkebaek, 1993
; Toppari et al., 1996
). In general, these compounds are much weaker estrogens than daidzein and humans are exposed to much lower quantities of these compounds. For example, the EC50 values for the transactivation of the rat estrogen receptors are in the range of 107 to 108 M for daidzein and related isoflavones, whereas EC50 values for bisphenol A, one of the most intensively studied "environmental estrogens," are in the range of 104 to 105 M (Casanova et al., 1999
; Kuiper et al., 1998
; Zava et al., 1997
). Environmental estrogens have been implicated in a variety of effects in humans such as increased incidences of breast cancer, decreased sperm counts, and developmental effects (Colborn et al., 1993
; Davis et al., 1993
). However, since human exposure to phytoestrogens is much larger as compared to industrial chemicals with estrogenic effects and phytoestrogens are more potent estrogens than most environmental estrogens, health risks of human exposure to environmental estrogens may be much lower as compared to risks of phytoestrogen exposure (Safe, 1995
) or phytoestrogens may also act as "endocrine disruptors" (Santti et al., 1998
; Setchell et al., 1997
).
In rodents, daidzein in vivo is only a relatively weak estrogen despite relatively high affinity to the estrogen receptor suggesting that toxicokinetics may be a major determinant of estrogenic potency in vivo. Therefore, the low potency of daidzein in vivo may be due to rapid biotransformation and excretion or inefficient absorption, therefore lowering the amount of daidzein available for receptor binding. The experimental toxicokinetics of daidzein and other endocrine modulators should be integrated into the process of extrapolation of toxicological data, from in vitro to in vivo, and from animals to humans, and into the risk assessment process of endocrine chemicals.
In this work we have studied the disposition, biotransformation, and kinetics of excretion of daidzein in rodents in order to elucidate the apparent discrepancies between estrogenic activities of daidzein in vivo and in vitro.
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MATERIALS AND METHODS |
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Instrumental analysis.
HPLC was performed with a Millipore Waters pump system (pumps 501 and 510 and gradient controller) and a HP 1050 UV-detector coupled to a Shimadzu-sil 9-A autosampler. Peak integration was performed by a Waters-Millipore data module 740 integrator. Separations were performed using a 4.6 x 250 mm steel column filled with Partisil ODS-3, 5 µm. Samples were analyzed by gradient elution using a linear gradient from 100% H2O (adjusted to pH 2 with trifluoroacetic acid) to 100% acetonitrile over 60 min at a flow rate of 1 ml/min. A different gradient (20% to 80% acetonitrile in water in 40 min) was used to identify unchanged daidzein. To record online UV-spectra, some urine samples were also analyzed by HPLC by a Hewlett-Packard 1040 series II diode array detector coupled to a Hewlett-Packard 1050 HPLC pump. Spectra were recorded from 220460 nm and HPLC separation conditions were identical as described above.
GC-MS in the electron impact mode (70 eV) was performed with a HP 5973 MSD using a DB-1 fused silica capillary column (30 m x 0.18 mm I.D., 0.5 µm film thickness; J & W Scientific, Folsom, CA) with a linear temperature gradient of 20°C/min from 100° to 320°C. Split injection with a split ratio of 20:1 and helium at a flow rate of 1 ml/min was used as carrier gas.
HPLC-MS/MS was performed with an Applied Biosystems 140B HPLC-pump coupled to a TSQ 7000 tandem mass spectrometer system equipped with an electrospray ionization interface (Finnigan MAT, Bremen, Germany). Data acquisition and evaluation were conducted on a DEC 5000/33 using the ICSI 8.1 software (Finnigan MAT). Samples were separated using a 100 x 2 mm ID steel column filled with Eurosphere 100-C18 (Macherey and Nagel, Düren, Germany) and a linear gradient from 100% H2O (adjusted to pH 2 with trifluoroacetic acid) to 100% acetonitrile over 60 min at a flow rate of 0.2 ml/min. Capillary temperature was 250°C and electrospray voltage was 3.5 kV. Spectra were collected in the positive ion mode.
