Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases. Soy and soy foods are rich sources of isoflavones, which have been shown to possess several biological activities. In this study, the metabolism of soy isoflavones daidzein, genistein and glycitein was investigated in human subjects. The aim was to find and identify urinary phase I metabolites of isoflavones, which have an intact isoflavonoid skeleton, and which might possess some bioactivity. Six volunteers included three soy bars per day into their normal western diet for a 2-week period. Daily urine samples were collected before, and after the supplementation period. Urine samples were hydrolyzed with Helix pomatia, extracted with diethyl ether, purified with Sephadex LH-20 chromatography, and analyzed as trimethylsilyl derivatives using gas chromatography–mass spectrometry (GC–MS). The structures of the isoflavone metabolites were identified using authentic reference compounds. The metabolites, for which authentic reference compounds were not available, were identified by the interpretation of mass spectra. Several new isoflavone metabolites were identified, and the presence of previously reported metabolites confirmed. The metabolic pathways of daidzein, genistein and glycitein are presented on the basis of the identification of the metabolites in human urine after soy supplementation.     

Genistein and daidzein induce cell proliferation and their metabolites cause oxidative DNA damage in relation to isoflavone-induced cancer of estrogen-sensitive organs

The soy isoflavones, genistein (5,7,4'-trihydroxyisoflavone) and daidzein (7,4'-dihydroxyisoflavone), are representative phytoestrogens that function as chemopreventive agents against cancers, cardiovascular disease, and osteoporosis. However, recent studies indicated that genistein and/or daidzein induced cancers of reproductive organs in rodents, such as the uterus and vulva. To clarify the molecular mechanisms underlying the induction of carcinogenesis by soy isoflavones, we examined the ability of genistein, daidzein, and their metabolites, 5,7,3',4'-tetrahydroxyisoflavone (orobol), 7,3',4'-trihydroxyisoflavone (7,3',4'-OH-IF), and 6,7,4'-trihydroxyisoflavone (6,7,4'-OH-IF), to cause DNA damage and cell proliferation. An E-screen assay revealed that genistein and daidzein enhanced proliferation of estrogen-sensitive breast cancer MCF-7 cells, while their metabolites had little or no effect. A surface plasmon resonance sensor showed that binding of isoflavone-liganded estrogen receptors (ER) to estrogen response elements (ERE) was largely consistent with cell proliferative activity of isoflavones. Orobol and 7,3',4'-OH-IF significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human mammary epithelial MCF-10A cells, while genistein, daidzein, and 6,7,4'-OH-IF did not. Experiments using isolated DNA revealed a metal-dependent mechanism of oxidative DNA damage induced by orobol and 7,3',4'-OH-IF. DNA damage was enhanced by the addition of endogenous reductant NADH, formed via the redox cycle. These findings suggest that oxidative DNA damage by isoflavone metabolites plays a role in tumor initiation and that cell proliferation by isoflavones via ER-ERE binding induces tumor promotion and/or progression, resulting in cancer of estrogen-sensitive organs.     

Metabolism of isoflavones in human subjects

Isoflavones form a group of plant compounds that occur mainly in legumes, soy being the most important source in human diet. The high levels of isoflavones in the diet have been associated with a lowered risk for hormone-dependent diseases, including breast and prostate cancers, osteoporosis and cardiovascular disease. The metabolism of isoflavones in humans has been studied to a certain extent, but detailed studies are lacking. This paper reviews the current knowledge on metabolism of isoflavones and presents some preliminary results of a comprehensive soy feeding study, in which the phase I metabolites of soy isoflavones, daidzein, genistein and glycitein, were identified by GC-MS. This revised version was published online in June 2006 with corrections to the Cover Date.     

Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.     

Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6'-OH-O-dma, and cis-4-OH-equol in human urine by gas chromatography-mass spectroscopy using authentic reference compounds.

The metabolic products of daidzein and genistein, the principal isoflavones of soy, were examined. Six volunteers included soy into their normal diet for a 2-week period and urine samples were analyzed before and after soy consumption. Isolation and characterization of the urinary metabolites were carried out with absorption chromatography on Sephadex LH-20 and gas chromatography-electron ionization mass spectrometry (GC-EIMS). The structures of the isoflavones isolated were confirmed by using authentic reference compounds. Dihydrogenistein, 6'-OH-O-desmethylangolensin, and cis-4-OH-equol were identified, in addition to known isoflavonoids daidzein, genistein, glycitein, and the known metabolites equol, O-desmethylangolensin, and dihydrodaidzein, by comparing the retention times and the spectra of the urinary compounds with those of the synthesized reference standards. The mammalian lignans enterolactone and enterodiol were also identified. Derivatization of the isoflavones for GC-MS was examined by comparing two silylating reagents, N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and pyridine:hexamethyldisilazan:trimethylchlorosilane (QSM), both used for the derivatization of these compounds. The silylation experiments revealed significant differences in the compositions of the derivatization products. Some corrections were made concerning the earlier published data of dihydrogenistein and 6'-OH-O-dma.     

Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.     

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.     

Metabolism of the soy isoflavones daidzein and genistein by fungi used in the preparation of various fermented soybean foods

The ability of fungi used in the preparation of fermented soybean foods to metabolize the soy isoflavones daidzein and genistein was investigated. A total of 21 fungal strains from dou-chi, miso, sake, soy sauce, and sufu were screened. The genera of the tested fungi included Actinomucor, Aspergillus, Candida, Debaryomyces, Monascus, Mucor, Rhizopus, Saccharomyces, and Zygosaccharomyces. The results were that all tested Aspergillus strains from these soybean foods, including five A. oryzae strains, one A. sojae strain, and one A. tamarii strain, metabolized both daidzein and genistein. In contrast, no other tested fungi from the fermented soybean foods metabolized either daidzein or genistein. The metabolites of daidzein and genistein by Aspergillus strains were identified as 8-hydroxydaidzein and 8-hydroxygenistein, respectively, based on their mass, (1)H-, and (13)C-NMR spectra.     

Comparison between daidzein and genistein antioxidant activity in primary and cancer lymphocytes

The main objective of this study was to compare the protective effect of daidzein and genistein against induced oxidative damage in Jurkat T-cell line and in peripheral blood lymphocytes of healthy subjects. After supplementation of cells with isoflavones (from 2.5 to 20micromol/L in Jurkat T-cell and from 0.01 to 2.5micromol/L in primary lymphocytes, 24h), we determined DNA damage induced by hydrogen peroxide using the comet assay and lipid peroxidation evaluating malondialdehyde (MDA) production after ferrous ion treatment. Supplementation of Jurkat cells and primary lymphocytes with both isoflavones significantly increased DNA protection from oxidative damage at concentrations between 0.1 and 5micromol/L (P<0.05), and with just daidzein, at concentrations higher than 2.5micromol/L, there was a decrease in the production of MDA (P<0.05). Our results seem to support that daidzein is just as effective as genistein in protecting cells against oxidative damage especially with respect to DNA. Moreover, since the protective effect was found at concentrations reachable in plasma after soy consumption (less than 2micromol/L), it can be assumed that the antioxidant activity of isoflavones could really contribute to the healthy properties of soy.     

Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates

The distribution of flavonoids, isoflavonoids, and their conjugates in developing soybean (Glycine max L.) seedling organs and in root and seed exudates has been examined. Conjugates of the isoflavones daidzein and genistein are major metabolites in all embryonic organs within the dry seed and in seedling roots, hypocotyl, and cotyledon tissues at all times after germination. Primary leaf tissues undergo a programmed shift from isoflavonoid to flavonoid metabolism 3 days after germination and become largely predominated by glycosides of the flavonols kampferol, quercetin, and isorhamnetin by 5 days. Cotyledons contain relatively constant and very high levels of conjugates of both daidzein and genistein. Hypocotyl tissues contain a third unidentified compound, P19.3, also present in multiple conjugated forms. Conjugates of daidzein, genistein, and P19.3 are at their highest levels in the hypocotyl hook and fall off progressively down the hypocotyl. These isoflavones also undergo a programmed and dramatic decrease between 2 and 4 days in the hypocotyl hook. All root sections are predominated by daidzein and its conjugates, particularly in the root tip, where they reach the highest levels in the seedling. Light has a pronounced effect on the distribution of the isoflavones; in the dark, isoflavone levels in the root tips are greatly reduced, while those in the cotyledons are higher. Finally, the conjugates of daidzein and genistein and several unidentified aromatic metabolites are selectively excreted into root and seed exudates. Analysis of seed exudates suggests that this is a continuous, but saturable event.     

Soybean isoflavonoids and their metabolic products inhibit in vitro lipoprotein oxidation in serum

Isoflavonoids are compounds present in many legumes, but are derived in the human diet mainly from soybeans and various soybean-based food products. The major isoflavonoids occurring in soy are the glycosides of genistein and daidzein. The metabolic products of genistein metabolism in humans have not been clearly shown. The two main products of daidzein metabolism in humans appear to be equol and O-desmethylangolensin. Increasing evidence suggests that oxidative modification to low-density lipoprotein is involved in atherogenesis, and that natural antioxidants that prevent or inhibit oxidative damage to low-density lipoprotein may beneficially influence atherogenesis. In the present experiments, the effects of genistein and daidzein, and the daidzein metabolites equol and O-desmethylangolensin on Cu²⁺-induced oxidation of lipoproteins in serum were examined. Three concentrations of each compound (0.1 μM, 1 μM, 10 μM) were tested for antioxidant activity in six individual serum samples. All compounds tested inhibited lipoprotein oxidation. The minimum concentration for significant inhibition was 1 μM for genistein and daidzein (P < 0.05), and 0.1 μM equol and O-desmethylangolensin (P < 0.05). Equol and O-desmethylangolensin were more potent inhibitors of in vitro lipoprotein oxidation in serum than the two major dietary isoflavonoids. This study has demonstrated that soybean isoflavonoids and metabolic products of daidzein metabolism inhibit lipoprotein oxidation in vitro. Human intervention studies are needed to determine if these compounds can influence oxidation in vivo.     

