Influence of polyphenols on macrophage cells. Zenon Czuba, Wojciech Krol; Department of Microbiology and Immunology, Medical University of Silesia, Zabrze-Rokitnica, Poland Macrophages are known to play an essential role in regulation of cellular immunity. Activation of the cells affects the generation of inflammatory products, such as cytokines, reactive nitrogen and oxygen intermediates. Flavonoids and cinnamic acid derivatives demonstrate a variety of biological activities. Earlier studies have shown that functions of activated macrophage such as release of cytokines and arachidonic acid metabolites, generation of reactive oxygen and nitrogen species, can be regulated by flavonoids. The activity of the compounds was dependent on their chemical structure (the number, location and type of substituents). In the study activated murine peritoneal macrophages and macrophage cell lines were used. The cells were stimulated with lipopolisaccharide and/or interferon ? in presence of tested flavonoids or cinnamic acid derivatives. After 24h culture of the cells the supernatants were removed and assayed for levels of cytokines (IL1, IL6, IL12, TNFa) and nitrite. The cytokines were measured by ELISA method. Nitrite as a result of nitric oxide generation was quantitative determined using Griess method.The chemiluminescence of these cells was measured in presence of luminol and tested compounds after stimulation with phorbol myristate acetate. The results were compared with controls without tested compounds. We observed differences in activity of tested compounds. Evidence for health benefits of flavonoids Peter C.H. Hollman; RIKILT; Bornsesteeg 45; 6708 PD Wageningen, The Netherlands e-mail: peter.hollman@wur.nl    Epidemiological studies suggest a protective effect of vegetables and fruits against cancer and coronary heart disease. An attractive hypothesis is that vegetables and fruits contain compounds that have a protective effect, independent of that of known nutrients and micro-nutrients. Plant polyphenols, a large group of natural antioxidants ubiquitous in a diet high in vegetables and fruits, certainly are serious candidates. In vitro studies have provided a wide array of potential mechanisms for protective effects. In addition, animal models also point to protective effects.    Although these results with polyphenols obtained in animal models and in vitro systems are promising, their relevance for humans remains to be proven. Epidemiology offers a complementary tool to study the health effects of polyphenols. The major advantage of epidemiology over the afore-menti-oned experimental studies is its ability to study associations between dietary components and the human disease, after prolonged exposure to physiological levels of these components. An important limitation of this approach is that only associations and no causal relations can be studied, involving many interacting variables, which may not be completely disentangled. Moreover, it is hard to accurately estimate the exposure to a food component in a population. Only flavonols and catechins have been subjected to epidemiological evaluations.    To date, 7 cohort studies on flavonols and CHD mortality have been published. Four of these prospective studies found a protective effect of flavonols on cardiovascular disease, and reduction of mortality risk was up to 50%. However, in 2 large cohorts no association between flavonol intake and coronary mortality was found. Increased mort-ality of ischaemic heart disease was found in a cohort of Welsh men. Residual confounding of lifestyle factors might have played a role in the evaluation of the results of this study. For catechins only two cohort studies on CHD mortality have been performed, one showing a effect, whereas another study found no clear associations with CHD mortality. For cancer, epidemiology does not yet support a protective role for flavonols or catechins.    Short-term intervention studies with flavonoids using intermediate endpoints of CHD or cancer would provide valuable information on the potential protective properties of flavonoids. Although a few of such intermediates are available, their relevance still has to be proven. The basic question to be answered remains by which biological mechanisms flavonoids would affect CHD. The rapid development of the functional genomics area will accelerate research on biological effects of flavonoids.    