ECNIS is a Network of Excellence within the European Union’s Sixth Framework Programme, Priority 5: Food Quality and Safety. It brings together some of the best European research groups in a concerted effort to achieve improved understanding of the environmental causes of cancer, of the potential of diet to prevent cancer and of the ways in which heredity can affect individual susceptibility to carcinogens, with the ultimate aim of reducing the cancer burden in Europe. ECNIS is coordinated by Prof. Konrad Rydzyƒski, Nofer Institute of Occupational Medicine, ul. Âw. Teresy 8, 91-348 ¸ódê, Poland. This review has been prepared as part of ECNIS Work Package 9: Mechanisms of modulation of cancer by dietary factors.

© ECNIS, 2007 All rights reserved. No part of this book may be reproduced in any form without the permission of the publisher.

Compiled and edited by Björn Åkesson and Per Mercke Biomedical Nutrition, Pure and Applied Biochemistry, Lund University PO Box 124 SE-221 00 Lund Tel: +46 (0) 46 222 45 23 Fax: +46 (0) 46 222 46 11

ISBN 978-83-60818-02-2

Technical editor: Katarzyna Rogowska Cover design, computer typesetting: Beata Grabska

Published by Nofer Institute of Occupational Medicine Âw. Teresy 8, 91-348 ¸ódê, Poland Tel.: +48 (0) 42 631 45 04 Fax: +48 (0) 42 656 83 31 E-mail: ecnis@ecnis.org Website: http://www.imp.lodz.pl

Contents

Executive summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.1. Introductory overview and future prospects Björn Åkesson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Appendix 1.2. Overview of possible anticarcinogenic food components Per Mercke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2. Vitamins and selenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.1. Biomarkers of exposure to and effects of vitamins A, C and E Lars O. Dragsted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2. Antioxidant vitamins and cancer risk: is oxidative DNA damage a relevant biomarker? Steffen Loft, Peter Møller, Marcus S. Cook, Rafa∏ Ró˝alski, Ryszard Oliƒski . . . . . . . . . . . . . . 51 2.3. Measurement of serum 25-hydroxycholecalciferol as marker of vitamin D status Jakob Linseisen, Sascha Abbas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 2.4. Selenium and cancer — selenoproteins as biomarkers in relation to selenium supplementation Björn Åkesson, Katharina Bruzelius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 2.5. Anticancerogenic activity of selenium — molecular mechanisms and epidemiological data Jolanta Gromadziƒska, Edyta Reszka, Wojciech Wàsowicz . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

3. Bioactive components in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 3.1. Anticarcinogenic effects of flavonoids Margaret M. Manson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 3.2. Biomarkers of dietary polyphenol intake for studying diet-cancer relationships Jakob Linseisen, Sabine Rohrmann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

. . . . . . . . . . . . . . . . . . . . . . Probiotics and prebiotics — potential anticarcinogenic food components Joseph Rafter . . . . . . .4. . . Kelleher. . . . . . Eggleston . . . . . . . . . . . . . . . . . . . Soterios A. . . 134 3. . . . . . . . de Kok. . . . . . . . . . . . van Breda . . . . . . Michael O. . Sotiroudis. 160 3. . . 179 . . . . . . . . .5. Hayes. . . . . . . 140 3. . . . . . . Combined action of different dietary compounds preventive of cancer Theo M. . . . . . . . . .3. . . . . . . . . . . . . . . . . . . . . . . 170 Annex ECNIS participants . . . . . . . . . . . .3. . Ian M. . . Kyrtopoulos . . . . . . . . . . . . . . . . Simone G. . . . . . . . . . . . . . . . . . . .6. . . . . . . . . . . . . . . . . . . . Anticarcinogenic compounds of olive oil and related biomarkers Theodore G. . Anticarcinogenic effects of glucosinolate breakdown products John D. . . . . . . . . .

Markers differ considerably in their characteristics. some of them mainly reflecting recent dietary intake and others indicating more the individual’s long-term nutritional status. the growth conditions and other factors.Executive summary The major aim of this review was to summarize the state-of-the-art in use of biomarkers for some anticarcinogenic food components and to identify knowledge gaps. Many compounds contained in the diet have been proposed as having anticarcinogenic properties. The ECNIS project aims at addressing important problems in this field and identifying knowledge gaps that exist. The microbial flora in the gut also play an important role in their action and composition through bioconversion and the release of various bioactive dietary components. that of reviewing the mechanisms of action of some of the most promising anticarcinogenic compounds. The area of biomarkers has progressed markedly with the advent of new analytical technology and the emergence of data showing that hitherto neglected food components may have important health effects and can be of strong interest. vitamins and selenium on one hand. The important links found between use of a substance as a biomarker and its mechanisms of action led to a further aim. particular progress has been made here recently. on the other. Since this is a vast area. which is a very complex field indeed in view of the large number of cancer diseases. Nutritional biomarkers are essential for studies of the links between dietary intake and the risk of cancer. Current research . The present review focuses on two major groups of food components. Vitamins and selenium Although the measurement of vitamins in body fluids is a classical area for nutritional biomarker research. pathogenetic mechanisms. The degree to which these compounds are present in plant tissue can vary markedly and be dependent on the plant variety. although plantbased foods appear to be the richest source. These compounds are found in foods of many different types. Also. especially those of relevance for the partners of the NoE ECNIS and its contacts. and bioactive compounds. Biomarkers are a key tool for assessing nutritional status and dietary exposure to specific substances. which to a large extent reflects the kinetics of metabolism and transport. the confounding factors affecting a given biomarker vary appreciably for different dietary components as well as in terms of the body fluid or tissue considered suitable for sampling. and pool sizes of the substance in question. as outlined below. Particular attention is directed at identifying dietary factors that modulate environmental and lifestyle factors related to cancer risk. only certain selected topics. are considered in detail. food components and food preparation methods involved.

