Validation of ELISAs for Isoflavones and Enterolactone for Phytoestrogen Intake Assessment in the French Population.

Phytoestrogens are dietary compounds with low estrogenic activity. The two main categories in the French diet are isoflavones from pulses and enterolignans metabolized by the gut flora from various lignans found in fruits, vegetables, grains, and beverages. Isoflavones and lignans have different effects on human physiology and can antagonize each other. Comprehensive lists of phytoestrogen sources were constructed based on measurements and literature data. The 24 h and 48 h dietary recalls were proposed to the volunteers of the ISOLED cohort (NCT03421184). Urine and plasma samples from these volunteers were assayed for genistein, daidzein, equol, and enterolactone. A dietary score was constructed considering the pharmacokinetic characteristics of these compounds. Correlation analyses were applied to fluid concentrations associated with dietary scores. Pearson correlations reached 0.921 (p < 0.001) for urineIF, 0.900 (p < 0.001) for plasmaIF, 0.764 (p < 0.001) for urineENL, and 0.723 (p < 0.001) for plasmaENL. ELISAs associated with careful intake assessments proved to be good tools for phytoestrogens' exposure estimation.

Phytoestrogens and Health Effects.

Phytoestrogens are literally estrogenic substances of plant origin. Although these substances are useful for plants in many aspects, their estrogenic properties are essentially relevant to their predators. As such, phytoestrogens can be considered to be substances potentially dedicated to plant-predator interaction. Therefore, it is not surprising to note that the word phytoestrogen comes from the early discovery of estrogenic effects in grazing animals and humans. Here, several compounds whose activities have been discovered at nutritional concentrations in animals and humans are examined. The substances analyzed belong to several chemical families, i.e., the flavanones, the coumestans, the resorcylic acid lactones, the isoflavones, and the enterolignans. Following their definition and the evocation of their role in plants, their metabolic transformations and bioavailabilities are discussed. A point is then made regarding their health effects, which can either be beneficial or adverse depending on the subject studied, the sex, the age, and the physiological status. Toxicological information is given based on official data. The effects are first presented in humans. Animal models are evoked when no data are available in humans. The effects are presented with a constant reference to doses and plausible exposure.

Endocrine disruptors on and in fruits and vegetables: Estimation of the potential exposure of the French population.

Fruits and vegetables are considered to be healthy compared to fats, carbohydrates, and meats. However, their production involves plant protection products (PPPs) or they can contain phytoestrogens which may exhibit endocrine effects. Thus, the exposure to the main PPPs and to phytoestrogens known as endocrine disruptors (EDs) is estimated. PPPs include fungicides, growth substances, herbicides, and insecticides authorised in France. ED-PPPs exposure is estimated from the maximum residue limits (MRLs) of 70 potential ED-PPPs used in France on 64 fruits and vegetables. The estimated exposure to potential ED-PPPs is 509 µg/d and involves agonist and antagonist substances in complex mixtures. Anti-androgens are preeminent, at 353 µg/d. Exposure to genistein and daidzein is calculated from 140 measurements in 9 categories of food-items containing soy. The global exposure to isoflavones in France is evaluated at 6700 µg/d. Phytoestrogen exposure is much higher than that of ED-PPPs. Their endocrine effects should be considered.

New Evaluation of Isoflavone Exposure in the French Population.

The study relates the present evaluation of exposure to estrogenic isoflavones of French consumers through two approaches: (1) identification of the isoflavone sources in the French food offering, (2) a consumption-survey on premenopausal women. For the foodstuff approach 150 food-items were analysed for genistein and daidzein. Additionally, 12,707 labels of processed-foods from French supermarket websites and a restaurant-supplier website were screened, and 1616 foodstuffs of interest were retained. The sources of phytoestrogens considered were soy, pea, broad bean and lupine. A price analysis was performed. A total of 270 premenopausal women from the French metropolitan territory were interviewed for their global diet habits and soy consumption and perception. In supermarkets, there were significantly less selected foodstuffs containing soy than in restaurant (11.76% vs. 25.71%, p < 0.01). There was significantly more soy in low price-foodstuff in supermarket (p < 0.01). Isoflavone levels ranged from 81 to 123,871 µg per portion of the analyzed soy containing foodstuff. Among the women inquired 46.3% claimed to have soy regularly. Isoflavone intake >45 mg/day is associated to vegan-diet (p < 0.01). In total, 11.9% of soy-consumers had a calculated isoflavone intake >50 mg/day. This dose can lengthen the menstrual cycles. The actual exposure to phytoestrogen is likely to have an effect in a part of the French population.