The ultimate evidence would come from long-term intervention studies with hard endpoints. However, such trials would be very expensive and not justified yet. The COMT-mediated metabolism of flavonoids and estrogen and its relevance to the cancer risk. Maria Kapiszewska: Department of General Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland Catechol-O-methyltransferase (COMT) is the Phase II enzyme which efficiently catalyze detoxification of endo- and exogenous compounds containing catechol groups which otherwise may become toxic, mutagenic or even carcinogenic. Therefore, the enzyme activity itself and the factors which influence the kinetic of such an inactivation are extensively studied. The COMT gene contains a single-nucleotide polymorphism (SNP), that is associated with an amino acid shift val -> met which is present up to 75 % of Caucasian. Such the mutation results in significant decrease in enzyme activity. It has been shown in several studies that the individuals having the low activity allele (COMTL) face a greater risk for developing breast cancer risk. The development of carcinogenesis may originate from catechol metabolites of estrogen which are not sufficiently O-methylated into inactive compounds because of the low COMT activity. Beside this genetic determinant, an expression of the enzyme in a given individuals or in a population is likely to be affected by environmental and lifestyle factors. Particularly, an exogenous compounds which compete for the enzyme activity like a dietary polyphenols with catechol motif, the drug being a direct or indirect COMT inhibitor or long-term exposure to exogenously administrated estrogen may contribute to the carcinogenicity in such individuals. COMT transfers a methyl group from S-adenyl-L-methionine to the catechol substrate. Therefore the differences in metabolism of folate may also affect the individual response to catechol inactivation and then the cancer risk. The relationship between the level of endogenous DNA modifications involved in risk cancer and polymorphism in COMT gene, dietary habits, particularly high-flavonoid diet, life style (estrogen levels and drugs) in an individuals will be discussed in the paper. Absorption and metabolism of polyphenols: What can we learn from in vitro models and how does it compare with human studies? Paul A Kroon; Nutrition Division, Institute of Food Research, Colney Lane, Norwich NR4 7UA, United Kingdom. tel: +44 (0)1603 255236; fax: +44 (0)1603 255038; email: paul.kroon@bbsrc.ac.uk Polyphenols have attracted considerable interest as phytoprotectants that may serve to prevent or delay the onset or development of degenerative diseases such as cancer, cataracts and cardiovasular disease. In vitro studies have shown that many polyphenols, especially the flavonoids, possess activities that are consistent with the health-protection afforded by diets rich in fruits and vegetables. However, the biological activity of polyphenols is strongly dependent upon their structure and on their concentration at target sites. The degree of absorption and the rate of clearance that define the exposure, and the effects of metabolism on the structure of polyphenols, are vital factors affecting the ability of dietary polyphenols to affect health, and must be considered before meaningful conclusions can be made concerning their relevance to health in vivo. Various model systems have been used to assess the absorption, metabolism and biological responses to polyphenols. These include various mammalian cell lines, whole animal studies and animal tissue experiments conducted ex vivo, and human primary cells cultured ex vivo. Mammalian cell cultures are convenient, widely available, and have been used extensively to study absorption and metabolism. Indeed, CaCo2, a human colon carcinoma-derived line that differentiates to mimic small intestinal enterocytes, is a widely used and highly regarded model of absorption used by drug companies. Nevertheless, there are several important differences between these cells and human enterocytes that need to be considered when trying to interpret data from in vitro experiments. For example, the expression of lactase in CaCo2 cells is virtually undetectable and this explains the differences in flavonoid uptake/metabolism observed between studies conducted with CaCo2 cells and human dietary interventions. Tumour-derived lines usually over-express one or more genes encoding transporters that are involved in drug resistance. Further, cultured cells are considered to be in a constant state of (oxidative) stress, and they respond rapidly to many treatments with increased production of hydrogen peroxide, raising questions as to the true cause of observed responses. There are also important differences between animals and humans that require care to be taken in the interpretation of data obtained using animal models. Nevertheless, human studies are expensive, time consuming, subject to large inter-individual variation, more difficult to control, and ethical considerations need to be taken into account and often limit the scope of studies. Hence although human studies are the desired approach for demonstrating absorption and/or biological effects associated with consumption of polyphenol-rich foods in vivo, there are strong arguments for using various model systems to evaluate certain aspects (e.g. plausibility and mechanisms, dose-response relationships, exposure limits and