Molecular mechanisms by which dietary isoflavones potentially prevent atherosclerosis

Dietary isoflavones are currently receiving much attention because of their potential role in preventing coronary artery disease and other chronic diseases. Accumulating evidence from cell culture and laboratory animal experiments indicates that isoflavones have the potential to prevent or delay atherogenesis. Suggested mechanisms of action include: a reduction in low-density lipoprotein (LDL) cholesterol and a potential reduction in the susceptibility of the LDL particle to oxidation; (2) an improvement in vascular reactivity; (3) an inhibition of pro-inflammatory cytokines, cell adhesion proteins and nitric oxide (NO) production; and (4) an inhibition of platelet aggregation. These mechanisms are consistent with the epidemiological evidence that a high consumption of isoflavone-rich soy products is associated with a reduced incidence of coronary artery disease. Biological effects of isoflavones are dependent on many factors, including dose consumed, duration of use, protein-binding affinity, and an individual's metabolism or intrinsic oestrogenic state. Further clinical studies are necessary to determine the potential health effects of isoflavones in specific population groups as we currently know little about age-related differences in exposure to these compounds and there are few guidelines on optimal dose for cardiovascular health benefits.

Critical review of health effects of soyabean phyto-oestrogens in post-menopausal women

A consensus view of soyabean phyto-oestrogens in clinical interventions in post-menopausal women is presented that is based on data from the EU-funded project Phytohealth. The phyto-oestrogens, primarily genistein and daidzein, were given as soyabean-protein isolates, whole-soyabean foods or extracts, supplements or pure compounds. A comprehensive literature search was conducted with well-defined inclusion or exclusion criteria. For areas for which substantial research exists only placebo-controlled double-blind randomised controlled trials (RCT) conducted on healthy post-menopausal women were included. For emerging areas all available human studies in post-menopausal women were reviewed. In order to make cross comparisons between studies the doses of isoflavones were calculated as aglycone equivalents. There is a suggestion, but no conclusive evidence, that isoflavones from the sources studied so far have a beneficial effect on bone health. The consumption of whole-soyabean foods and soyabean-protein isolates has some beneficial effects on lipid markers of cardiovascular risk. The consumption of isolated isoflavones does not affect blood lipid levels or blood pressure, although it may improve endothelial function. For menopausal symptoms there is currently limited evidence that soyabean-protein isolates, soyabean foods or red-clover (Trifolium pratense L.) extract are effective but soyabean isoflavone extracts may be effective in reducing hot flushes. There are too few RCT studies to reach conclusions on the effects of isoflavones on breast cancer, colon cancer, diabetes or cognitive function. The health benefits of soyabean phyto-oestrogens in healthy post-menopausal women are subtle and even some well-designed studies do not show protective effects. Future studies should focus on high-risk post-menopausal women, especially in the areas of diabetes, CVD, breast cancer and bone health.

Genistein affects the expression of genes involved in blood pressure regulation and angiogenesis in primary human endothelial cells

Background: Several lines of evidence suggest that the dietary isoflavone genistein (Gen) has beneficial effects with regard to cardiovascular disease and in particular on aspects related to blood pressure and angiogenesis. The biological action of Gen may be, at Least in part, attributed to its ability to affect cell signalling and response. However, so far, most of the molecular mechanisms underlying the activity of Gen in the endothelium are unknown. Methods and results: To examine the transcriptional response to 2.5 mu M Gen on primary human endothelial cells (HUVEC), we applied cDNA array technology both under baseline condition and after treatment with the pro-atherogenic stimulus, copper-oxidized LDL. The alteration of the expression patterns of individual transcripts was substantiated using either RT-PCR or Northern blotting. Gen significantly affected the expression of genes encoding for proteins centrally involved in the vascular tone such as endothelin-converting enzyme-1, endothetin-2, estrogen related receptor a and atria[ natriuretic peptide receptor A precursor. Furthermore, Gen countered the effect of oxLDL on mRNA levels encoding for vascular endothelial growth factor receptor 165, types 1 and 2. Conclusions: Our data indicate that physiologically achievable levels of Gen change the expression of mRNA encoding for proteins involved in the control of blood pressure under baseline conditions and reduce the angiogenic response to oxLDL in the endothelium. (c) 2005 Elsevier B.V. All rights reserved.