and since metabolites of vitamin D have important biological effects. strong research efforts in this area should be encouraged. Regarding intervention studies using antioxidant supplementation. C and E are an important part of the diet-cancer field. probably higher accuracy as well as the possibility it to determine ascorbate and dehydroascorbate simultaneously. The effects that have been postulated of high levels of vitamin E supplementation on exercise-induced lipid oxidation. epidemio-logic variations in vitamin D status and its determinants. there are six investigations that have reported beneficial effects . Regarding vitamin E. Vitamin C. Simpler tests using fluorescence or immunological techniques have a high level of accuracy and precision. In contrast. clear short-term pro-oxidant effects on lipid oxidation have been observed following the infusion of high-dose vitamin C into the bloodstream. Biomarkers of vitamins A. the variations in measurement between different assays and different laboratories. including cancer. using oxidative DNA damage as a biomarker. The antioxidant vitamins have been studied very much in relation to risk of cancer. polyphenols. it appears to have only limited capacity as a direct antioxidant in vivo. Although it is likely that severe oxidative stress is a consequence rather than a cause of the development of many types of cancer. The latter has the advantage of possessing lower detection limits. No studies are currently available on the relationship between lipid or protein oxidation markers and chronic disease. Serum retinol is still the most reliable indicator for evaluating vitamin A deficiency.Dietary vitamins. but there is a need of developing simpler methodologies. as determined by isoprostanes and by inflammatory markers. HPLC methods are those which are most sensitive. and that the intake of high doses of vitamin C can be used to decrease oxidative stress. both HPLC and GC-MS methodology have high precision and accuracy for its detection in plasma. at present it is impossible to assess the quantitative involvement of oxidative stress in the origin of cancer. can readily be determined in plasma by automated colorimetric assays or by HPLC. Vitamin E intervention has not been shown to decrease the oxidation of plasma proteins. The determination of vitamin D status is still a challenging task. Concerning the functions of vitamin A. selenium and probiotics: biomarkers of exposure and mechanisms of anticarcinogenic action 6 on vitamin D has shown its links to a broader spectrum of health matters and diseases. The measurement of vitamin D metabolites presents a number of problems including the occurrence of vitamin D in different forms. as assessed by currently available markers of lipid or protein oxidation in humans. There is limited evidence that plasma ascorbate is a functional antioxidant. which is water-soluble. The handling and storage of plasma for vitamin C determination has a strong impact on its accuracy. but colorimetric assays have a much higher throughput. Epidemiologic studies in particular can help advance our knowledge of the association between vitamin D insufficiency and the risk of disease including that of cancer at various sites. and the problems in defining the appropriate threshold for vitamin D sufficiency. Since DNA mutations are a crucial step in carcinogenesis and elevated levels of oxidative DNA lesions have been noted in many tumours. are controversial. but they are not useful as yet for large screenings. such damage is strongly implicated in the aetiology of cancer. There is limited evidence that of a protective effect of vitamin E supplementation at levels of 200-2000 IU/d as judged from its effects on markers of lipid oxidation.

Like other types of dietary chemopreventive agents. Several large human intervention studies have indicated that selenium supplementation can prevent the development of several different forms of cancer (prostate. release of cytochrome c and the activation of caspases. Most studies have involved healthy individuals. but the few well-controlled studies that have reported realistic appearing levels of oxidative DNA damage in leucocytes isolated from oxidatively stressed subjects do not lend much support to the notion that such a population benefits more from antioxidant supplementation than a normal study population would. Some recent mechanistic findings on the flavonoids apigenin. The relationship of low selenium status and selenium supplementation to cancer disease is another important segment of focus in the diet-cancer field. There is also a need of clarifying the mechanistic role of selenium in the etiology of cancer. The optimal type and dose of selenium supplements needs to also be defined. and the induction of apoptosis. flavonoids exhibit a wide range of potentially beneficial activities in terms of cancer prevention. Several epidemiologic case-control studies have indicated a protective role of selenium in helping prevent the development of cancer. These are usually divided up into blocking and suppressing activities. DNA repair and signal transduction. It is also important to determine whether there is an increased risk of certain forms of cancer after selenium supplementation. Both organic and inorganic forms of selenium have been evaluated. mitochondrial membrane depolarisation. Flavonoids can also induce cell cycle arrest by modulating key components of cell cycle regulation. The suppressing activities include the inhibition of signalling pathways responsible for cell proliferation and survival. Bioactive compounds in foods Much direction has been directed at the beneficial health effects of flavonoids and other polyphenolic components that occur in the diet. The blocking activities include antioxidant effects and the modulation of drugmetabolising enzymes and multidrug resistant genes. epigallo- . There is little support for the notion that ingestion of antioxidant-rich foods is associated with a lower level of spontaneous oxidative DNA damage in leucocytes than produced by the intake of single antioxidants. use has been made of several biomarkers such as the concentration of total selenium in different body fluids as well as different selenoproteins. It is important to explore the possibility of using other selenoproteins as biomarkers too. lung. colon). whereas there are 13 studies that have reported a null effect. In these studies. the cell cycle. mainly through intrinsic pathways involving members of the Bcl family.Executive summary 7 of it on oxidative DNA damage. primarily glutathione peroxidase. including cyclins. cyclin-dependent kinases and inhibitors. Further studies are underway. In the future greater attention should also be directed at alternative chemopreventive mechanisms such as upregulation of DNA repair systems and the chemopreventive effects of antioxidants on non-lymphatic tissue. Experimental studies have shown that selenium compounds affect cell growth. methylated selenocompounds having been found to have particularly strong chemoprotective effects.