Soy protein isolate induces CYP3A1 and CYP3A2 in prepubertal rats

Feeding soy diets has been shown to induce cytochrome P450s in gene family CYP3A in Sprague-Dawley rat liver. We compared expression of CYP3A enzymes on postnatal Day 33 (PND33) rats fed casein or soy protein isolate (SPI+)-based AIN-93G diets continuously from gestational Day 4 through PND33 or the diets were switched on PND15 (n = 3-6 litters) to examine the potential imprinting effects of soy on drug metabolism. In addition rats were fed casein, SPI+, SPI+ stripped of phytochemicals (SPI-), or casein diets supplemented with the soy-associated isoflavones genistein or daidzein from weaning through PND33 to examine the hypothesis that the isoflavones are responsible for CYP3A induction by soy feeding. Feeding SPI either continuously or from weaning induced hepatic CYP3A1 and CYP3A2 mRNA, apoprotein, and CYP3A-dependent testosterone 6beta-hydroxylase activity in liver microsomes 2- to 5-fold (P < 0.05). CYP3A mRNA expression was also elevated 2- to 3-fold in the jejunum of SPI-fed rats (P < 0.05). CYP3A was not induced in livers of rats switched to casein from soy at weaning. Induction of CYP3A1 also did not occur in rats fed SPI-, but CYP3A2 mRNA and apoprotein were induced (P < 0.05) in females fed SPI-. Offspring weaned onto genistein-supplemented diets had no elevation of CYP3A mRNAs or apoproteins. Weaning onto daidzein diets increased CYP3A2 mRNA and apoprotein expression in male rats (P < 0.05). These data suggest that early soy consumption may increase the metabolism of a wide variety of CYP3A substrates, but that soy does not imprint the expression of CYP3A enzymes. Effects on CYP3A1 expression appear to be primarily due to phytochemical components of SPI other than isoflavones. In contrast, consumption of soy protein and daidzein appear to be associated with the induction of CYP3A2.     

Glucuronidation of the soyabean isoflavones genistein and daidzein by human liver is related to levels of UGT1A1 and UGT1A9 activity and alters isoflavone response in the MCF-7 human breast cancer cell line

The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 microM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.     

Proteome analysis suggests that mitochondrial dysfunction in stressed endothelial cells is reversed by a soy extract and isolated isoflavones

Apoptosis is a driving force in atherosclerosis development. A soy extract or a combination of the soy isoflavones genistein and daidzein inhibited apoptosis induced by oxidized LDL in endothelial cells. Proteome analysis revealed that the LDL-induced alterations of numerous proteins were reversed by the extract and the genistein/daidzein mixture but only three protein entities, all functionally linked to mitochondrial dysfunction, were regulated in common by both treatments.     

Soy protein with or without isoflavones, soy germ and soy germ extract, and daidzein lessen plasma cholesterol levels in golden Syrian hamsters

Dietary isolated soy protein (ISP, containing approximately equal amounts of daidzein and genistein), ethanol-extracted ISP (ISP (-)), soygerm or soygerm extract (containing large amounts of daidzein and glycitein and little genistein) and the isoflavone, daidzein, were hypothesized to lessen plasma cholesterol in comparison with casein. Sixty male and 60 female golden Syrian hamsters (6-8 weeks of age) were randomly assigned to six treatments fed for 10 weeks. Four of the experimental diets (ISP, daidzein, soygerm, and soygerm extract) contained 1.3 mmol total isoflavones/kg. The ISP (-) diet contained 0.013 mmol isoflavone/kg, whereas the casein diet contained no isoflavones. Hamsters fed ISP, ISP (-), daidzein, soygerm, and soygerm extract had significantly less plasma total cholesterol (by 16%-28%), less non-HDL cholesterol (by 15%-50%) and less non-HDL/HDL cholesterol ratios compared with hamsters fed casein (P < 0.01). For male hamsters, there were no differences among treatments in plasma HDL concentrations. Female hamsters fed ISP (-) had significantly greater HDL levels (P < 0.01) than females fed casein or daidzein. Triglyceride concentration was significantly less in hamsters fed ISP (-) compared with the casein-fed females. Because soy protein with or without isoflavones, soygerm and soygerm extract, and daidzein lessened plasma cholesterol to an approximately equal extent, soy protein alone, varying mixtures of isoflavones, and other extractable components of soy are responsible for cholesterol-lessening effects of soy foods, mainly due to their effects to lessen LDL cholesterol.     