Dietary intakes and food sources of phytoestrogens in the European Prospective Investigation into Cancer and Nutrition (EPIC) 24-hour dietary recall cohort

BACKGROUND/OBJECTIVES: Phytoestrogens are estradiol-like natural compounds found in plants that have been associated with protective effects against chronic diseases, including some cancers, cardiovascular diseases and osteoporosis. The purpose of this study was to estimate the dietary intake of phytoestrogens, identify their food sources and their association with lifestyle factors in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. SUBJECTS/METHODS: Single 24-hour dietary recalls were collected from 36 037 individuals from 10 European countries, aged 35–74 years using a standardized computerized interview programe (EPIC-Soft). An ad hoc food composition database on phytoestrogens (isoflavones, lignans, coumestans, enterolignans and equol) was compiled using data from available databases, in order to obtain and describe phytoestrogen intakes and their food sources across 27 redefined EPIC centres. RESULTS: Mean total phytoestrogen intake was the highest in the UK health-conscious group (24.9 mg/day in men and 21.1 mg/day in women) whereas lowest in Greece (1.3 mg/day) in men and Spain-Granada (1.0 mg/day) in women. Northern European countries had higher intakes than southern countries. The main phytoestrogen contributors were isoflavones in both UK centres and lignans in the other EPIC cohorts. Age, body mass index, educational level, smoking status and physical activity were related to increased intakes of lignans, enterolignans and equol, but not to total phytoestrogen, isoflavone or coumestan intakes. In the UK cohorts, the major food sources of phytoestrogens were soy products. In the other EPIC cohorts the dietary sources were more distributed, among fruits, vegetables, soy products, cereal products, non-alcoholic and alcoholic beverages. CONCLUSIONS: There was a high variability in the dietary intake of total and phytoestrogen subclasses and their food sources across European regions.

Estimated intake of dietary phytoestrogens in Australian women and evaluation of correlates of phytoestrogen intake

The role of dietary phytoestrogens in health has been of continued interest and debate, but available data on the distribution of intake in the Australian diet is scarce. Therefore, we aimed to estimate phytoestrogen consumption in Australian women, describe the pattern of intake and identify correlates of high phytoestrogen intake. Study participants were 2078 control women (18-79 y) from two population-based case-control studies on gyneacological cancers (2002-2007). Dietary information was obtained using a 135-item semiquantitative FFQ and intakes of isoflavones, lignans, enterolignans and coumestans, including their individual components, were estimated using a database of phytoestrogen content in food developed in the UK. Median total intake (energy-adjusted) of phytoestrogens was 1.29 mg/d, of isoflavones 611 μg/d, of lignans 639 μg/d, of enterolignans 21μg/d and of coumestrol 8 μg/d. Both isoflavone and lignan intake were strongly skewed towards higher values and positively correlated with age. Women consumed on average 2 serves of soy foods/week. Compared to low phytoestrogen consumers (≤1.29 mg/d, median split), high phytoestrogen consumers (>1.29 mg/d) were slightly older, less likely to be smokers, had a higher educational and physical activity level, lower BMI, lower intake of dietary fat, and higher intake of fibre, selected micronutrient and soy food (all p<0.03). The daily intake of phytoestrogens in Australian women with predominantly Caucasian ethnicity is approximately 1 mg, similar to other Western populations, but considerably lower than among Asian women. However, those with a relatively high phytoestrogen diet seem to have healthier lifestyle and more favourable dietary profile compared to others.

Intakes and sources of isoflavones, lignans, enterolignans, coumestrol and soya-containing foods in the Norfolk arm of the European Prospective Investigation into Cancer and Nutrition (EPIC-Norfolk), from 7-day food diaries, using a newly updated database