The more recently developed metabolomic techniques may offer new possibilities in the future. but further study regarding this will be needed. The bioavailability of different compounds varies markedly and for many compounds information on this point is scarce. promotion and progression of multistage carcinogenesis. the measurement of polyphenols in body fluids is a difficult task. squalene and β-sitosterol) are reviewed. These include tocopherol and carotenoid antioxidants. quercetin. which reduces the formation of isothiocyanates from glucosinolates. These compounds have a number of different mechanisms of action. such as isoflavones and lignans. cyano-epithioalkanes and oxazolidine-2-thiones from glucosinolates. but their utilisation in this way is hampered by several factors. which occurs at the expense of their forming isothiocyanates. Without such information. A major impediment to our understanding of the chemopreventative mechanisms stimulated by glucosinolates is that relatively little is known regarding the biological effects of glucosinolate breakdown products other than the isothiocyanates and the indole-containing derivatives. at low doses of phytochemicals cytoprotective adaptive responses . hydroxytyrosol. polyphenols. For some compounds. Hydrolytic and clean-up steps are usually needed. its becoming increasingly clear that it is their breakdown products that can influence the initiation and progression of carcinogenesis. It is unclear whether the formation of thiocyanates. An especially interesting matter here is that intake of olive oil can increase the bioavailability of anticarcinogenic compounds. the availability of immunoassays is of great value. the chalone. as well as squalene and β-sitosterol. a number of recent studies on the bioavailability of certain minor but important olive oil components (polyphenols. xanthohumol and the novel flavonol tricin have been summarised. and the sensitivity of detection may be a limiting factor although the increasing use of detection methods based on mass spectrometry will solve some of these problems including the identification of analytes. is undesirable. In addition. The half-life of many compounds in plasma is less than one day and measurements of their plasma levels mainly reflect short-term dietary intake. nitriles. certain phenolic compounds (tyrosol. To clarify the matter. which also applies to analyses of these compounds in urine. selenium and probiotics: biomarkers of exposure and mechanisms of anticarcinogenic action 8 catechin gallate. genistein. resveratrol. They also appear to influence apoptotic responses to chemotherapeutic agents. It is also unclear whether the activity of the epithiospecifier protein (ESP). Mammalian cells display marked dose responsiveness to phytochemicals. is undesirable in terms of chemoprevention of cancer. The anticancerogenic properties of the glucosinolates have received a great deal of attention. Olive oil is one of the foods at which special interest has been directed in the diet-cancer field because of its containing a special mixture of agents that affect the initiation. it is difficult to relate responses of cells in culture to particular concentrations of phytochemicals to the situation in vivo. relatively little is known of the pharmacokinetic properties of glucosinolate breakdown products in humans. Also. secoiridoids and lignans).Dietary vitamins. From an analytical point of view. It has also been pointed out that their efficacy is dependent on their bioavailability. lignans. the use of polyphenols as biomarkers of dietary intake has attracted considerable interest. These are areas in need of further investigation.

Even with the above reservations and the limited number of human studies available in mind. It is important here to have insight into recent developments in analytical technology and thorough knowledge of biomarker kinetics. . Different strains of the bacteria may possibly target different mechanisms. An important goal for the future should be to conduct carefully designed human clinical trials with the aim of corroborating insofar as possible results of the wealth of experimental studies that have been carried out. Many healthful diet effects are attributed to probiotic bacteria. More work needs to be done to identify the specific strains and strain characteristics responsible for particular antitumour effects and the mediating mechanisms involved. Although our understanding of the molecular mechanisms behind the observed combinational effects is still limited. The development of new dietary supplement regimens and nutraceuticals can also benefit from improved insight into the mechanisms behind the synergistic effects of both natural and synthetic chemopreventive compounds. A growing number of in vitro and in vivo studies indicate that combinations of dietary chemopreventive agents can sometimes result in significant levels of activity at concentrations at which any single agent is inactive. This can well contribute to cancer prevention. one can regard the use of lactic cultures for human cancer suppression as interesting. Much hope is attached in this connection to the powerful techniques available today within the field of nutritional genomics. In some systems. promising and as deserving of closer scrutiny. As has been indicated here. The synergistic effects of dietary phytochemicals need to be investigated further. Several possible mechanisms may explain how lactic acid bacteria can protect against tumour development in the colon. The development of ideas regarding this has also been stimulated by findings that dietary supplements of only a single compound may have negative effects. An understanding of the mechanisms involved is a necessary basis for the development and validation of new biomarkers of nutritional status. The strongest evidence for the anticancer effects these can have comes from animal studies. considerable information of detailed character concerning the mechanisms governing the effects of bioactive food compounds and of nutrients on the cellular processes that relate to carcinogenesis has accumulated. evidence from human studies still being limited.Executive summary 9 being activated. and green tea flavonoids can also show increased activity when present together with other phytochemicals. It is a highly challenging task to integrate this knowledge in such a way that useful hypotheses can be formulated that ultimately can be tested in human dietary intervention trials. growth arrest or apoptosis is to occur are determined. whereas at higher doses cell cycle arrest and apoptosis occur. This may explain why some food items or diets may show cancer preventive effects that cannot be explained on the basis of the individual bioactive ingredients alone. It is presently unclear how these different types of responses are coordinated by the cell and how matters of whether adaptation. it appears that many combinations of complementary modes of action may be involved. Identification of the mechanisms that control such outcomes would be very useful. combinations of green tea flavonoids are more active than single compounds of this sort.

.

Sweden Dietary assessment methodology and the development and validation of biomarkers of nutritional status are key scientific fields within nutritional science.g. Lund University and Department of Clinical Nutrition. Pure and Applied Biochemistry. Introductory overview and future prospects Björn Åkesson Biomedical Nutrition. Lund University Hospital. 1–6] dealing with the basic theories and methodology. A brief overview of food components that appear to have an anticarcinogenic effect is provided in Appendix 1. A further aim is to examine and explore the mechanisms responsible for the action for different promising anticarcinogenic compounds. Various properties of biomarkers are summarised in Table 1.2. Table 1. A main concern is to identify dietary factors that modulate environmental and lifestyle factors that can affect risk of cancer and which in the long run can also provide support for the development of functional foods. markers of nutritional status Markers of differing kinetics (response times) Markers of nutrients vs. This is a very complex field in view of the large number of different types of cancer.1. that role that various food components and food preparation methods play in connection with this. their physiological basis and the use of biomarkers in studies of different types.1. The major aim of this review is to summarize the state-of-the-art of biomarkers that can be applied to various anticarcinogenic food components and to highlight different knowledge gaps. A number of textbooks and reviews are available within the field of nutritional biomarkers [see e. . The ECNIS project aims at addressing important problems in this field and identifying various knowledge gaps. the pathogenetic mechanisms involved.1. Lund.1. They have expanded markedly with the advent of new analytical technology and the emergence of scientific findings showing that hitherto neglected food components may have interesting and important health effects. Introduction 1. markers of non-nutrient dietary components Types of confounding factors and errors affecting the assessment of dietary intake and nutritional status Choice of body fluids or tissues for sampling and the handling of samples Analytical methodology and throughput A major area of use of nutritional biomarkers is in studying the risk of different types of cancer diseases in relation to dietary intake and nutritional status. Some major issues regarding nutritional biomarkers Markers of dietary intake vs.