Use of dietary supplements containing soy isoflavones and breast cancer risk among women aged >50 y: a prospective study.

BACKGROUND: Soy-based dietary supplements have been promoted as natural alternatives to menopausal hormone therapy, but their potential effect on breast cancer development is controversial. OBJECTIVES: We examined the relation between the consumption of soy supplements and the risk of breast cancer, overall and by tumor hormone receptor status, among women aged >50 y. METHODS: In total, 76,442 women from the Etude Epidemiologique aupres de Femmes de la Mutuelle Generale de l'Education Nationale (E3N) cohort, born between 1925 and 1950, were followed from 2000 to 2011 (11.2 y on average, starting at a mean age of 59.5 y; 3608 incident breast cancers), with soy supplement use assessed every 2-3 y. HRs of breast cancer were estimated with the use of multivariable Cox models. RESULTS: Compared with never using soy supplements, the HRs associated with current use of soy supplements were 0.92 (95% CI: 0.76, 1.11) for all, 0.78 (95% CI: 0.60, 0.99) for estrogen receptor (ER)-positive, and 2.01 (95% CI: 1.41, 2.86) for ER-negative breast cancers. There was no association between past use of soy supplements and breast cancer. HRs for current use were 1.36 (95% CI: 0.95, 1.93) and 0.82 (95% CI: 0.65, 1.02) among women with and without a family history of breast cancer, respectively (P-interaction = 0.03) and 1.06 (95% CI: 0.87, 1.30) ≥5 y after menopause compared with 0.50 (95% CI: 0.31, 0.81) in premenopause or ≤5 y postmenopause (P-interaction = 0.04). CONCLUSIONS: In this cohort of women aged >50 y, we report opposing associations of soy supplements with ER-positive and ER-negative breast cancer risk. Our results also caution against the use of these supplements in women with a family history of breast cancer. Whether the risk profile of soy supplements could be more favorable among premenopausal or recently postmenopausal women deserves further investigation.

Risks and benefits of phytoestrogens: where are we now?

PURPOSE OF REVIEW: The estrogenic effects of genistein, as reconfirmed by the American National Toxicology Program (USA-NTP), have led to several new clinical studies being undertaken. Here, we highlight the most relevant recent data, reporting either beneficial or adverse effects. RECENT FINDINGS: Phytoestrogens are natural molecules from edible plants exhibiting estrogenic activities. Post-USA-NTP studies investigated both human and animal reproductive and other physiological issues. These studies showed that estrogens can be either deleterious for reproduction and estrogen-dependent diseases, or beneficial for those with steroid deficiencies, that is more than 50. The specific outcome depends on exposure level and on the estrogenic status of the patients exposed. Recently, it was reported that, with the industrialization of soybean process, phytoestrogen exposure dramatically increased in both humans and cattle, whereas traditional Asian soy-food-processing empirically removed isoflavones. Phytoestrogen exposure has also become more widespread with the progressive internationalization of soybean use in human and cattle food. SUMMARY: Phytoestrogens should be considered as modern endocrine disruptors and studied as such.

Bioavailability of glycitein relatively to other soy isoflavones in healthy young Caucasian men.