Objective: A phytoestrogen-rich diet has been suggested to protect against a variety of common diseases but UK intake data on phytoestrogens or their food sources is sparse. This study aims to estimate the average intake of isoflavones, lignans, enterolignans and coumestrol from 7-day food diaries (7dFD), and to provide data on total isoflavone, lignan and phytoestrogen consumption by food group. Design: Development of a food composition database for twelve phytoestrogens and analysis of soya food and phytoestrogen consumption in a population-based study. Setting: Men and women, aged 40-79 years from the general population participating in EPIC-Norfolk between 1993 and 1997, with nutrient and food data from 7dFD. Subjects: A subset of 20 437 participants. Results: The median daily phytoestrogen intake for men was 1.20mg (interquartile range (IQR) 0.93-1.54 mg; mean 1.50 mg, SD 1.50 mg) and 0.89 mg for women (IQR 0.71-1.14 mg; mean 1.20 mg, SD 1.70 mg). In soya-consumers (SC), median daily intakes were higher: 2.86 mg in men (IQR – 1.30-7.27mg; mean 5.05 mg, SD 5.03 mg) and 3.14 mg in women (IQR – 1.09-7.33mg; mean 5.40 mg, SD 6.09 mg). In both men and women, bread made the greatest contribution to phytoestrogen intake – 40.7% and 35.7% respectively. In SC men and women, vegetable dishes and soya/goat’s/sheep’s milks were the main contributors – 42.6% and 18.9% in men and 38.8% and 29.1% in women, respectively. Conclusions: The ability to estimate phytoestrogen intake in Western populations more accurately will aid investigations into their suggested effects on health.

Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro

It has been postulated that the R- and S-equol enantiomers have different biological properties given their different binding affinities for the estrogen receptor. S-(-)equol is produced via the bacterial conversion of the soy isoflavone daidzein in the gut. We have compared the biological effects of purified S-equol to that of racemic (R and S) equol on breast and prostate cancer cells of varying receptor status in vitro. Both racemic and S-equol inhibited the growth of the breast cancer cell line MDA-MB-231 (> or = 10 microM) and the prostate cancer cell lines LNCaP (> or = 5 microM) and LAPC-4 (> or = 2.5 microM). The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30 microM) was unable to prevent DNA damage in MCF-7 or MCF-10A breast cells following exposure to 2-hydroxy-4-nonenal, menadione, or benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. In contrast, racemic equol (10, 30 microM) prevented DNA damage in MCF-10A cells following exposure to 2-hydroxy-4-nonenal or menadione. These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol.

Genistein protects prostate cells against hydrogen peroxide-induced DNA damage and induces expression of genes involved in the defence against oxidative stress

Prostate cancer is one of the most frequent cancer types in Western societies and predominately occurs in the elderly male. The strong age-related increase of prostate cancer is associated with a progressive accumulation of oxidative DNA damage which is presumably supported by a decline of the cellular antioxidative defence during ageing. Risk of developing prostate cancer is much lower in many Asian countries where soy food is an integral part of diet. Therefore, isoflavones from soy were suggested to have chemopreventive activities in prostate cells. Here, we have investigated the hypothesis that the soy-isoflavone genistein could protect DNA of LAPC-4 prostate cells from oxidative stress-related damage by enhancing the expression of antioxidative genes and proteins. A 24 h preincubation with genistein (1-30 microM) protected cells from hydrogen peroxide-induced DNA damage, as determined by the comet assay. Analysis of two cDNA macroarrays, each containing 96 genes of biotransformation and stress response, revealed a modulated expression of 3 genes at 1 microM and of 19 genes at 10 microM genistein. Real-time PCR confirmed the induction of three genes encoding products with antioxidant activities, namely glutathione reductase (2.7-fold), microsomal glutathione S-transferase 1 (1.9-fold) and metallothionein 1X (6.3-fold), at 1-30 microM genistein. 17Beta-estradiol, in contrast, decreased the expression of metallothionein 1X at 0.3 microM (2.0-fold), possibly pointing to an estrogen receptor-mediated regulation of this gene. Immunocytochemical staining revealed an induction of metallothionein proteins at 30 microM genistein, while their intracellular localization was unaffected. Metallothioneins were previously found to protect cells from hydrogen peroxide-induced DNA damage. Hence, our findings indicate that genistein protects prostate cells from oxidative stress-related DNA damage presumably by inducing the expression of antioxidative products, such as metallothioneins. Genistein, therefore, might counteract the age-related decline of important antioxidative defence systems which in turn maintain DNA integrity.