The amount of a substance occurring in the erythrocytes. urine or hair. since the erythrocyte cells have a lifetime of about 120 days [3].Björn Åkesson 12 Dietary components as biomarkers Many biomarkers involve the measurement of a nutrient or some other dietary component in the blood. often reflects much more the long-term intake if it than the plasma level does. Various diet-related compounds in the plasma also differ considerably in turnover times. such as the retinol-binding protein and ceruloplasmin. In some cases a biomarker or a set of biomarkers may reflect the intake of a specific food containing a specific pattern of particular substances. the main function of some markers being that of serving as indicators of malnutrition. whereas others are performed only in a few specialised research laboratories. A large number of confounding factors such as hormones. and inflammatory and other diseases can influence the levels of different biomarkers [3]. Also. . different nutrient-dependent physiological tests can be employed [2]. Biomarkers show temporal variation. diurnal variations. gender and gene polymorphism (as outlined below). Use of nutritional biomarkers It is often the case that a biomarker does not respond to changes in the status of a nutrient equally well over the entire range of nutritional status. Some methods. such as the analysis of cobalamine and folate in plasma. interactions between dietary or endogenous components or xenobiotics. A classification of feasibility (predictive power) and of degree of responsiveness to different ranges of intake for various biomarkers is provided by Bates et al. plasma. In addition. are in routine daily use in clinical laboratories. As reviewed in this volume there is a strong link between selenium content and glutathione peroxidase activity since it is covalently bound in the peptide chain. The level of a biomarker often varies as well with age. for example zinc and copper to superoxide dismutase activity and selenium to the activity of glutathione peroxidase and other selenoproteins. The selection of the type of sampling to carry out depends very much on the components to be studied and the sampling facilities available. One such example is olive oil discussed in this volume by Sotiroudis and Kyrtopoulos. [3]. Functional biomarkers of nutrients Since many nutrients are essential for the activity of enzymes (as coenzymes for example) the levels of activity involved are sometimes used as nutritional biomarkers such as in activation assays for thiamine and riboflavin [3]. and some of them reflect recent dietary intake strongly such as the amounts of various components consumed a short time before that occur in the urine. variations in metabolism or in excretory pathways. Also proteins with a transport function as well as other functions are used as biomarkers. and they may not respond to overexposure of nutrients at all. trace elements are coupled to the levels of enzyme activities. the gut flora.

There are particularly good reasons for using the 25-hydroxy vitamin D as a biomarker in extended studies in the future. Also. thus strengthening dietary associations. adipose tissue or biopies from other organs have been developed. faeces. although epidemiological findings on the association between the selenium level and cancer are reviewed by Gromadziƒska et al. 2) help establish causality of food and nutrient associations in epidemiology studies. it is known generally that the levels of nutrients in extracellular fluids do not necessarily reflect their level or saturation at crucial cellular sites or in storage tissues. methods for measuring nutrients or nutrient-dependent variables in cells in the blood (mainly leucocytes). however. in the case of some analyses carried out on samples of the plasma the main bottlenecks are linked with the precision or the throughput of the analytical methods involved. urine. information on the interactions between dietary intake and gene polymorphisms “can serve to 1) define susceptible subpopulations. This topic is only taken up briefly in the present volume. The assessment of dietary intake in epidemiological studies has been a major use of biochemical markers of nutritional status. as exemplified in the present volume by Linseisen and his colleagues in connection with vitamin D and polyphenols. There are also very interesting hypotheses regarding the role of folate and other dietary components involved in methyl transfer in the epigenetic regulation of methylation status and its association with the risk of disease [11. Although the use of the biomarker concept for these purposes is very attractive. if the analytical methodology improves so that smaller amounts of sample suffice. For this reason. An overview of this matter is provided in [7]. together with an assessment of dietary intake by a food frequency questionnaire or by some other methods. Biomarkers in studies of that type have been used either alone. A number of statistical methods for the correction and evaluation of such data are available [8].12]. or for the calibration of the dietary assessment methods [8]. Some of these samplings require much skill and effort and can only be used in small studies. it should be borne in mind that there are very few (if any) “ideal” biomarkers that have been validated for studies of different types. Folate has served as a prototype here for demonstrating how genetic make-up can influence the nutrient status of an individual. 3) aid in distinguishing causal components of complex dietary mixtures. and 4) eventually provide the basis for gene-based screening tests”. . Nutrient biomarkers in relation to genetic polymorphism It is generally assumed that genetic polymorphism influences the response of biomarkers in different individuals but for most biomarkers this has not yet been much studied.Introductory overview and future prospects 13 Although several of the biomarkers mentioned here are widely used. The two contributions mentioned also illustrate the difficulty in defining valid cut-off points. Their use may increase. buccal mucosa. The influence that the C677T polymorphism found in methylene tetrahydrofolate reductase (MTHFR) has on folate physiology and the risk of disease is a much studied example of this [10–12]. As summarised by Hunter [9]. These matters are outside the scope of the present review.

such enzymes as cyclo-oxygenase 2. It was also stated that “in four trials (three with unclear/inadequate methodology). Biomarkers of this category include tumours and the mortality in animal models. The results of the first generation of nutritional intervention studies concerned with prevention of cancer were summarized recently [14]. vitamin A and/or vitamin E [13]. Biomarkers for the assessment of dietary effects on carcinogenesis Biomarkers used as a surrogate endpoint in studies of the effect that dietary manipulation has at different stages of carcinogenesis are another type of biomarkers of importance in the diet-cancer field. α-tocopherol. cell proliferation and differentiation. had negative effects.15]. The content or profile in the blood of β-carotene. gut-lumen enzymes and carcinogen-metabolising enzymes. The potential preventive effect of selenium should be studied in adequate randomised trials” [13]. review the evidence that individual antioxidants as well as for antioxidant-rich diets affecting oxidative DNA damage. selenium showed significant beneficial effect on the incidence of gastrointestinal cancer. apoptosis. the criteria to be employed for the evaluation of efficacy in the future being proposed [14. Studies in which nutrient antioxidants or combinations of those are employed are of special relevance for various topics taken up in the present volume. In the present volume Loft et al. especially β-carotene. A possible further use to which such biomarkers could be put would be to substantiate ‘anticancer ’ claims regarding various food components. the existence of a renal threshold or other factors. . a meta-analysis showing there to be an increase in mortality in studies of antioxidant supplementation by β-carotene. adenomatous polyps. The finding of negative effects after long-term dietary supplementation of various antioxidant nutrients has led to the increased study instead of the effects and mechanisms of action of non-nutrient antioxidants or bioactive components.17]. Hayes. usually responds to an increase in the supply of them. C and E on biomarkers of oxidative stress. Other aspects of cancer-related biomarkers have been the subject of a previous review conducted within the ECNIS project [18]. although for other nutrients and their biomarkers there may be little or no response due to homeostatic regulation of their plasma levels. To the surprise of the scientific community it was found that some antioxidants. work that is as reviewed by Manson.Björn Åkesson 14 Biomarkers and response to dietary supplements An important use of biomarkers is to aid in the assessment of compliance in meal studies and dietary intervention studies. a matter reviewed recently [16. de Kok and their colleagues in this volume. and Dragsted summarises the effects of vitamins A. as well as precancerous lesions. Biomarkers are also used in long-term human intervention studies in which there are clinical endpoints. selenium and polyenoic fatty acids for example. certain metabolites and various effects on DNA and its metabolism [16].