Chlorination and nitration of soy isoflavones.

Diets enriched in soy foods containing a high concentration of isoflavonoids are associated with a decrease in the incidence of several chronic inflammatory diseases. Studies with experimental models of diseases, such as atherosclerosis, suggest that these effects can be ascribed to the biological properties of the isoflavones. Since the isoflavones and tyrosine have structural similarities and modifications to tyrosine by inflammatory oxidants such as hypochlorous acid (HOCl) and peroxynitrite (ONOO(-)) have been recently recognized, we hypothesized that the isoflavones also react with HOCl and ONOO(-). Using an in vitro approach, we demonstrate in the present study that the isoflavones genistein, daidzein, and biochanin-A can be chlorinated and nitrated by these oxidants. These reactions were investigated using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. In the reaction with HOCl, both mono- and dichlorinated derivatives of genistein and biochanin-A are formed, whereas with daidzein only a monochlorinated derivative was detected. The reaction between genistein or daidzein and ONOO(-) yielded a mononitrated product. However, no nitrated product was detected with biochanin-A. Furthermore, the reaction between genistein and sodium nitrite and HOCl yielded a chloronitrogenistein derivative, as well as a dichloronitrogenistein derivative. These results indicate that the ability of the isoflavones to react with these oxidant species depends on their structure and suggest that they could be formed under conditions where these reactive species are generated under pathological conditions.     

大豆异黄酮对大鼠乳腺癌细胞内cAMP/PKA信号途径的影响

本实验研究了大豆异黄酮对SHZ-88大鼠乳腺癌细胞内cAMP／PKA信号途径的影响。实验设3组：空白对照组、50μg／ml大豆黄酮及15μg／ml染料木素组。采用放射免疫测定法（RIA）检测了胞内cAMP的浓度、腺苷酸环化酶（adenylate cyclase,AC）和磷酸二酯酶（phosphodiesterase,PDE）的活性,用（γ-^32P）ATP掺入法测定cAMP依赖性PKA的活性,半定量RT-PCR法分析cAMP反应元件结合蛋白（cAMP response element binding protein,CREB）mRNA表达的变化。结果表明：在处理后5min,大豆黄酮组和染料木素组细胞的cAMP浓度分别比对照组升高了9．5％和11．0％（P〈0．05）：10min时,分别比对照组升高31．0％和40.3％（P〈0．01）。3组细胞的AC活性在处理时间内没有明显变化。但在处理后5min,大豆黄酮组和染料木素组细胞的PDE活性分别降至对照组的71．8％和71．6％（P〈0．05）。处理后20min,大豆黄酮组和染料木素组细胞PKA活性分别上升到对照组的125．8％和122．3％（P〈0．05）;到40min时仍维持在高水平。大豆黄酮组和染料木素组细胞CREB mRNA的表达量在处理后3h分别比对照组增加31．6％和51．1％（P〈0．05）;6h后开始下降。这些结果提示,大豆异黄酮能够激活大鼠乳腺癌细胞内cAMP／PKA信号途径;而且是通过抑制磷酸二酯酶的活性,导致胞内cAMP浓度升高而实现的。     

Daidzein and genistein content of fruits and nuts

Dietary phytoestrogens such as the isoflavones daidzein and genistein are thought to protect against chronic diseases that are common in Western societies, such as cancer, osteoporosis, and ischemic heart disease. In addition, there are concerns regarding the deleterious effects of hormone-like compounds, especially with respect to the development of infants. However, there is little information regarding the phytoestrogen content of foods, and therefore epidemiologic investigations of phytoestrogens are limited. As part of a study quantifying the consumption of phytoestrogens, the objective of this work was to assess the daidzein and genistein content of fruits and nuts commonly eaten in Europe. Eighty different fruits and nuts were sampled, prepared for eating, and freeze-dried. Daidzein and genistein were extracted from the dried foods, and the two isoflavones were quantified after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) as internal standards. Of the 80 foods assayed, 43 contained no detectable daidzein or genistein, at a limit of quantification of 1 microg/kg dry weight of food. Nine foods contained more than 100 microg of the two isoflavones combined per kilogram wet weight, and 28 contained less than this amount. Currants and raisins were the richest sources of the isoflavones, containing 2,250 microg and 1,840 microg of the two isoflavones combined per kilogram of wet weight of food. Although fruits and nuts are not as rich in isoflavone phytoestrogens as are soy and other legumes, this is the first documentation of levels of daidzein and genistein occurring in these foods.