Influence of 10 wk of soy consumption on plasma concentrations and excretion of isoflavonoids and on gut microflora metabolism in healthy adults

Although interindividual variation in isoflavone metabolism was high, intraindividual variation was low. Only concentrations of O-DMA in plasma and urine appeared to be influenced by sex. Chronic soy consumption does not appear to induce many significant changes to the gut metabolism of isoflavones other than higher beta-glucosidase activity.

Soy products in the management of breast cancer

Purpose of review: Meta-analyses of epidemiological studies of soy consumption and breast cancer risk have demonstrated modest protective effects, usually attributed to isoflavones. Concern has been expressed, however, that the estrogenic activity of isoflavones may have adverse effects on breast cancer recurrence. Recent findings: The review covers epidemiological studies that have investigated the impact of soy consumption in breast cancer patients on recurrence and mortality. There are preliminary data to suggest that soy has differential effects on recurrence in human epidermal growth factor receptor-2 positive and human epidermal growth factor receptor-2 negative tumours. Recent studies on mechanisms of action of soy in breast cancer provide insights into epigenetic effects and the interaction of isoflavones with IGF-1 and with a number of polymorphisms of genes associated with breast cancer risk such as MDM2 and CYP1B1. Summary: Overall, these studies indicate that soy foods consumed at levels comparable to those in Asian populations have no detrimental effects on risk of breast cancer recurrence and in some cases significantly reduce the risk. Importantly, soy does not appear to interfere with tamoxifen or anastrozole therapy. Recent research suggests that women who are at increased risk of breast cancer due to polymorphisms in genes associated with the disease may especially benefit from high soy isoflavone intake.

Daidzein, R-(+)equol and S-(-)equol inhibit the invasion of MDA-MB-231 breast cancer cells potentially via the down-regulation of matrix metalloproteinase-2

PURPOSE: Soy isoflavones may inhibit tumor cell invasion and metastasis via their effects on matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The current study investigates the effects of daidzein, R- and S-equol on the invasion of MDA-MB-231 human breast cancer cells and the effects of these compounds on MMP/TIMP expression at the mRNA level. METHODS: The anti-invasive effects of daidzein, R- and S-equol (0, 2.5, 10, 50 μM) on MDA-MB-231 cells were determined using the Matrigel invasion assay following 48-h exposure. Effects on MMP-2, MMP-9, TIMP-1 and TIMP-2 expression were assessed using real-time PCR. Chiral HPLC analysis was used to determine intracellular concentrations of R- and S-equol. RESULTS: The invasive capacity of MDA-MB-231 cells was significantly reduced (by approximately 50-60 %) following treatment with 50 μM daidzein, R- or S-equol. Anti-invasive effects were also observed with R-equol at 2.5 and 10 μM though overall equipotent effects were induced by all compounds. Inhibition of invasion induced by all three compounds at 50 μM was associated with the down-regulation of MMP-2, while none of the compounds tested significantly affected the expression levels of MMP-9, TIMP-1 or TIMP-2 at this concentration. Following exposure to media containing 50 μM R- or S-equol for 48-h intracellular concentrations of R- and S-equol were 4.38 ± 1.17 and 3.22 ± 0.47 nM, respectively. CONCLUSION: Daidzein, R- and S-equol inhibit the invasion of MDA-MB-231 human breast cancer cells in part via the down-regulation of MMP-2 expression, with equipotent effects observed for the parent isoflavone daidzein and the equol enantiomers.

The effects of flavonoid and other polyphenol consumption on cognitive performance: A systematic research review of human experimental and epidemiological studies

Literature reviews suggest flavonoids, a sub-class of polyphenols, are beneficial for cognition. This is the first review examining the effect of consumption of all polyphenol groups on cognitive function. Inclusion criteria were polyphenol vs. control interventions and epidemiological studies with an objective measure of cognitive function. Participants were healthy or mildly cognitively impaired adults. Studies were excluded if clinical assessment or diagnosis of Alzheimer’s disease, dementia, or cognitive impairment was the sole measure of cognitive function, or if the polyphenol was present with potentially confounding compounds such as caffeine (e.g. tea studies) or Ginkgo Biloba. 28 studies were identified; 4 berry juice studies, 4 cocoa studies, 13 isoflavone supplement studies, 3 other supplement studies, and 4 epidemiological surveys. Overall, 16 studies reported cognitive benefits following polyphenol consumption. Evidence suggests that consuming additional polyphenols in the diet can lead to cognitive benefits, however, the observed effects were small. Declarative memory and particularly spatial memory appear most sensitive to polyphenol consumption and effects may differ depending on polyphenol source. Polyphenol berry fruit juice consumption was most beneficial for immediate verbal memory, whereas isoflavone based interventions were associated with significant improvements for delayed spatial memory and executive function. Comparison between studies was hampered by methodological inconsistencies. Hence, there was no clear evidence for an association between cognitive outcomes and polyphenol dose response, duration of intervention, or population studied. In conclusion, however, the findings do imply that polyphenol consumption has potential to benefit cognition both acutely and chronically.

Effects of isoflavones on the coagulation and fibrinolytic system of postmenopausal women

Objective: We evaluated the effects of soy isoflavone supplementation on hemostasis in healthy postmenopausal women. Methods: In this double-blinded, placebo-controlled study, 47 postmenopausal women 47-66 y of age received 40 mg of soy isoflavone (n = 25) or 40 mg of casein placebo (n = 22) once a day for 6 mo. Levels of factors VII and X. fibrinogen, thrombin-antithrombin complex, prothrombin fragments I plus 2, antithrombin, protein C, total and free protein S, plasminogen, plasminogen activator inhibitor-1, and D-dimers were measured at baseline and 6 mo. Urinary isoflavone concentrations (genistein and daidzein) were measured as a marker of compliance and absorption using high-performance liquid chromatography. Baseline characteristics were compared by unpaired Student`s t test. Within-group changes and comparison between the isoflavone and casein placebo groups were determined by a mixed effects model. Results: The levels of hemostatic variables did not change significantly throughout the study in the isoflavone group; however, the isoflavone group showed a statistically significant reduction in plasma concentration of prothrombin fragments I plus 2; both groups showed a statistically significant reduction in antithrombin, protein C, and free protein S levels. A significant increase in D-dimers was observed only in the isoflavone group. Plasminogen activator inhibitor-l levels increased significantly in the placebo group. However, these changes were not statistically different between groups. Conclusion: The results of the present study do not support a biologically significant estrogenic effect of soy isoflavone on coagulation and fibrinolysis in postmenopausal women. However, further research will be necessary to definitively assess the safety and efficacy of isoflavone. (D 2008 Elsevier Inc. All rights reserved.

Lack of effects of isoflavones on the lipid profile of Brazilian postmenopausal women

Objective: To evaluate the effects of soy isoflavone supplementation on profile lipid and endogenous hormone levels. Methods: In this double-blind, placebo-controlled Study, 47 post menopausal women 47-66 v of age received 40 mg of isoflavone (n = 25) or 40 mg of casein placebo (11 = 22). Cardiovascular risk factors were assessed by evaluating lipid profile at baseline and after 6 mo of treatment. To examine the effects of this regime on endogenous hormone levels, follicle-stimulating hormone and beta-estradiol were measured. Urinary isoflavone concentrations (genistein and daidzein) were measured as markers of both compliance and absorption using high performance liquid chromatography. Baseline characteristics were compared by the unpaired Student`s t-test. Within-group changes were determined by paired Student`s t-test and comparison between the isoflavone and casein placebo groups were determined by analysis of variance. Results: Lipid levels (low-density lipoprotein and total cholesterol) similarly decreased in both,groups. High-density lipoprotein increased significantly in both groups and cannot thus be attributable to treatment: the reason for Such variation is unknown and can be attributed to chance or to bias (even that of a real placebo effect in both groups or perhaps in spontaneous changes in exercise and dietary habits of patients after their inclusion). Furthermore, in both groups very low-density lipoprotein and triacylglycerol levels increased in a non-significant manner. Conclusion: The results of the present Study do not support any biologically significant estrogenic effects of isoflavone on the parameters assessed. Further research will he necessary to definitively assess the safety and efficacy of isoflavone. (C) 2008 Elsevier Inc. All rights reserved.

Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: Implications to tetramer stability and ligand-binding

Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis. (C) 2010 Elsevier Inc. All rights reserved.