Introductory overview and future prospects
15

Application of omics technologies Emerging “omics” technologies — transcriptomics, proteomics, genomics and metabolomics — will probably have a strong influence on research on relations between nutrition and cancer [12,19]. The use of transcriptomics opens up the possibility of monitoring the changes in global gene expression caused by a wide array of dietary components in different experimental systems. Proteomics, in turn, makes it possible for the same type of experiments to be carried out at the protein level, providing unique and possibly more detailed information. Transcriptomics and proteomics can both be expected to become major tools in gaining a better understanding of the cancerprotective effects of different nutrients. The metabolomics approach involves the multiparametric measurement of metabolites and provides a comprehensive account of the metabolic profile of different biological systems, such as various body fluids [20,21]. Genomics, finally, includes techniques for the global genotypic characterization of individuals with the aim of discovering polymorphisms associated with susceptibility [9]. SNP arrays are one powerful genomic tool enabling whole-genome association studies of up to 500 000 SNPs (single nucleotide polymorphisms) to be carried out in a single run. The “omics” technologies as a whole will surely play a key role in future nutritional research regarding the protective role of diet in connection with cancer. The use of results obtained by various of these techniques involves complex ethical considerations [22].

Concluding remarks The contributions the present volume contains clearly indicate that a deeper understanding is needed of the mechanisms underlying the protective effects that various food components can have in preventing or counteracting carcinogenesis. Considering the links to the susceptibility the individual has inherited is becoming increasingly important too. At the same time it is often difficult to single out the mediating components in the protective food patterns emerging from epidemiological studies diet-cancer relationships. It is important, therefore, to identify the compounds that are most active in different experimental systems, and to study variations in their activity when they are ingested as a part of different foods, together with the combinatorial effects of phytochemicals and other food components. There is also much uncertainty regarding the relevance of observed in vitro or short-term effects in humans in relation to the long-term effects documented in chemopreventative human studies [7,23]. The newly obtained findings in the molecular nutrition area can be used to make more rapid progress in the development and validation of biomarkers. Such biomarkers should reflect not only exposure to food components but also damage to biological components (DNA, protein and lipids) and other targets of the diet-carcinogenesis interaction [18].

Björn Åkesson
16

References
1. Hunter D. Biochemical indicators of dietary intake. In: Willett W, editor. Nutritional epidemiology. Oxford University Press;1990, p. 143–216. 2. Gibson RS. Principles of nutritional assessment. Oxford University Press, 1990. 3. Bates CJ, Thurnham DI, Bingham SA, Margetts BM, Nelson M. Biochemical markers of nutrient intake. In: Margetts BM, Nelson M, editors. Design concepts in nutritional epidemiology. Oxford University Press;1991, p. 192–265. 4. Crews H, Alink G, Andersen R, Braesco V, Holst B, Maiani G, et al. A critical assessment of some biomarker approaches linked with dietary intake. Br J Nutr 2001;86 Suppl 1:S5–S35. 5. EUROFEDA consortium. European research on the functional effects of dietary antioxidants — EUROFEDA. Mol Aspects Med 2002;23:1–285. 6. Alfthan G, editor. Nordic Biomarker Seminar. Nordic Council of Ministers, Tema Nord 2005;554:1–6. 7. Heber D, editor. Nutritional oncology. 2nd ed. San Diego (CA): Academic Press-Elsevier; 2006. 8. Kaaks RJ. Biochemical markers as additional measurements in studies of the accuracy of dietary questionnaire measurements: conceptual issues. Am J Clin Nutr 1997;65 Suppl:1232S–9S. 9. Hunter DJ. The influence of genetic polymorphism. J Nutr 2006;136 Suppl:2711S–3S. 10. Molloy AM. Genetic variation and nutritional requirements. World Rev Nutr Diet 2004;93:153–63. 11. Powers HJ. Interaction among folate, riboflavin, genotype, and cancer, with reference to colorectal and cervical cancer. J Nutr 2005;135 Suppl:2960S–6S. 12. Davis CD, Hord NG. Nutritional “omics“ technologies for elucidating the role(s) of bioactive food components in colon cancer prevention. J Nutr 2005;135:2694–7. 13. Bjelakovic G, Nikolova D, Simonetti RG, Gluud C. Antioxidant supplements for prevention of gastrointestinal cancers: a systematic review and meta-analysis. Lancet 2004;364:1219–28. 14. Taylor PR, Greenwald P. Nutritional interventions in cancer prevention. J Clin Oncol 2005;23:333–45. 15. Combs Jr GF. Current evidence and research needs to support a health claim for selenium and cancer prevention. J Nutr 2005;135:343–7. 16. Rafter J, Govers M, Martel P, Pannemans D, Pool-Zobel B, Rechkemmer G, et al. PASSCLAIM — diet-related cancer. Eur J Nutr 2004;43 Suppl 2:II47–84. 17. Kavanaugh C, Seifried H, Ellwood K, Yetley E, Swanson C, Milner J. A research agenda for biomarkers as indicators of cancer risk reduction following dietary manipulation. J Nutr 2006;136 Suppl:2666S–7S. 18. Farmer PB, Emeny JM, editors. Biomarkers of carcinogen exposure and early effects. ECNIS. ¸ódê, Poland: The Nofer Institute of Occupational Medicine; 2006. 19. Mathers JC. The biological revolution — towards a mechanistic understanding of the impact of diet on cancer risk. Mutat Res 2004;551:43–9. 20. Kaput J, Rodriguez R, editors. Nutritional genomics. Discovering the path to personalized nutrition. Hoboken (NJ): Wiley; 2006.

Introductory overview and future prospects
17

21. Brigelius-Flohé R, Joost H-G, editors. Nutritional genomics. Impact on health and disease. Weinheim: Wiley-VCH; 2006. 22. Görman U. Ethical issues raised by personalized nutrition based on genetic information. Genes Nutr 2006;1:13–22. 23. Collins AR, Ferguson LR. Nutrition and carcinogenesis. Mutat Res 2004;551:1–8.