Glycitein is a Selective Estradiol Receptor Modulator (SERM) from soy. The study reports plasma bioavailability and urine excretion of glycitein compared to other soy isoflavones after a unique intake of food supplement based on soy germ containing 55.24mg isoflavones. Eighteen plasma and urinary sampling profiles collected over 48h from healthy young Caucasian men were analysed using specific ELISAs. Eight profiles contained equol. Glycitein T(max), C(max), AUC(0→24h) and T(½) in plasma were calculated. Urine T(max), % of excretion at 24h and clearance were assessed. Glycitein is one of the best absorbed flavonoids. Plasma steady-state level can be achieved by several intakes a day. Glycitein bioavailability is similar to that of daidzein and its urinary excretion is significantly higher than that of genistein. Equol does not affect glycitein bioavailability. Knowing glycitein bioavailability in man is essential for the development of soy-germ-based food supplements for health applications.

Comparative effects of R- and S-equol and implication of transactivation functions (AF-1 and AF-2) in estrogen receptor-induced transcriptional activity.

Equol, one of the main metabolites of daidzein, is a chiral compound with pleiotropic effects on cellular signaling. This property may induce activation/inhibition of the estrogen receptors (ER) a or b, and therefore, explain the beneficial/deleterious effects of equol on estrogen-dependent diseases. With its asymmetric centre at position C-3, equol can exist in two enantiomeric forms (R- and S-equol). To elucidate the yet unclear mechanisms of ER activation/inhibition by equol, we performed a comprehensive analysis of ERa and ERb transactivation by racemic equol, as well as by enantiomerically pure forms. Racemic equol was prepared by catalytic hydrogenation from daidzein and separated into enantiomers by chiral HPLC. The configuration assignment was performed by optical rotatory power measurements. The ER-induced transactivation by R- and S-equol (0.1-10 µM) and 17b-estradiol (E2, 10 nM) was studied using transient transfections of ERα and ERß in CHO, HepG2 and HeLa cell lines. R- and S-equol induce ER transactivation in an opposite fashion according to the cellular context. R-equol and S-equol are more potent in inducing ERα in an AF-2 and AF-1 permissive cell line, respectively. Involvement of ERα transactivation functions (AF-1 and AF-2) in these effects has been examined. Both AF-1 and AF-2 are involved in racemic equol, R-equol and S-equol induced ERα transcriptional activity. These results could be of interest to find a specific ligand modulating ER transactivation and could contribute to explaining the diversity of equol actions in vivo.

Synthesis of haptens and conjugates for ELISA of glycitein: development and validation of an immunological test.

Two carboxylic acid haptens of glycitein were synthesized, with a spacer arm at the C2 position. They differed in the length of the spacer arm, with the length of the spacer arms being three or four carbon atoms, and were named Delta3-glycitein and Delta4-glycitein haptens, respectively. The different haptens were coupled to bovine serum albumin (BSA), and the coupling efficiency was assessed by MALDI mass spectrometry. Polyclonal antibodies were generated against the BSA conjugates. An additional conjugate of Delta4-glycitein hapten was generated with swine thyroglobulin (Thyr). Enzyme-linked immunosorbent assays (ELISAs) based on the competition between free glycitein and Delta4-glycitein-Thyr conjugates for specific antibodies were developed. The IC50 of the standard curves was 15.6 ng mL(-1) with anti-Delta3-glycitein and 62.5 ng mL(-1) with anti-Delta4-glycitein, that is, 10.9 and 44 pmol/well, respectively. With the Delta3-glycitein antibody, interassay and intra-assay variations were 12.2 and 11.5%, respectively. Specificity tests did not show any significant cross-reaction with any other soy isoflavone. This specificity is not influenced by the length of the spacer arm. The assay was validated by measurements performed on plasma samples as well as on soy-based foodstuffs and on soy-based food supplements.

Enterodiol and enterolactone, two major diet-derived polyphenol metabolites have different impact on ERalpha transcriptional activation in human breast cancer cells.

