Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Oral Communication Abstract - S2g

BIOLOGICAL EFFECTS OF PHYTOESTROGENS

BRANCA F., LORENZETTI S.

Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione – INRAN, Via Ardeatina 546, 00178 Roma

f.branca@agora.it ; lorenzetti_s@hotmail.com

Phyto-oestrogens are naturally occurring plant-derived phyto-chemicals. Although composed by a wide group of nonsteroidal compounds of diverse structure, phyto-oestrogens have been shown to bind estrogen receptors and to behave as weak agonist/antagonist in both animals and humans.

Among several classes of phyto-oestrogens, lignans (found in fruits, cereals and flaxseeds) and isoflavones (mainly found in legumes) are the most interesting as potentially beneficial in alternative therapies in a broad range of hormone-dependent conditions. Indeed, epidemiological studies showing an inverse association between vegetables and fruits consumption and the risk of human cancers at many tissues pointed primarily attention to soybeans. For instance, the incidence of hormone-related cancers such as breast and prostate cancers vary as much as 20-fold between Asian and Western populations.

Isoflavones are the best studied phyto-oestrogens. It has been estimated that soy foods contain 0.1-3.0 mg isoflavones per gram of dry weight. The minimal requirement of isoflavones suggested to have a physiological effect is about 45 mg/day. Indeed, such a value is reached only in typical Asian diets, with a peak at about 100 mg/day in the Hong-Kong Chinese and in Japanese populations. In the Western countries, the isoflavone intake is much lower: the most recent evaluations in USA, as well as in different European countries, indicate in less of 1 mg/day the mean intake of isoflavones. Even in the EU soy food consumers, the mean isoflavones intake does not reach more than 6-10 mg/day.

The most recent reports on the pharmacokinetics of pure isoflavones are suggesting that for 50 mg of isoflavone intake, the expected plasma concentrations will be about 1 mM, whereas the tissue absorbable form, the unconjugated one, will correspond to about 1-2 %. Even considering interindividual variations, it is reasonable to consider that plasma concentrations of unconjugated isoflavones will not reach more than 50 nM, a value that has to be taken into account in the cell culture models set up to unravel isoflavones activities and their mechanisms of action.

Recently, a consensus panel - established to set a proper isoflavones food fortification level - after reviewing human studies in the area of menopause, osteoporosis, cardiovascular diseases, and cancer, reached the conclusion that for health benefit the recommended isoflavone intakes should fall between 60 to 100 mg aglycones/person/day, with the lower dose considered as “reasonable and responsible”.

In particular, for improvement in bone mineral density (BMD), 60-100 mg aglycones/day for a period of at least 6-12 months should determine a significant advantage as suggested by a study on postmenopausal women in which a consumption of 90 mg IF/day, given with soy protein, determined an increase in vertebral bone density. It is also suggested that the contemporary addition of soy protein might ameliorate all parameters of calcium metabolism including absorption and urinary calcium excretion.

For the relief of menopausal symptoms a consumption of 60 mg aglycones/day has been suggested since some reduction was already seen at about 50 mg aglycones/day.

Related to cancer prevention a consumption between 50 to 110 mg aglycones/day is considered beneficial to reduce risks of breast, colon and prostate tumours in humans.

Finally, to decrease serum LDL-cholesterol a minimal intake of about 40-60 mg aglycones/day, depending on prior cholesterol status, is suggested to be used together with about 25 grams of soy protein that is absolutely necessary at the level of LDL metabolism.

Metabolism of isoflavones and lignans is complex and requires both mammalian and gut microbial processes. In plants and, consequently, in soy food products, isoflavones occur in three main biologically active forms (genistein, daidzein and glycitein), present either as aglycones or as sugar-conjugated glycosides (genistin, daidzin, and glycitin), the latter ones being the predominant naturally occurring forms. Although in humans flavonoids were showed to be absorbed in the naturally occurring glycosidic forms, isoflavones are not. Their bioavailability seems to require an initial hydrolysis of the sugar moiety by intestinal B-glucosidases to allow the following uptake by entyerocytes and the flow through the peripheral circulation. Following absorption, isoflavones are then reconjugated mainly to glucuronic acid and to a lesser degree to sulphuric acid. The demonstration that the rate of conjugation is high comes from the low levels of free aglycone detected in blood. Further, gut bacteria operate an extensive metabolism of isoflavones (to equol and O-desmethyl-angolensin/O-DMA) and lignans (to enterodiol and enterolactone). In humans, a large individual variation of isoflavones and lignans is recognized. Factors affecting absorption and metabolism include diet and gut microflora and the main affected step is the production of the metabolite equol (from daidzein) by the gut bacteria. Beside their absorption, the bioavailability of phytoestrogens depends on post-absorptive processes such as distribution, metabolism and excretion. Overall, human studies showed that: a) the absorption of aglycones depends on the intestinal hydrolysis of the glucosides, b) the peak plasma level is reached 6-8 hours after consumption, c) the urinary excretion is rapid and almost complete within 24-48 hours.

In Asian countries, isoflavones consuming has a long history of safe use as a natural component of human diet. It has been estimated that approximatively 10% of Asian populations consume routinely about 100 mg isoflavones/day. Further, in soy-based formula for infants, the maximum intake of isoflavones has been reported as 32-47 mg/day.

In humans, many clinical studies were performed to assess soy isoflavones effect on chronic disease risk factors. Anyway, the safety profile of isoflavones is difficult to be drawn due to limited sample sizes and short periods of investigation. At the moment, anyway, we might assume as upper tested limits: a) an ingestion of up to 9 mg of total isoflavones (3 mg/kg body weight) as consumed by some human infants without significant clinical effects, and b) up to 16 mg/kg body weight as used in human adults with little significant toxicity observed in any subject at any dose. Notably, genistein and daidzein were demonstrated as not mutagenic in the Salmonella/mammalian microsome reverse mutation assay (Ames test).

Suggested mechanism(s) of action of phyto-oestrogens and reports on the most reliable clinical trials will be presented with a particular focus on the analysis of the diseases known or suspected to be modulated by estrogen-like compounds.

Finally, since the ability of dietary phyto-oestrogens to substitute for endogenous estrogens to modulate bone resorption in humans is not well documented, we are presently making use of an ex vivo model of osteoclastogenesis. We cultured human peripheral blood mononuclear cells (PBMC) in vitro with soluble RANKL and M-CSF. Since putative osteoclast precursors express strongly the CD14 marker, we positively selected CD14(+) cells from PBMC. Now, we are going to use this human primary monocyte-derived osteoclasts model to: a) investigate the gene expression profile of several molecular markers of osteoclast differentiation during exposure to genistein compared to 17b-estradiol; and b) evaluate the ability to resorb bone of terminally differentiated osteoclast treated with several different photo-oestrogens.

Up to now, our preliminary results shown that, when cells are treated with 10 nM 17b-estradiol or with the mice-oestrogen zearalenone, a mitogenic effect at the level of the osteoclast precursors is present but not with the same concentration of the isoflavone genistein. Notwithstanding this, we observed that both genistein and zearalenone as well as estradiol are able to inhibit the secretion of matrix metalloproteinases, proteins responsible of bone resorption and known markers of osteoclast differentiation.

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