Overview of possible anticarcinogenic food components Per Mercke Biomedical Nutrition.).3.Per Mercke 18 Appendix 1. but plant-based foods appear to be their richest source. These protective compounds are found basically in all types of food. The criteria used for the selection were their relevance as adjudged from their title. The microbial flora in the gut has also been shown to play an important role in the modification of the composition and bioavailability of ingested compounds by mediating bioconversion and release of different dietary components. Different plant families harbor different mixtures of compounds of this sort. Plant tissues can also vary enormously in the store of such compounds. A step-wise search in the database PubMed for reviews linking biomarkers of nutrition and diet to cancer PubMed search Biomarker Biomarker AND diet Biomarker AND (diet OR nutrition) Biomarker AND (diet OR nutrition) AND cancer Biomarker AND (diet OR nutrition) AND cancer /reviews/ Biomarker AND (diet OR nutrition) AND cancer /reviews/last 10 years No. Sweden There is a vast range of dietary compounds proclaimed to have anti-carcinogenic properties as investigated by the scientific community. a list of putative anticarcinogenic food components has been generated (Table 1. and avoiding of similar articles by any given author. .2. emphasis on recent reviews. depending on the plant variety and the growth conditions. Lund University. To provide an overview of the cancer-protective compounds at which special attention is directed in the literature and assemble various criteria used in selecting such compounds. This information was collected with use of a PubMed search conducted in 2005. of refs 305256 3351 4173 869 181 146 The forty-seven reviews listed below were selected from among the 146 reviews arrived at last in this search. Table 1. Lund.2. for example.

Sharma and Farmer [22]. Milner [10]. Maruvada and Srivastava [19]. pyridoxine. [9]. Kelloff et al. Rafter et al. Mayne [20]. Rafter [11]. Seifried et al. [9]. Granado et al. [2]. [7]. Milner [10]. Ferguson [3]. Manson [18]. Wild et al. [14]. Konety and Getzenberg [25]. Manson et al. [2]. [1]. [2]. [23] Branca et al. Branca et al. Mason [26]. Sanderson et al. Vlastos et al.and probiotics Butyrate Vitamins Vitamin A including retinol and retinoic acids α. [2]. Kelloff et al. [21]. Seifried et al. Milner [10]. Vlastos et al. Greenwald [4]. [2]. Greenwald [4]. [7]. Crews et al.and β-Carotene Lycopene Lutein Zeaxanthin β-Cryptoxanthin Carotenoids Orange vegetables Tomatoes Green vegetables Branca et al. [21]. Manson et al. Gill and Rowland [6]. Sanderson et al. [8]. [12] Branca et al. [12] Pre. Sanderson et al. Greenwald [4]. Loft and Paulsen [17]. Mayne [20]. [23]. [15]. [23] Branca et al. [24]. Gill and Rowland [6]. Sharma and Farmer [22]. [23] Branca et al. [14]. Ferguson [3]. [8]. Wild et al. Maruvada and Srivastava [19]. Loft and Paulsen [17]. Kelloff et al. [9] Branca et al. Milner [10]. Mayne [20]. [16]. [16]. Vlastos et al. [16]. Turini and DuBois [5] Branca et al. Rafter et al. [2]. Wild et al. Kelloff et al. Ferguson [3] Starch and resistant starch Fibre/non-starch polysaccaride Vegetables Branca et al.Overview of possible anticarcinogenic food components 19 Table 1. Gill and Rowland [6]. Milner [10]. [12]. [13]. [8]. Rafter et al. Seifried et al. Ferguson [3]. Vitamin E Vitamin D Vitamin C B vitamins (Folic acid cobalamine. [7]. [12]. Ferguson [3]. [12]. [8]. [2]. Crews et al. Bowen et al. Loft and Paulsen [17]. [2]. [16]. Ferguson [3]. Vlastos et al. choline. An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected Class of compound Fatty acids Example of bioactive substance Omega-3/omega-6 Food source Vegetable oils Reference Bartsch et al. Greenwald [4]. Manson et al. [9]. [2]. Turini and DuBois [5] Branca et al. [16]. [2]. Vlastos et al. Wild et al. [21]. Rafter et al. methionine) . Sanderson et al.3. riboflavin. Ferguson [3]. Ferguson [3]. Sharma and Farmer [22]. Turini and DuBois [5]. Kelloff et al. Wagner et al. Ferguson [3].

Manson [18] Iron Iodine Isothiocyanates Benzyl isothiocyanate Cruciferous vegetables Phenethyl isothiocyanate Sulphoraphane Oltipraz (synthetic) Cruciferous vegetables Sulphones Dithiolthiones Glucosinolates Indole-3-carbinol 3. Rafter et al. Branca et al. Kelloff et al. Kelloff et al. Ferguson [3]. broad beans. Wiseman [33] . Gill and Rowland [6]. [16]. Zhang [28] Ferguson [3]. onion Radish. [30]. chocolate Epicatechin Epigallocatechin (EGC) Epigallocatechin gallate Caffeic acid Ferulic acid Ellagic acid Genistein Cereals. Ferguson [3]. Manson [18]. Manson et al. [21]. Manson [18] Phenolic acids Isoflavones Adlercreutz [31]. [21]. Milner [10]. Kelloff et al. Kelloff et al. [14]. Mayne [20]. Sharma and Farmer [22] Branca et al. Greenwald [4]. potatoes. [2]. [16]. [2]. [23] Bianchini and Vainio [27]. [7]. Rafter et al. [9]. Sharma and Farmer [22] Flavonoid polyphenolics Catechins Catechin Tea. [2]. garlic. Manson [18]. kale. [2]. Seifried et al. An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected — cont. [2]. [14]. [2]. Milner [10]. Manson [18]. Seifried et al. Branca et al. Wild et al. scallions. Manson [18] Crews et al. endive Citrus fruits Branca et al. Manson [18]. Greenwald [4]. squash. Atkinson and Bingham [32]. Sanderson et al.3. Rafter et al. Manson et al. [9]. Manson [18]. broccoli. horse-radish. Sharma and Farmer [22]. tomatoes. Milner [10]. Sanderson et al. [16]. Ferguson [3]. [7] Allium compounds Onions. chives Citrus fruits. Sanderson et al. Kelloff et al. [7]. Crews et al. [21]. Rafter et al. [16]. Seifried et al. Sanderson et al. [23]. Seifried et al. Gill and Rowland [6]. [8]. Kelloff et al. Manson et al. Wild et al. Manson et al. [16]. [9]. soybeans) Branca et al. [9].Per Mercke 20 Table 1. Class of compound Minerals and trace elements Example of bioactive substance Selenium (including selenomethionine and other selenium compounds) Calcium Food source Reference Branca et al. Maruvada and Srivastava [19]. Ferguson [3]. [21] Ferguson [3]. [7]. Ferguson [3]. [8]. berries.3’-Diindolylmethane Indole-3-acetonitrile Diallyl sulphide Allylmethyl trisulphide Tangeretin Apigenin Nobiletin Rutin Quercetin Kaempferol Taxifolin Cruciferous vegetables Bianchini and Vainio [27]. [8]. Saleem et al. [16]. [8]. Duthie and Crozier [29]. pulses (millet. Ferguson [3]. sorghum. Turini and DuBois [5] Maruvada and Srivastava [19].