toxicity) that would be difficult to achieve in vivo. This lecture will serve to highlight the advantages and limitations of in vitro studies, animal models and human studies, with regard to assessing the absorption, metabolism and responses of human tissues to dietary polyphenols. Ethical aspects of human nutritional intervention studies - a British view point. Elizabeth Lund, Dr; Institute of Food Research, Norwich, NR4 7UA, United Kingdom Much of the evidence of biological activity associated with phytochemicals has been based on cell culture studies, usually using cancer cell lines and often taking little heed of the amount or biological form presented to a particular cell type. Only by undertaking studies on whole organisms, including humans, can we really assess the importance of foods in relation to health benefits. But, undertaking human intervention studies presents the scientist with a number of scientific and ethical dilemmas which are often in conflict. It is not, however, ethically acceptable to undertake a human study which is not scientifically valid, so before designing an intervention study the aim of that study must be clearly defined and then the limitations of any markers, or effect or practicality of execution, evaluated in the light of the original aim. Once the science of a study has been determined then the degree of benefit to both the individual and society must be weighed up against the inconvenience or risk for the individual. Nutritional intervention studies should be considered with the same degree of care as pharmaceutical trials, particularly as healthy volunteers are frequently involved who are unlikely to gain any personal benefit from the study. Additionally, when considering the biological activity of phytochemicals, especially those associated with herbal medicines, and with the development of neutraceuticals the line between nutrition and pharmacy is blurred. In Britain, human intervention studies involving healthy volunteers, not conducted within The National Health Service, must still be subject to ethical review and increasingly the use of Hospital Local Research Ethics Committees representing a wide range of professionals is being encouraged by The Department of Health in their guidelines for research governance. The ethical aspects of these guidelines are ultimately based on the Helsinki Declaration. Volunteers who take part in a study must understand the purpose of that study; to this end a full written information sheet must be provided and a verbal explanation given well in advance of the start of the intervention. Only once a volunteer understands the study can his or her consent be considered fully informed and thus have some legal status. Sometimes this can cause conflict with the science. For example, you tell the volunteers that eating a specific vegetable may reduce your risk of a succumbing to a particular disease but they are in the control group. You may ask them 'please don't eat the vegetable' but they may still eat it. The scientist may wish to introduce a low level of deception by not defining which part of the diet they are particularly interested in. It would then be for the ethics committee to decide what level of information must be revealed to the participant in the study. In this lecture I will consider a number of theoretical examples of intervention studies and the ethical and scientific dilemmas which would be associated with them. Application of Chemometrics to HPLC Data Processing Aimed at the Prediction of Xenobiotics Bioactivity A. Nasal1, A. Buciński2, A. Wojdełko1, T. Bączek1, R. Kaliszan1; 1Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Gen. J. Hallera 107, 80-416 Gdańsk, Poland; 2Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Division of Food Science, Tuwima 10, 10-747 Olsztyn, Poland The same basic intermolecular interactions determine the behavior of chemical compounds in both biological and chromatographic environments [1]. HPLC is a unique method that can readily yield a great amount of diversified, precise, and reproducible data. Based on linear free-energy relationships (LFER), it has been assumed that systematic information on the behavior of agents in a number of well designed chromatographic systems should help to predict the differences in their biological activity. Extracting such systematic information from large sets of diverse, often mutually interrelated data, became feasible due to the computerized methods of multivariate data processing, like principal component analysis (PCA) PCA of HPLC retention data of a series of a -adrenoceptor binding drugs allowed the classification of the agents in accordance with their affinity to the a 1 and a 2 receptors [2]. Similarly, subclasses of H1- and H2-histamine receptor antagonists [3] were separated. A series of 83 drugs belonging to five pharmacological classes was subjected to HPLC analysis in eight chromatographic systems. As a