Lignans are plant compounds metabolized in the mammalian gut to produce the estrogenic enterolignans, enterodiol (ED) and enterolactone (EL). Because estrogens have been linked to breast cancer etiology, enterolignans could affect breast cancer risk, but to our knowledge, the mechanisms by which they exert their estrogenic and/or anti-estrogenic effects in humans are still unclear. To better understand how estrogenic compounds from the food, such as the enterolignans, might influence breast cancer progression and their mechanisms to interfere with human estrogen receptor (ER) signalling in hormone-dependant diseases, we examined and compared the ability of ED, EL and 17beta-estradiol (E2) to induce the transactivation of ERalpha and ERbeta, to modulate ERalpha target genes, to exert either growth stimulatory or anti-proliferative effects and finally to modulate MCF-7 cell migration by acting on matrix metalloproteases (MMP)-2 and -9, at concentrations that are achievable through a lignan-rich diet. This study indicates that enterolignans show distinct properties for transactivation of ERalpha and ERbeta. ED, as E2, induces ERalpha transcriptional activation through transactivation functions AF-1 and AF-2, while EL is less efficient in inducing AF-1, acting predominantly through AF-2. Furthermore, ED and EL modulate ERalpha mRNA and protein contents as well as MCF-7 cell proliferation and secreted MMP activities in a different way. Enterolignans are compounds of wide interest nowadays and our results help to unveil their mechanisms of action on ER, emphasizing the fact that the dietary load in lignans could be of importance in the balance between being risk or chemopreventive factors for breast cancer and women's health.

Higher bioavailability of isoflavones after a single ingestion of a soya-based supplement than a soya-based food in young healthy males.

Soya isoflavones, genistein and daidzein, are the focus of numerous studies investigating their potential effects on health and results remain controversial. Bioavailability is clearly a crucial factor influencing their bioefficacy and could explain these discrepancies. This study aimed at assessing: (1) the isoflavone content of sixty-nine European soya-derivative products sold on the French market; (2) the bioavailability of isoflavones comparing supplement with food. Twelve healthy volunteers were recruited in a randomized two-way crossover trial and received 35 mg isoflavones equivalent aglycone either through supplements or through cheese, both containing different patterns of isoflavone conjugates and different daidzein:genistein ratios. A specific ELISA method was used to assess the plasma and urinary concentrations of isoflavones and thus the pharmacokinetic parameters, which were then normalized to mg of each isoflavone ingested. Results showed that the normalized Cmax of daidzein (P = 0.002) and similarly the normalized AUC0 --> infinity and Cmax of genistein (P = 0.002) from soya-based capsules were higher than that from soya-based cheese. In conclusion, this work completes studies on isoflavone bioavailability and presents new data regarding isoflavone concentrations in soya-derivative products. Assuming that isoflavone conjugation patterns do not influence isoflavone bioavailability, this study shows that isoflavones contained in capsules are more bioavailable than those contained in soya-based cheese. Although the supplement is more bioavailable, the relative importance of this is difficult to interpret as there is little evidence that supplements are biologically active in human subjects to date and further studies will be necessary for this specific supplement to prove its efficacy.

Isoflavonoid-based bone-sparing treatments exert a low activity on reproductive organs and on hepatic metabolism of estradiol in ovariectomized rats.

The use of soy isoflavones is a potential alternative to hormone replacement therapy in post-menopausal bone-loss prevention. Nevertheless, phytoestrogens can target other organs and may disrupt cell proliferation, or could modify endogenous steroid hormone metabolism. These mechanisms could be linked to an increased risk of developing cancer. We therefore studied the possible side effects of such treatments in an experimental model of menopause. Forty adult female Wistar rats were ovariectomized and fed with a genistein-, daidzein- or equol-supplemented diet at bone-sparing levels (10 mg/kg BW/day) for 3 months. The estrogenic effects were assessed by histological and molecular analyses on reproductive organs. The impact on the oxidative metabolism of estradiol and on associated cytochrome P450 (CYP) activities was evaluated in liver microsomes. The relative wet weights of both the uterus and the vagina were increased in the equol group, but no significant changes in proliferating cell nuclear antigen or hormone receptor mRNA expression were noticed. In contrast, genistein and daidzein did not induce uterotrophy but caused an overexpression of estrogen receptor alpha mRNA which could correspond to a long-lasting effect of physiological concentrations of estrogens. The hepatic metabolism of estradiol was influenced by daidzein which increased the synthesis of putative mutagenic derivatives. At the same time, genistein favored estrogen 2-hydroxylation, and equol decreased 4-hydroxyestrogen production. Surprisingly, no significant alteration in hepatic CYP activities was detected. Taken together, these results demonstrate that isoflavonoid-based bone-sparing treatments are able to cause side effects on other estrogen-sensitive target organs when given in the long-term.