Half-lives were calculated using exponential regression in Microsoft Excel® spreadsheets using the curve fitting function of the program. Excretion data correlated with first-order kinetics with r2 values > 0.8.
Animals and treatment.
Daidzein (100 mg/kg, dissolved in corn oil) was administered by gavage to 4 male (280300 g) and 4 female (160190 g) Fischer F344 rats (Charles-River-Wiga, Sulzfeld, Germany). After administration, the animals were individually kept in metabolic cages and urine and feces were collected at 4°C at predetermined intervals for 96 h. For acclimatization, animals were kept in metabolic cages for 72 h before daidzein administration and control urine and feces were collected during this time. Animals had free access to food and tap water during the sample collection period and were given a diet low in soy proteins and in isoflavones (No 1324, Altromin, Langen, Germany) for 1 week before daidzein administration and during the sampling period. Daidzein and genistein, 2 major isoflavones usually present in soy, could not be detected in this diet by HPLC or by GC/MS after methanol extraction (Casanova et al., 1999). Only very low levels (less than 2 nmol/ml) of daidzein and daidzein-metabolites were detected in urine and feces collected from the animals for 24 h before daidzein administration.
Separation and quantification of daidzein-metabolites.
A solution (10 µl) of the internal standard flavone (2-phenyl-4H-1-benzopyran-4-one, 40 µmol/ml in methanol) was added to rat urine samples (20 µl) and the obtained solution was diluted with 770 µl of methanol. After centrifugation, 50 µl of this solution were separated by HPLC. For quantitation of daidzein and metabolites, the eluate from the column was monitored at 302 nm. Quantitation was performed relative to the content of flavone and referenced to calibration curves with fortified aliquots of urine samples from controls containing 080 nmol/ml daidzein. The method was linear in the range of concentrations used and calibration standards were analyzed with every sample series (usually 2030 samples). The method permitted the quantitation of 2 nmoles of daidzein/ml of urine with a signal to noise ratio of 5:1. When identical samples were repeatedly analyzed, deviations of the obtained quantitative results were lower than 10%. Daidzein-glucuronide and daidzein-sulfate were quantified based on calibration curves obtained with daidzein due to identical UV-absorption.
Quantitation of daidzein excretion with feces.
Feces collected at different time points were lyophilized. To extract daidzein and daidzein-metabolites, 250 mg of freeze-dried feces samples were suspended in 20 ml of ethyl acetate and agitated at 60°C for 48 h in closed vials. After filtration, the ethyl acetate extracts were adjusted to a volume of 25 ml, 10 µl of the internal standard flavone were added to aliquots of the extract (0.79 ml) and 50 µl of the obtained solution were separated by HPLC as described above. Recovery of daidzein from spiked samples of control feces collected from the rats before daidzein administration was >90%.
Identification of minor daidzein-metabolites in feces.
Minor daidzein-metabolites in feces were identified by GC/MS in the ethyl acetate extracts prepared as described above. The solvent from aliquots (5 ml) of the extracts was removed under reduced pressure and the obtained residues were treated with 0.5 ml of bis-trimethylsilyl trifluoroacetamide at 100°C. After 1 h, 2 µl of the solutions were analyzed by GC/MS. Standards of O-desmethylangolensin, equol, 4-hydroxybenzoic acid, and 2-(4-hydroxyphenyl)propanoic acid were added to feces obtained from control rats to demonstrate recovery.
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RESULTS |
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DISCUSSION |
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Of the applied daidzein dose, only a minor part was recovered in urine both as unchanged parent compound, and, as expected, in the form of conjugates. No indication for phase I oxidation reactions in daidzein biotransformation is indicated by the structure of metabolites found in urine; due to the presence of hydroxyl groups, daidzein is efficiently conjugated to glucuronic acid and to sulfate and the highly water soluble conjugates are rapidly excreted. The exact regiochemistry of the conjugates was determined previously, glucuronic acid is coupled to the oxygen atom in position 7 to give daidzein-7-O-ß-D-glucuronide and sulfate is coupled to the oxygen atom in 4'-position to daidzein-4'-O-sulfate (Yasuda et al., 1994) (Fig. 6
).
