Phytoestrogen Research in the European Union

Ian Rowland, Northern Ireland Centre for Food and Health University of Ulster, Coleraine, Northern Ireland

INTRODUCTION

The European Commission’s Fifth Framework programme, entitled ‘Quality of Life and Management of Living Resources’ running from 1998­2002 encompassed 6 ‘Key Actions’ the first of which was Food Nutrition and Health. Important objectives under this area are to understand the role of nutrition in promoting and sustaining health and preventing disease and to facilitate the development of health promoting products and diets. As part of that Key Action, a number of projects on the health effects of phytoestrogens have been commissioned. These comprise three shared cost projects (Phytos, Isoheart and Phytoprevent) covering the role of phytoestrogens in osteoporosis, heart disease and cancer respectively, and two concerted actions (Venus and Phytohealth) covering various health-related aspects of phytoestrogens. Most of these projects have been underway for two years or less, so the following account will concentrate on their main aims, approaches and expected results.

PHYTOS

The objective of this project is to study the effects of phytoestrogen-rich diets on bone turnover in post­menopausal women. This project is co­ordinated by The Instituto Nazionale della Nutrizione, Italy, (Dr Francesco Branca), with partner institutions in six countries of the EU. It will run from March 2001 to February 2005.

The incidence of osteoporosis and bone fractures is dramatically increasing in Western countries, especially among postmenopausal women (Cooper & Melton, 1996). The drastic reduction in circulating oestrogens at menopause initiates a phase of accelerated bone loss, which is maximal in the first five years of menopause (Riggs & Melton 1986; Nguyen et al 1995). Although estrogen replacement therapies are the first choice to treat hormone-related osteoporosis, less than 25% of women opt for this treatment, because of fear of side effects and safety, and subsequently more than 60% those withdraw (Colditz et al 1995; Beresford et al 1997). Natural compounds with low estrogenic activity are found in foods of plant origin (mainly soy) and potentially could help prevent the loss of bone density during menopause (Valtuena et al 2003).

The overall aims of the project are to test the efficacy of phytoestrogen enriched foods in reducing hormone-dependent bone loss in menopause and to assess safety of phytoestrogen consumption in healthy post­menopausal women.

The work plan of the project has three main tasks:

The first task of the project is the production and evaluation of isoflavone (IF) rich foods products (biscuits and cereal bars) suitable for EU consumers. There will chemical and organoleptic evaluation of the various products and the effect of processing and storage on IF level in the foods will be assessed. Finally, each food will be tested in an IF bioavailability study and the one yielding the highest plasma IF will be used in the second part of the project.

The second task is to evaluate the effects of IF foods on bone mineral density and bone metabolism.

A randomized, placebo controlled trial is planned in which post­menopausal women (100 each in Italy, France and The Netherlands) will be assigned to PE-rich or PE-free foods for 6 months. The end points to be determined are: bone mineral density at various sites in the skeleton and markers of bone turnover. Any side effects of the phytoestrogen treatment will be evaluated. Since hormones are only one determinant of osteoporosis risk, the study will take into account various lifestyle and genetic modifiers such as diet, exercise, ER receptor status and vitamin D receptor status.

The third area of work is intended to provide a consumer perspective. This will be achieved by conducting surveys in five EU member states to assess acceptability of phytoestrogen rich foods for the consumer, the best substitution strategy, the expectations created by different nutritional claims in relation to phytoestrogen-enriched food products among women aged 45 and over.

ISOHEART

This project considers the role of isoflavones for reducing risk of coronary heart disease among postmenopausal women. Co-ordinated by the Royal Veterinary & Agric University, Denmark, with partners in four other EU countries, the project runs from January 2002 to December 2005.

Prevalence and mortality of cardiovascular disease (CVD) increase in women after menopause has been attributed to the loss of the cardio protective actions of oestrogen at menopause (Williams, 1997). There is considerable evidence from animal models and epidemiology to suggest that soy and in particular soy protein containing isoflavones, could reduce risk of CVD (Anderson et al 1995; Kirk et al 1998; Knight & Eden 1996). The majority of the studies in human volunteers however have focussed on serum cholesterol reduction as the marker of reduced CVD risk (Anderson et al 1995;Wangan et al 2001). Animal data and preliminary clinical evidence also suggest that isoflavones can improve systemic vascular compliance and reduce blood pressure in women (Williams and Clarkson 1998, Nestel et al 1997; Washburn et al 1999). This project evaluates the impact of soy derived isoflavones and comprises a wide range of measurements of potential cardiovascular risk factors including blood lipids, lipoproteins and key factors in the blood coagulation and fibrinolytic system, measurements of in vivo vascular compliance and blood pressure, ex vivo measurements of markers of endothelial function, vascular inflammatory events, and oxidative damage.

Genomic and proteomic techniques will be applied to identify novel biomarkers of physiological response. A further, no less important, aim is to address the consumer perspective - specifically to provide quantitative, qualitative and comparative documentation of social, cultural and socio-psychological factors that impact upon consumer acceptability of isoflavone enhanced foods among postmenopausal European women.