result of PCA of the retention data, a grouping of drugs was obtained which was in accordance with their pharmacological classification [4]. Detroyer et al. [5] reported the application of several chemometric techniques complementary to PCA, such as hierarchical cluster analysis or sequential projection pursuit, to the HPLC data of Nasal et al. [4]. Original observations were thus confirmed and the inputs of individual HPLC systems to the assignment of drugs to individual pharmacological groups were determined. Retention data of 65 new buspirone analogues analyzed in nine HPLC systems were subjected to PCA [6]. A grouping of analytes was obtained which was exclusively due to a systematic similarity of their behavior in the HPLC systems studied. The grouping was related to the affinity of the agents to the brain serotonin 5-HT1A receptors. It has been concluded that chemometrically processed HPLC parameters may help to guide the biotesting strategy and to limit the number of biological assays in the search for new drugs. [1] R. Kaliszan, Structure and Retention in Chromatography.A Chemometric Approach, Harwood, Amsterdam, 1997; [2] R. Gami-Yilinkou, R. Kaliszan. J.Chromatogr. 1991, 550, 573; [3] R. Gami-Yilinkou et al., J.Chromatogr. 1993, 633, 57; [4] A. Nasal et al., Int.J.Pharm. 1997, 159, 43; [5] A. Detroyer et al., J.Chromatogr.A 2000, 897, 23; [6] A. Nasal et al., J.Sep.Sci. 2002, 25, 273. The use of estrogen receptors for evaluation of phytoestrogens activities in mammals Marek Snochowski, Katarzyna Romanowicz; The Kielanowski Institute of Animal Physiology and Nutrition, PL-05-110 Jablonna, Poland tel.: +48 (22) 7824422; fax: +48 (22) 7742038, m.snochowski@ifzz.pan.pl Phytoestrogens form a distinguished group of phytochemicals which biological effect in mammalian organism is clinically similar to that of exogenous estrogens. The cellular response to estrogens is realized by binding to estrogen receptors (ERa and/or ERb) which function as ligand inducible nuclear transcription factors either by direct interaction with estrogen response element (ERE; cognate DNA binding sites) or interact with other DNA-bound transcription factors of the RNA polymerase II transcription initiation complex. Thus, ERs are suitable proteins for in vitro evaluation of biological activity of phytoestrogens based on determination of (1) ligand binding affinity (ligand competition assay), (2) ligand-induced changes in ERs interaction with nuclear coactivators (in vitro functional assay), (3) the specific regulatory effect of liganded ERs on particular gene expression using native EREs (cell transient transfection assays). Such complex analysis revealed that e.g. genistein and coumestrol are ER agonists with higher affinity to ERb (often considered to be ERb specific) exhibiting high (also with ERb) ligand specific transcriptional activity. These characteristics differ form that of resveratrol, which was recognized as, mixed agonist (on ERb) and antagonist (on ERa). In order to evaluate tissue specific response we have used ER extracts from ovine tissues (anoestrous ewe) known to express predominantly ERa (pituitary, uterus) or both forms equally (thyroid) the relative binding affinities (RBA) to common dietary phytoestrogens. Using ligand binding competition assay and 17b-estradiol (E2) as a standard we have found similar order of RBAs in all three tissues being E2 >> coumestrol = genistein > equol > daidzein > biochanin A = formonoetin. However, the RBA of genistein was higher in thyroid (7.5%) and pituitary (4.9%) then in uterine (0.8%) preparation. It was also noted that the metabolic conversion of daidzein to equol increases more the 5 times its RBA. The obtained results indicate that tissue susceptibility to phytoestrogens in anoestrous ewe is higher for thyroid and pituitary then for uterus. The general effects of phytoestrogens on ERs will be briefly discussed in view of recent reports on their physiological functions (growth, development, aging) and the role in hormone-dependent diseases (cancer, osteoporosis, arteriosclerosis). Antioxidant activity of flavonoids in different animal model systems -In vitro, ex vivo and in vivo- Junji Terao; Department of Nutrition, The University of Tokushima School of Medicine, Japan Antioxidant activity of dietary flavonoids has attracted much attention in relation to their possible role in the prevention of oxidative stress-induced chronic diseases such as ischemic heart disease. However, there seems to be little evidence for the involvement of dietary flavonoids in antioxidative defense in vivo. Using a biomembrane model, we previously pointed out that flavonoids are interfacial antioxidants whose activities appear at the interface between lipid-phase and water-phase (1). Nevertheless, recent works clarified that dietary flavonoids are mostly subject to metabolic conversion during absorption and are present as their conjugated metabolites in blood