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The high recovery of administered daidzein in feces may indicate an inefficient absorption of daidzein from the gastrointestinal tract of rats or an intensive conjugation of daidzein in rat liver, enterohepatic circulation of the formed conjugates and cleavage of the conjugates by glucuronidase, and/or sulfatase in intestinal bacteria resulting in daidzein excretion with feces. Likely, an inefficient absorption of daidzein is responsible for the high percentage of administered dose excreted with feces. This assumption is supported by the expected elimination of the highly water-soluble daidzein-sulfate from the liver into the blood stream to be excreted in urine and therefore not subjected to enterohepatic circulation. In case of enterohepatic circulation, the differences in the extent of daidzein-sulfate formation between male and female rats should therefore result in differences in the extent of daidzein excretion with feces and a lower recovery of the administered dose in feces of male animals. The lack of gender difference in fecal excretion suggest that enterohepatic circulation of daidzein-conjugates is unlikely to have a major impact on daidzein disposition. Moreover, enterohepatic circulation of daidzein-conjugates is also not supported by the rapid excretion of unchanged daidzein with feces and of daidzein and daidzein-conjugates with urine. Structurally similar compounds to daidzein such as bisphenol A, which undergo enterohepatic circulation in rats, are only slowly excreted with urine and feces over a prolonged period of time (Pottenger et al., 2000).
A minor part of the applied daidzein in rats was reduced to O-desmethylangolensin and to equol, likely by intestinal bacteria (Adlercreutz et al., 1986). Reductive metabolism of daidzein by intestinal bacteria has also been observed in humans. Species differences in the content and type of intestinal bacteria may explain species differences in the extent of daidzein reduction. A reductive cleavage of daidzein resulting in ring fission as observed with genistein (Coldham and Sauer, 2000
) was not seen in our studies with daidzein. Genistein is reduced to give 4-hydroxyphenyl-2-propionic acid and dihydrogenistein, the expected products from daidzein to be formed by an identical mechanism, 4-hydroxybenzoic acid and 4-hydroxyphenyl-2-propionic acid were not detected in the feces of rats despite application of sensitive and specific methods.
In humans, disposition of daidzein from dietary sources was reported to be different from the disposition data observed in this study in rats. Daidzein in human diet may be slowly but more efficiently absorbed from the gastrointestinal tract in humans (Lu et al., 1995, 1996b
; Xu et al., 1995
, 1994
). Therefore, higher blood levels of daidzein in humans as compared to rats may be the result. Indeed, daidzein and other isoflavones seem to be relatively potent estrogens in humans since estrogenic effects were seen in people consuming diets rich in isoflavones for a prolonged time (Lu et al., 1996a
).
In summary, our results show that daidzein absorption from the gastrointestinal tract in rodents is not efficiently absorbed and absorbed daidzein is rapidly conjugated and excreted. Thus, only low concentrations of daidzein in rat blood and in organs are to be expected resulting in only weak estrogenic effects despite administration of relatively high doses and a relatively high affinity of daidzein to the estrogen receptor. The inefficient absorption of daidzein is therefore likely correlated to the low potency of this compound as an estrogen in vivo; a low estrogenic potency (Hopert et al., 1998) and low rate of absorption of daidzein in comparison to other estrogenic isoflavonoids was also observed (Breinholt et al., 2000
).
Disposition and biotransformation data as described here may be important contributors to the risk assessment process of "environmental estrogens" and will have to be included in comparisons of relative contributions of estrogenic chemicals from different sources to the total "estrogen load" of an organism. Species differences in extent of absorption, disposition, and biotransformation may further complicate the risk assessment process for these compounds. As a conclusion, in vitro data on estrogenicity such as the E-screen (Andersen et al., 1999; Arnold et al., 1996
; Soto et al., 1995
) and other systems should only be used as a prescreen to obtain information on potential estrogenicity of a compound. An assessment of estrogenic effects in vivo in rodents and comparative toxicokinetics in rodents and humans need to be included in the assessment of potentially adverse effects of chemicals with possible endocrine modulating activity (Kavlock et al., 1996
; Safe et al., 1997
).
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ACKNOWLEDGMENTS |
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NOTES |
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