The major long-term objective of the project is to facilitate the development of health-promoting isoflavone enhanced foods for the consumption of older women by:

1. Establishing a multidisciplinary scientific basis for nutritionally enhanced food products designed to promote women’s health by reducing the risk of CVD in postmenopausal women
2. Documenting the presumed health benefits of isoflavone enhanced foods consumed by older women as identified by positive changes of markers of cardiovascular disease risk
3. Exploring the metabolism of isoflavones including factors determining individual response to a defined intake of isoflavones
4. Revealing molecular mechanisms for the mode of action of isoflavones using global genomic and proteomic approaches
5. Providing quantitative and qualitative documentation of social and cultural factors that impact upon consumer acceptability of isoflavone enhanced foods among post-menopausal European women
6. Disseminating the results of this project in a manner that can promote product development and marketing of isoflavone enhanced food products.

The work programme of the project comprises; Measurements of in vivo availability of soy derived isoflavones when added to various foods; Studies of molecular mechanisms for the mode of action of isoflavones; Determination of the response to a daily intake of isoflavone enhanced foods on a wide range of markers of coronary heart disease risk; Studies of the social, cultural and socio-psychological factors that impact upon dietary behaviour among the target group of consumers with specific regard to enhanced food products.

The core study is a placebo-controlled dietary intervention in postmenopausal women with soy isoflavones incorporated into commonly eaten foods. Food processing and product formulation will be based on technological and sensoric evaluation and the in vivo bio-availability assessed. One hundred twenty post­menopausal women (60 - 70 yr old) with moderately elevated risk for CVD will be recruited. The intervention design will be randomised cross over trial with provision of isoflavone-enhanced foods for 8 wk and the same foods without isoflavones for another period of 8 wk. A wide range of established and recently developed biological markers related to cardiovascular risk and mechanisms involved in atherogenesis and thrombogenesis will be measured. In addition, biomarkers of endothelial cell function identified by proteomic and genomic technology will be evaluated. Consumer perspectives on isoflavone enhanced foods will be assessed in random samples of the target group of consumers.

PHYTOPREVENT

This project is investigating the role of dietary phytoestrogens in the prevention of breast and prostate cancer. It is co-ordinated by the University of Ulster with partners in eight other EU countries. It is scheduled to run from April 2001 to March 2004.

Hormone-related cancers such as breast and prostate cancer among the most common causes of death from malignancy in Europe (Globocan 2000). However, incidences vary by as much as twenty-fold between Asian and Western populations,. The lower rates, typically seen in Asian populations have been associated with low fat, high fibre and particularly high soy diets (Tham et al., 1998). The association with soy is suggestive of a role for phytoestrogens in prevention of these malignancies. Interestingly, in the case of prostate cancer, the incidence of latent or small prostate tumours is SE Asian populations is similar to that in the West, suggesting that diet affects the progression of tumours to malignancy (Adlercreutz & Mazur 1997. Animal and in vitro studies strongly support a role for phytoestrogens (isoflavonoids and lignans) in preventing mammary and prostate cancer (Barnes et al 1990; Wang and Kurzer 1997; 1998) but epidemiological studies have yielded somewhat inconsistent results, suggesting that other factors such as genetics, dietary background and interindividual differences in phytoestrogen metabolism may influence the outcome (Lee et al 1991; Hirose et al 1995; COT 2003).

The overall aim of the project is to investigate the preventive role of phytoestrogens on breast and prostate cancers and to assess the influence, on cancer risk, of interidividual variation in phytoestrogen metabolism, with special emphasis on the metabolites generated by the gut microflora.

The work plan comprises:

1. The isolation and identification of new isoflavone and lignan metabolites to give a comprehensive picture of phytoestrogen metabolism. The development and validation of rapid imunoassays for equol and O-Dma and the development of new urinary ’stix’ methods for detection of genistein and enterolactone
2. A comparison of the activities of the parent isoflavones and lignans and their mammalian metabolites in a series of in vitro assays designed to mimic the process of carcinogenesis in breast and prostate from initiation, through tumour promotion to metastasis and angiogenesis.
3. The use of transgenic and conventional animal models to study the influence of isoflavones and lignans on breast and prostate tumour development in vivo.
4. The application of molecular techniques to study cancer biomarkers in human breast and prostate tumours
5. An intervention study with soy, flaxseed and rye products to investigate effects interindividual differences in phytoestrogen metabolism on bio-markers of cancer risk.

VENUS

This project was designed to consider the dietary exposure to phytoestrogens and related compounds and their effects on skeletal tissues. Co-ordinated by The Instituto Nazionale della Nutrizione, Rome, Italy, ( Dr Francesco Branca), this project ran for two years from 1999 to 2001.

The Venus project is a concerted action that encompassed a series of plenary meetings and workshops designed to define the current state of the art and suggest future directions in a number of specific areas related to phytoestrogen intake, metabolism and health effects. The partners in the project prepared a series of papers that are in process of being published in a supplement to the Britsh Journal of Nutrition.