Bioavailability and urinary excretion of isoflavones in humans: effects of soy-based supplements formulation and equol production.

Soy isoflavones (IF) are of particular interest for their possible estrogenic effects on the symptoms of menopause. The bioavailability of IF is clearly a factor influencing their biological activity. The first aim of this study was to elucidate the impact of the matrix process and especially the formulation of soy-based capsules on IF bioavailability. Twelve healthy volunteers were recruited for a randomized, double-blind, two-way crossover trial and received a single dose of the two soy-based formulations, one containing a pure soy standardized extract of IF, and the other containing soy flour in addition to the standardized extract of IF. Using a new and validated ELISA method, we measured the plasma and urinary concentrations of genistein, daidzein and its metabolite equol. Based on European Medicine Evaluation Agency recommendations, the main pharmacokinetic parameters allowed us to demonstrate the bioequivalence of the two formulations, indicating that the presence or absence of soy flour did not alter either the absorption or the elimination of daidzein and genistein. As bioequivalence was demonstrated, we pooled data collected during the two study-periods to address another original issue: Did the ability to produce equol affect the bioavailability of daidzein? We demonstrated that daidzein excretion was significantly lower in equol producers compared with equol non producers over the entire elimination period of the soy IF. This difference disappeared when equol excretion was added to daidzein excretion in equol producers. Our results indicated that the production of equol could partly explain the difference in daidzein bioavailability after IF ingestion.

Isoflavones and functional foods alter the dominant intestinal microbiota in postmenopausal women.

Dietary phytoestrogens, such as isoflavones, are used as food additives to prevent menopause-related disorders. In addition to other factors, their bioavailability strongly depends on the activity of intestinal bacteria but the underlying interactions remain poorly understood. A randomized, double-blind, placebo-controlled study was undertaken with 39 postmenopausal women to characterize changes in the dominant microbial communities of the intestinal tract after 2 mo of isoflavone supplementation with and without pro- or prebiotic. The diversity and composition of the dominant microbiota were analyzed by temporal temperature-gradient gel electrophoresis (TTGE) and fluorescent in situ hybridization. Isoflavones alone stimulated dominant microorganisms of the Clostridium coccoides-Eubacterium rectale cluster, Lactobacillus-Enterococcus group, Faecalibacterium prausnitzii subgroup, and Bifidobacterium genus. The stimulation of the Clostridium coccoides-Eubacterium rectale cluster depended on the women's equol excretion and was transient, with the exception of a prolonged bifidogenic effect. Lasting changes in the diversity of the dominant species were also observed. The probiotic strain supplied could be detected by TTGE during its passage through the intestinal tract, and ingestion of fructooligosaccharides triggered a marked and specific bifidogenic effect. In conclusion, this is the first human study that shows changes in the diversity and composition of dominant bacterial communities in response to dietary supplementation with hormone-related compounds combined with functional foods.

Risks and safety of polyphenol consumption.

This article gives an overview of the potential hazards of polyphenol consumption, as reported during the round-table discussion at the 1st International Conference on Polyphenols and Health, held in Vichy, France, November 2003. Adverse effects of polyphenols have been evaluated primarily in experimental studies. It is known, for example, that certain polyphenols may have carcinogenic/genotoxic effects or may interfere with thyroid hormone biosynthesis. Isoflavones are of particular interest because of their estrogenic activity, for which beneficial as well as detrimental effects have been observed. Furthermore, consumption of polyphenols inhibits nonheme iron absorption and may lead to iron depletion in populations with marginal iron stores. Finally, polyphenols may interact with certain pharmaceutical agents and enhance their biologic effects. It is important to consider the doses at which these effects occur, in relation to the concentrations that naturally occur in the human body. Future studies evaluating either beneficial or adverse effects should therefore include relevant forms and doses of polyphenols and, before the development of fortified foods or supplements with pharmacologic doses, safety assessments of the applied doses should be performed.