stream.. Our ex vivo studies showed that oral intake of either (-)-epicatechin or quercetin elevated the antioxidative capacity of rat plasma (2, 3). In addition, we identified that an antioxidative metabolite, quercetin 3-O-ß-D-glucuronide (Q3GA) from quercetin-administered rat plasma (4). Q3GA was shown to suppress peroxynitrite-induced human LDL degradation (5). Q3GA was found to be more effective than quercetin aglycone in the inhibition of H2O2-induced intracellular ROS production in mouse fibroblast cultured cells, although it scarcely diffused into the cytoplasm or cell nucleus compartment (6). It is plausible that some conjugated metabolites exert an antioxidant activity by interacting with the cellular membranes. It is therefore likely that dietary flavonoids possess a power for elevating the antioxidant defense in vivo, although they are mostly converted to conjugated metabolites. Conjugation reaction is involved in detoxification pathway of xenobiotics. We demonstrated that intake of quercetin at extremely high level induced 8-OHdG production in rat urine. It seems that some flavonoids are capable of acting as prooxidants in vivo, depending on the strength of oxidative stress. Here we will discuss the role of metabolism for regulating the antioxidative effect of dietary flavonoids. (1)Terao Piskula Nutrition 19:790 (1999) (2) da Silva, Piskula Terao FRBM 24:1209 (1998) (3)da Silva Piskula Terao FEBS Lett 430:405 (1998) (4) Moon Tsushida Nakahara Terao FRBM 30:1274 (2001) (5)Terao Yamaguchi Shirai Miyoshi Moon Oshima Inakuma Tsushida Kato Free Radical Res. 35:925 (2001) (6) Shirai Yamanishi Moon Murota Terao Biosci. Biotechnol. Biocherm. 66:1015 (2002) Evaluation of physiological functionalities of some flavonoids using animal cell culture Tojiro Tsushida; Food Function Division, National Food Research Institute, Japan Introduction The tertiary function of food to regulate the physiological system of human body and to prevent chronic diseases has been investigated by many researchers in the world in recent years, and phenolic compounds as well as other phytochemicals showing antioxidant activity have attracted a great deal of public attention. We also have been interested in the bioactivities of phenolic compounds and especially enchanted by flavonoids. Then to estimate some physiological activities and to elucidate the mechanisms of the activities of flavonoids, we employed the animal cell culture methods as simplified and rapid measuring model systems and obtained some results as follows. Effects on leukemia cell differentiation The flavones, apigenin and luteolin, strongly inhibited the growth of HL60 human leukemia cells and induced morphological differentiation into NBT-positive granulocytes. The flavonol quercetin inhibited the cell growth and induced a differentiation marker of granulocytes but not morphologically differentiated into it. The dihydrochalcone phloretin weakly induced a marker of monocytic differentiation alph-naphtyl butyrate esterase activity in the cells. Quercetin and phloretin appeared to induce the differentiation of HL60 cells into monocytes. Proanthocyanidin prodelphinidin B-3, T1, T2, and T3 from barley bran induced 26-40% NBT-positive cells and 22-32% alpha-naphtyl butyrate esterase-positive cells. Moreover, proanthocyanidins intensify retinoic acid-induced granulocytic and sodium butyrate-induced monocytic differentiation in HL60 cells. Effects on differentiation of pre-adipocytic 3T3-L1 cells The inhibitors for differentiation of fibroblast cells into adipocytic cells are expected to be effective in the prevention of obesity. Glycerol-3-phosphate dehydrogenase (GPDH) activity was measured as a hallmark of 3TS-L1 differentiation into adipocytic cells. Quercetin, kaempherol and isorhamnetin significantly lowered the GPDH activity of 3T3-L1 cells during cultivation. The inhibitory action of quercetin on the differentiation of 3T3-L1 cells into adopocytic cells estimate to involve the direct inhibition of GPDH, which is a key enzyme of lipid synthesis. Effects on apoptosis of cancer cells Dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma cells and HL60 human leukemia cells. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased but the level of p53, Bcl-2 proteins did not changed. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, because phloretin inhibited protein kinase C activity in HL60 more than that in B16 cells. And phloretin reduced the level of caspase 3 protein, but not the level of the Bcl-2 protein. Further experiment showed that isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. Flow cytometric analysis showed that these chalcones increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that the chalcones inhibit cell proliferation and induce apoptosis in B16 melanoma cells. In addition, anthocyanine delphinidine and malvidine were also found to induce apoptosis in HL60 cells. Effects of polyphenol metabolism on gene expression in vivo Gary Williamson; Nutrient Bioavailability; Nestlé Research Center, Vers-Chez-Les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland; phone: + 41 21 785 8546, fax: + 41 21 785 8544, mobile: +41 79 201 9902, e-mail: gary.williamson@rdls.nestle.com Pathways of polyphenol metabolism The understanding of the pathways, mechanisms and rate limiting steps of polyphenol metabolism have advanced dramatically in the last 10 years. A combination of in vitro, animal and human intervention studies have demonstrated that common pathways are involved in flavonoid metabolism; in highly simplified form, the predominant reactions are deglycosylation, conjugation with glucuronide in the enterocyte, transfer back to the lumen or into blood, circulation in the blood, transfer to tissues or further metabolism in the liver, biliary excretion or urinary excretion, colon microflora metabolism to smaller phenolics, and entry of these phenolics into the circulation. At all stages, the pathways are controlled by the specificity of metabolizing enzymes and of transporters. Human studies on polyphenols There are now more than 60 reported studies on the biological effects of polyphenols, and the major target from these studies is clearly vascular function. There are very few studies on humans where biomarkers of carcinogenesis have been measured as a result of polyphenol intake. There are now also a significant number of studies on humans in vivo demonstrating polyphenol "bioavailability", but using measurements in plasma. Effects on cell function in vivo The time has come to address the difficult issue of how polyphenols affect cellular function in vivo in humans. Most of the human studies to date have measured biomarkers in plasma, but most in vitro studies have demonstrated effects at the cellular level. The next challenge is to combine these two aspects and demonstrate the effect on polyphenol intake on cells in situ in humans. As a first step to addressing this question, we have used an intestinal perfusion system in humans, which can measure changes in epithelial cell function, and at the same time, allow measurement of the metabolism of the polyphenol. Data from this study will be presented in the context of other information in the literature on humans concerning the biological effects of polyphenols in vivo. Ethical and Legal Conditions for Using Animals in Research Zenon Zduńczyk1, Klaus-Dieter Jany2;1Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Division of Food Science, 10 Tuwima Str., 10-747 Olsztyn, Poland 2Federal Research Centre for Nutrition, Haid-und Neu-Str. 9, 76131 Karlsruhe, Germany In European countries, conditions regarding the use of animals for scientific purposes have been outlined in the course of evolution of viewpoints and moral norms, which has been observed in Western civilisation over the last decades. Current animal-oriented humanitarian beliefs are based on the thought of Michel De Montaigne (a French writer and philosopher of the XVIth century): "If we condemn cruelty to people, then we should also condemn cruelty to other creatures, as we are somehow one family, the children of Mother nature. Nowadays, biocentrally -oriented environmental ethics, formulated by Taylor (1986) assumes that life is the basis and point of reference to all other vales valid for humans and animals as well. The resulting rule of neutrality obliges to non-instrumental treatment of animals being a part sphere of values and moral evaluations. This rule is to a high extent respected in legislation of the European Union and Member States. The first legal act concerning that issue was the Act against cruelty to animals adopted in Great Britain in 1876. Current legal and administrative regulations of the EU regarding protection of animals used for experimental and other scientific purposes in the Member Countries of the Community have been compiled in a council Directive of November 24, 1986, the so-called Directive 86/609/EEC. Its contents are fully reflected in two rules: (1) when seeking for knowledge, health and safety a man cannot use animals under justified grounds that the results of these studies will provide extension of knowledge or general advantage to humans or animals; (2) a man has a moral duty to respect all animals and should bear in mind and avoid their suffering. Experiments on animals should therefore be carried out only when indispensable for higher purposes, verified by appropriate institution established by the EU Member States to respect Directive provisions. One of the conditions of using animals for scientific purposes is respecting the 3R rule of Russel and Burch (1959), i.e. consideration of the possibility of replacing in vivo method with in vitro technique (replacement), reduction of a number of animals to a necessary minimum (reduction), and refinement of experimental method so as to limit its invasiveness (refinement).