The output of the project includes the following areas:

1. Composition table of main European foods indicating content in isoflavonoids, lignans, coumestrol and indols.
2. Report showing estimated intake of phytoestrogens and related compounds in the diets in the Netherlands, UK, Ireland and Italy
3. Literature review on factors affecting bioavailability of phytoestrogens and related compounds.
4. Protocol for human studies on bioavailability and metabolism of phytoestrogens and related compounds.
5. Menus with high content of phytoestrogens and related compounds.
6. Feasibility study on the production of phytoestrogen rich products.
7. Guidelines for in vitro testing. The Guidelines will provide a literature and project result review on the responsiveness of different cell systems to challenge with phytoestrogenic compounds, the different experimental conditions used, the biomarkers and their expected sensitivity.
8. Database of genes. A database of genes known to be up and down regulated by phytoestrogens will be compiled through literature research. The information will be used to consider whether such genes could be used to monitor the effect of phytoestrogens in cell culture systems.
9. Report on animal experiments.
10. Protocols for human studies. The report will illustrate possible diets, the characteristics of functional foods or mixture of compounds, the characteristics of the subjects, the sample size required and the bio-markers to be measured.

PHYTOHEALTH

This EC Concerted Action programme looks at ways of improving health through dietary phytoestrogens with the objective of establishing a pan-European network on consumers’ issues and opportunities for producers. The project is co-ordinated by The Instituto Nazionale della Nutrizione, Rome, Italy, (Dr Francesco Branca).

This project began in 2003 and will lead to the establishment of a pan-European network of institutions dealing with safety and health effects of phytoestrogens, identification of optimal sources and processing technologies, building around a cluster of three EU funded projects PHYTOS, PHYTOPREVENT and ISOHEART. The network involves key nutrition and agro-industri-al research Institutes in Europe, academic institutions, leading food manufacturers and SMEs in a total of 11 European countries (Austria, Denmark, France, Finland, Germany, Greece, Italy, Netherlands, Spain, Sweden, United Kingdom) and two associated States (Poland, Switzerland).

The project envisages the organisation of scientific meetings, preparation of consensus papers, fact sheet for regulators, producers and consumers, online communication with all stakeholders, i.e. scientists, product manufacturers, consumers targeted to receive products, and regulatory authorities.

The PHYTOHEALTH network will be a forum for scientific and technological exchange, for generation of innovative projects and for communication between stakeholders of the food system. It will concentrate on phytoestrogens in the first stage of the project, but will extend to other categories of phytoprotectants in the near future. As such, PHYTOHEALTH is an open structure, that will allow future incorporation of new partners.

The work will be organised and implemented by three expert platforms – consumers / regulatory, industrial and research. The consumers’ platform will:

1. Assess the exposure to PE from supplements, natural and processed foods in different European countries
2. Develop the methodology to profile the purchasing decisions and attitudes of consumers in the partners countries of origin
3. Use all available data to evaluate risks of PE consumption at documented “health-promoting” doses in different population groups, and
4. Review the existing regulations regarding marketing, sale and publicity of PE-enriched foods, supplements and other preparations. How to optimise the production of raw materials for PE extraction and purification, the identification of new sources of PE that are commonly grown in Europe and the set up of techniques and laboratory methods to evaluate PE compounds present in foods with regard to their estrogenic potency, all will be addressed by the industrial platform.

Finally, the research platform, in the view past and ongoing research projects in the PE field, will identify the present evidence and future required information regarding the effects of these compounds in humans by population subgroup.

The network will allow pooling of scientific data in various areas (phytoestrogen content in food and plants, consumption of phytoestrogen rich foods, presence of endocrine disrupters in the environment, effects of phytoestrogens on health, toxicological evaluation of phytoestrogens, agrobiotechnological techniques, processing technologies), the standardisation of research methodologies (calculation of exposure to phytoestrogens from food, risk assessment models, analytical methodologies, exploration of consumers’ attitudes, bio-assays for evaluation of potency of compounds) and the establishment of a common information basis for consumers, industry and regulators.

ACKNOWLEDGEMENT

This review has been carried out with the financial support of the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living resources”, Key Action 1-Health Food and Environment, QLRT-2000-266: The role of dietary phytoestrogens in the prevention of breast and prostate cancer. It does not necessarily reflect its views and in no way anticipates the Commission’s future policy in this area.

WEBSITES

PHYTOS - www.phytos.org PHYTOPREVENT - www.phytoprevent.org  VENUS - www.venus-ca.org ISOHEART – www.isoheart.kvl.dk

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BIOGRAPHY

Ian Rowland is Professor of Human Nutrition and Director of the Northern Ireland Centre for Food and Health (NICHE) in the School of Biomedical Sciences, University of Ulster Coleraine, Northern Ireland. His main areas of research are diet and cancer prevention, gut function and gut microflora, with specific interests in probiotics, carbohydrates and phytoestrogens. He is a member of the UK Advisory Committee on Novel Foods and Processes and the UK Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment.