Catherine Bennetau-Pelissero

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.

Mass Spectrometry-based Metabolomics for the Discovery of Biomarkers of Fruit and Vegetable Intake: Citrus Fruit as a Case Study

Elucidation of the relationships between genotype, diet, and health requires accurate dietary assessment. In intervention and epidemiological studies, dietary assessment usually relies on questionnaires, which are susceptible to recall bias. An alternative approach is to quantify biomarkers of intake in biofluids, but few such markers have been validated so far. Here we describe the use of metabolomics for the discovery of nutritional biomarkers, using citrus fruits as a case study. Three study designs were compared. Urinary metabolomes were profiled for volunteers that had (a) consumed an acute dose of orange or grapefruit juice, (b) consumed orange juice regularly for one month, and (c) reported high or low consumption of citrus products for a large cohort study. Some signals were found to reflect citrus consumption in all three studies. Proline betaine and flavanone glucuronides were identified as known biomarkers, but various other biomarkers were revealed. Further, many signals that increased after citrus intake in the acute study were not sensitive enough to discriminate high and low citrus consumers in the cohort study. We propose that urine profiling of cohort subjects stratified by consumption is an effective strategy for discovery of sensitive biomarkers of consumption for a wide range of foods.

Dose-dependent bone-sparing effects of dietary isoflavones in the ovariectomised rat

The dose-dependent bone-sparing effects of dietary isoflavones (IF) were investigated in adult (7-month-old) Wistar rats. Forty animals were ovariectomised, allocated into four groups of ten rats each, and immediately treated orally with IF at 0 (OVX), 20 (IF20), 40 (IF40) or 80 (IF80) microg/g body weight per d for 91 d; ten sham-operated (SH) controls received the same diet without added IF. Animals were killed on day 91. Both femoral failure load and total femoral, diaphyseal or metaphyseal bone mineral densities (BMD) were lower in OVX animals than in SH animals. Urinary deoxypyridinoline (DPD) excretion, a marker of bone resorption, and plasma osteocalcin (OC) levels, a marker of osteoblast activity, were higher in OVX animals than in SH animals. Total femoral and diaphyseal BMD and femoral failure load were similar in IF-treated rats and SH rats. Although metaphyseal BMD in IF40 or IF80 rats was similar to that in SH rats, its value was lower in IF20 rats than in controls. The day 91 urinary DPD excretion in IF40 and IF80 rats, but not in IF20 rats, was similar to that in SH rats. Day 91 plasma OC concentrations in IF-treated rats were similar to day 45 values, but were decreased in OVX and SH rats. Thus, daily IF consumption prevented ovariectomy-induced bone loss, both by depressing bone resorption and stimulating osteoblast activity. Moreover, as only the highest IF level induced a weak uterotrophic activity, the optimal IF dose which preserves both cancellous and cortical bone, but exhibits no oestrogen-like effects on the uterus, was 40 microg/g body weight per d.

Naringin, the major grapefruit flavonoid, specifically affects atherosclerosis development in diet-induced hypercholesterolemia in mice

Naringin (NAR) from grapefruit has exhibited potential protective effects against atherosclerosis development. However, specific mechanisms responsible for such effects are poorly understood. Thus, we aimed to investigate the antiatherogenic effects of NAR in different mouse models of hypercholesterolemia and decipher its molecular targets in the aorta using transcriptomic approach. Two mouse models of hypercholesterolemia, wild-type mice fed a high-fat/high-cholesterol diet and apolipoprotein E-deficient mice fed a semisynthetic diet, were studied. Mice were fed a respective control diets supplemented or not for 18 weeks with 0.02% of NAR, that is, nutritional supplementation. NAR supplementation reduced plaque progression only in wild-type mice fed the high-fat/high-cholesterol diet (-41%). Consistent with this protective effect, NAR reduced plasma non-high-density lipoprotein cholesterol concentrations as well as biomarkers of endothelial dysfunction. Microarray studies performed on aortas demonstrated differentially expressed genes encoding proteins involved in cell adhesion, actin cytoskeleton organization and cell division. Thus, the changes in gene expression induced by NAR could suggest a limited atherosclerosis progression by preventing immune cell adhesion and infiltration in the intima of vascular wall, as well as smooth muscle cell proliferation. Furthermore, this hypothesis was strengthened by in vitro experiments, which showed the ability of naringenin to reduce monocyte adhesion to endothelial cells and smooth muscle cell proliferation. In conclusion, this study revealed the antiatherogenic effect of NAR supplemented at a nutritionally achievable dose, specifically toward diet-induced atherosclerosis, and depicted its multitarget mode of action at the vascular level.

Binding affinities of hepatic nuclear estrogen receptors for phytoestrogens in rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baeri)

Phytoestrogens are dietary estrogenic contaminants capable of inducing vitellogenin synthesis in rainbow trout and Siberian sturgeon. A competitive-binding assay on their hepatic estrogen receptors (ER) was performed to determine the relative affinity of phytoestrogens compared to estradiol (E(2)). Phytoestrogen concentrations used were 1000 times higher than for E(2), except for genistein and formononetin. For each compound, the competition with 50%-bound labelled E(2) (DC(50)) was considered in order to classify phytoestrogens according to their affinity for ER. The affinities are compared for each species. In rainbow trout, estradiol (DC(50): 7 nM)>formononetin (DC(50): 260 nM)>genistein (DC(50): 570 nM)>equol (DC(50): 5.3 microM)>daidzein (DC(50): 9 microM)>biochanin A (DC(50): 100 microM). In sturgeon, estradiol (DC(50): 5 nM)>genistein (DC(50): 220)>formononetin (DC(50): 1 microM)>equol>(DC(50): 8.3 microM)>daidzein>(DC(50): 80 microM)>biochanin A (DC(50): 100 microM). These results demonstrate that phytoestrogens, mimicking estradiol, can disturb the endocrine system by competing for ER. Also, the higher sensitivity to genistein observed in vivo in Siberian sturgeon (vitellogenin synthesis), compared to rainbow trout, is not due to a higher affinity of genistein for the hepatic ER. Thus, the metabolism of phytoestrogen could be species dependent and affect sensitivity.

Enterodiol and enterolactone, two major diet-derived polyphenol metabolites have different impact on ERα 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 womens health.

ELISA as a new method to measure genistein and daidzein in food and human fluids

Widely distributed in the plant kingdom, phytoestrogens, like soy isoflavones are found in plant protein extracts at varying levels depending on culture conditions and cultivars. Their increasingly used in human, as natural estrogens and their varying levels in raw materials raise the question concerning the measurement of isoflavones both in food or food supplements as well as in human fluid. To validate our new ELISA technique, isoflavones measurements in food-supplements, in soy food and in human fluid from volunteers participating to a kinetic study or a survey, were done. Our method was also compared with other physico-chemical techniques in an inter-laboratory assay (23 laboratories). In conclusion, our new ELISA technique is reliable, sensitive, cheap, rapid and can be used either for a great number of human fluid analysis, for crude matter or food analysis as long as the appropriate extraction technique is performed prior to ELISA procedure. # 2003 Elsevier Ltd. All rights reserved.

Occurrence and in vitro biosynthesis of 11-ketotestosterone in Siberian sturgeon, Acipenser baeri Brandt maturing females.

High levels of 11-ketotestosterone (11KT) were found (49 to 160 ng ml−1) in plasma of Siberian sturgeon females during the end of their reproductive cycle. These levels were measured either by specific radioimmunoassay, or both by specific radioimmunoassay and by UV absorption after HPLC (isocratic conditions, 33% methanol, 26% acetonitrile, 41% water). In order to find the origin of 11KT synthesis, ovaries were incubated (30 min and 2h at 20°C) with tritiated 17-hydroxyprogesterone (17OHP) or with tritiated androstenedione (A4). Testosterone (conversion rate from tritiated 17OHP: 4%) and 11-ketotestosterone (conversion rate from tritiated A4: 1.6%) were identified as metabolites of respectively 17OHP and A4 (TLC, HPLC and crystallization). 11β-hydroxyandrostenedione (11βOHA4) and 11β-hydroxytestosterone (11βOHT) were suggested to be intermediate metabolites. Besides interrenal and blood cells were incubated respectively with tritiated cortisol and tritiated A4. 11βOHA4 was identified in interrenal incubation (yield from tritiated cortisol: 1.2%). 11KT in interrenal (yield from tritiated cortisol: 0.14%), and 11βOHA4 and 11KT in blood cells (yield from tritiated A4: 1.6%), were suspected to be synthesized (TLC, HPLC, acetylation). No significant metabolization of tritiated cortisol could be found in liver. The possible contribution of each of these tissues to high 11KT levels found in plasma is discussed.

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.

Respective contribution exerted by AF-1 and AF-2 transactivation functions in estrogen receptor α induced transcriptional activity by isoflavones and equol: Consequence on breast cancer cell proliferation

Estrogens used in hormone replacement therapy regimens may increase the risk of developing breast cancer. Paradoxically, high consumption of plant-derived phytoestrogens, particularly soybean isoflavones, is associated with a low incidence of breast cancer. To explore the molecular basis for these potentially different experimental/clinical outcomes, we investigated whether soybean isoflavones elicit distinct transcriptional actions from estrogens by performing transient transfections in different cell lines. Our results demonstrate that 17beta estradiol (E2), isoflavones, and equol (EQ) effectively trigger the transcriptional activation with both estrogen receptors (ER), ER alpha and ER beta. ER alpha transcriptional activity is mediated through two transactivation domains AF-1 and AF-2, whose activity is tightly regulated in a cell-type and promoter-specific manner. Isoflavones, genistein, and daidzein (DAI), and EQ, the main estrogenic metabolite of DAI, are ER alpha agonists for transcriptional activation. The molecular mechanisms for ER alpha-induced transcriptional activity by isoflavones and EQ involve their capacity to act mainly through AF-1 regardless of the cell type. Therefore, our data indicate that estrogenic ligands, such as isoflavones and EQ, exert their effects on ER alpha transactivation similarly to the endogenous ligand E2, and suggest that the risk of estrogen-related diseases might not be reduced by soy-rich food and/or isoflavone- or EQ-based supplements.