Rafter JJ. Bartsch et al.43 Suppl 2:II47–84. 11.88 Suppl 1:S73–87. Johnson IT. 3. J Cell Biochem 1996. Rowland IR. strawberries. DuBois RN. Kelloff et al. Manson MM. Rafter J. Branca F. Emerging dietrelated surrogate end points for colorectal cancer: UK Food Standards Agency diet and colonic health workshop report.Overview of possible anticarcinogenic food components 21 Table 1. 10. 2. Bartsch H. cacao Manson [18] Reference Monoterpenes Citrus fruits (peel) Ferguson [3]. PASSCLAIM — dietrelated cancer. Biomarkers in disease and health. Prospects for cancer prevention. Gill CI. McGlynn AP.383:915–21. et al. J Nutr 2003. Turini ME. [8] Other non-flavonoid phenolics Lignans Olive oil Adlercreutz [31]. sis: potential role of lipid peroxidation. Diet and cancer: assessing the risk. Powers HJ. Nair J. raspberries.86 Suppl 1:S55–S92. pecans. coffee. Pool-Zobel B. Turmeric Branca et al. Hematol Oncol Clin North Am 2002.88 Suppl 2:S219–24. [2]. Milner JA. Br J Nutr 2001. Hanley AB. walnuts. 6. Class of compound Methylxanthines Example of bioactive substance Caffeine Theophylline Theobromine Limonene Perillyl alcohol Geraniol Menthol Carvone Hydroxytyrosol Curcumin Resveratrol Food source Tea. Primary prevention: phytoprevention and chemoprevention of colorectal cancer. An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected — cont. Biol Chem 2002. black-berries. Innovative agents in cancer prevention. 7. Martel P. 8.25 Suppl:29–36. Pool-Zobel B.166:257–75. Eur J Nutr 2004. Rechkemmer G. olive oil References (selected from the PubMed search described above) 1. Br J Nutr 2002. . Ferguson LR. Wiseman [33] Grapes. Br J Nutr 2002. Manson [18]. Scientific basis of biomarkers and benefits of functional foods for reduction of disease risk: cancer. [1]. Gescher A.428:329–38. Sanderson P. 9. Mutat Res 1999. Recent Results Cancer Res 2005. Steward WP.91:315–23. Owen RW Exocyclic DNA adducts as oxidative stress markers in colon carcinogene. Br J Nutr 2004. Cereals. Incorporating basic nutrition science into health interventions for cancer prevention. et al. dietary fat and antioxidants. cola. Govers M. [16].3. 5. Downes CS. Greenwald P. Farmer PB.133 Suppl 1:3820S–6S. 4. Rafter et al. Mathers JC.16:811–40. Pannemans D. Verhagen H. Cancer risk factors for selecting cohorts for large-scale chemoprevention trials.

36:655–67. Maruvada P. Br J Nutr 2001. Stacewicz-Sapuntzakis M.48:169–88. Follen M. J Mol Med 1996. A critical assessment of some biomarker approaches linked with dietary intake. et al. J Nutr 2003. Br J Nutr 2001.4:1–4. 18. Biological relevance of adduct detection to the chemoprevention of cancer. Mammographic breast density as a biomarker of effects of isoflavones on the female breast.130 Suppl:467S–71S. Wild CP.46:261–73. Loft S. Phytoestrogens and breast cancer. O'Brien NM. Zhang Y.133 Suppl 3:941S–7S.86 Suppl 1:S37–53. 25.63:4295–8. Olmedilla B. Tea beverage in chemoprevention of prostate cancer: a mini-review. . J Nutr 2004. Srivastava S. Cancer prevention — the potential for diet to modulate molecular signalling. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. 16. Braesco V. Farmer PB. Granado F. Breast Cancer Res.29:95–106. Isothiocyanates in cancer prevention. Bowen P. Steele VE. Manson MM. 24. Schottenfeld D. Cancer Res 2003. Wagner KH. Gamma-tocopherol — an underestimated vitamin? Ann Nutr Metab 2004. McDonald SS.Per Mercke 22 12. Andersson C. J Steroid Biochem Mol Biol 2002.3:447–51. Milner JA. et al. J Nutr 2003. Exp Biol Med (Maywood) 2002. Progress in cancer chemoprevention: development of diet-derived chemopreventive agents. Biomarkers for cancer diagnosis: implications for nutritional research. Sharma RA. Vitamin D and prostate cancer.74:297–312. Siddiqui IA. Lubet RA. 21.90:487–502.10:4901–12. 28. 19. Duthie G.227:886–93.9:11–8.83:113–8. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Getzenberg RH. 30. Alink G.86 Suppl 1:S5–35. Wilson L. Mukhtar H. Crowell JA. 22. Mayne ST. 2002. Curr Opin Clin Nutr Metab Care 2000. 23. Boone CW. 26. Kelloff GJ. Vlastos AT. 32. 20. Seifried HE. Ghosh L. Drug Metab Rev 2004. Elmadfa I. Nutr Cancer 2003. 29. Clin Cancer Res 2004. Sharifi R. Kamal-Eldin A.47:13–23. Trends Mol Med 2003. Woods JA. A critical evaluation of the application of biomarkers in epidemiological studies on diet and health. 13. Poulsen HE. The antioxidant conundrum in cancer. 15. Bianchini F. Br J Nutr 2003. Anderson DE. Saleem M. Crews H. Holst B. Chen L. Greenwald P. et al. J Nutr 2000. 31. Plant-derived phenolic antioxidants. Andersen R. Maiani G. Adhami VM. Crit Rev Oncol Hematol 2003. Cancer risk and oxidative DNA damage in man.133 Suppl 3:933S–40S. Bingham SA. 14.134 Suppl:1640S–5S. Biomarkers and their use in cervical cancer chemoprevention. Mutat Res 2004. Urol Clin North Am 2002. Nutritional and clinical relevance of lutein in human health.555:173–90. Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis. 27. Mason JB. 17. Konety BR. Vainio H. Duncan C. Blanco I. Malone WA. Atkinson C. Adlercreutz H. Biomarkers of nutrient exposure and status in one-carbon (methyl) metabolism. Crozier A.

Lifestyle/dietary supplement partial androgen suppression and/or estrogen manipulation. Antioxidant vitamins: evidence from biomarkers in humans. Kampman E. Biomarkers. Public Health Nutr 2002. Biochemical markers as additional measurements in dietary validity studies: application of the method of triads with examples from the European Prospective Investigation into Cancer and Nutrition. part I: time to redirect our attention? Urol Clin North Am 2004.6:147–51. Limburg PJ. low-dose carcinogenesis and dietary exposures.55:46–67. Frei B. Moyad MA. Future directions. Tahir A.5:977–84.31:289–300. Who's afraid of n-6 polyunsaturated fatty acids? Methodological considerations for assessing whether they are harmful. 38. 44. Sarhill N. Molecular epidemiological approaches to the study of the genotoxic effects of urban air pollution. 37. 45. 39.11:181–8. Mahmoud FA. Nutr Rev 1999. 43. Nutr Metab Cardiovasc Dis 2001. Surg Clin North Am 2002.73:70–8. 42. Carroll PR.82:905–41. Sugar E. Viner JL.492:255–62. 40. Arts IC. observational versus experimental human studies.20:465–73. Bell SM.29:115–24. Neuhouser M. Patterson R. Establishing the significance and optimal intake of dietary antioxidants: the biomarker concept. Am J Clin Nutr 1997. 41. Ocke MC.9:1829–40.Overview of possible anticarcinogenic food components 23 33. Rivlin RS. 46. Wang CY. Prentice RL. Expert Opin Investig Drugs 2000. Am J Hosp Palliat Care 2003. The therapeutic potential of phytoestrogens. Research strategies and the use of nutrient biomarkers in studies of diet and chronic disease. Georgiadis P. Kyrtopoulos SA.428:91–8. Int J Vitam Nutr Res 2003. Moyad MA. Christie R. Lifestyle recommendations to prevent prostate cancer. Vineis P.57:104–13. Nutrition and cancer prevention: new insights into the role of phytochemicals. McCall MR.65 Suppl 4:1240S–5S. Halliwell B. 34. Eur J Cancer Prev 1997. Quirke P. Plant foods versus compounds in carcinogenesis. 47. Hawk ET.195:111–34. . Epidemiology and prevention of colorectal cancer. Kaaks RJ. Hollman PC. 36. Bibl Nutr Dieta 2001. Berry EM. J Pathol 2001. Mutat Res 1999. Adv Exp Med Biol 2001. Methylation and colorectal cancer. A novel PSA reducer and preventive/treatment option for prostate cancer? Urol Clin North Am 2002. Jubb AM. Assessment of nutritional status and fluid deficits in advanced cancer. 35. Wiseman H.

.

such as pro-vitamin A carotenoids. has also been demonstrated [1]. except for markers of DNA damage. The possibility of using dried blood spots. Direct fluorescence methods for assessing the retinol level in plasma or in dried blood are feasible because of the high intensity of retinol fluorescence when it is bound to its transporter. The direct-phase methods can also typically measure a large number of other lipid-soluble vitamin isomers in the same run. tocopherols and tocotrienols. menadione and phylloquinones. C and E Lars O.1. the retinol binding protein (RBP) [1. the development of fast and simple methods for the determination of vitamin A status has long been given a high priority. a technique that was later superseded by various chromatographic and fluorescent techniques.4] or reversed-phase [5–7] liquid chromatography . which is advantageous from a collection standpoint. these techniques took over and today retinol can be determined in serum routinely by direct. Vitamins and selenium 2. biological markers of the dietary exposure to these vitamins is of importance. Biomarkers of exposure to and effects of vitamins A.[3. which are dealt with elsewhere in this volume.2. Dragsted University of Copenhagen. With the advent of high performance liquid chromatography (HPLC). The structure of vitamin A and pro-vitamin A carotenoids is shown in Figure 2. The reversed-phase techniques are faster and smaller sample volumes suffice but they are generally unable to discriminate between the various isomers of vitamin A to the same extent as the direct-phase methods can. xanthophylls. The observation that RBP occurs in plasma in a virtually 1:1 ratio to retinol has prompted the development of radial diffusion assays and enzyme immunoassays for RBP as a surrogate marker for plasma or serum retinol [9–11].2]. Vitamin A Biomarkers for vitamin A in body fluids The vitamin A (all-trans-retinol and its esters) level was originally determined by bioefficiency assay. Copenhagen. There is also a need of effect biomarkers for determining the ability of these and other antioxidants to increase the overall antioxidant capacity and decrease the oxidative damage occurring in biological samples. Due to worldwide concern for vitamin A deficiency (VAD).1. This chapter is concerned with such markers. Denmark Since antioxidant vitamins can affect an organism’s capacity for defence against reactive oxygen species. although reversed-phase methods able to separate certain of the retinol isomers have been published [8]. .

Lars O. Dragsted
26

A comparison of the various tests in terms of price, speed of performance and coefficient of intraindividual variability is shown in Table 2.1. Although the accuracy for each of the methods taken up in Table 2.1. is good (less than 5% deviation from the standards) and the interday variability is low, the correlation coefficient between the HPLC methods and ELISA is only around 0.8, probably due to differences in linearity. Since the latter methods are less demanding in terms of equipment they have considerable potential for screening purposes in the less developed countries where VAD is still causing blindness, growth retardation and decreased resistance to infections in large numbers of children.

Fig. 2.1. Structures of retinol (vitamin A) and of the most important pro-vitamin A carotenoids.

Table 2.1. The performance of different methods for determining serum vitamin A

Test method Reversed-phase HPLC Direct-phase HPLC Fluorescence ELISA (RBP)
a

CV% 4 4 <10 9

Cost/sample (relative) 20 30a 2a 1

Speed (samples/day) 25 20 50–100 150
a

Reference [7] [4] [1] [9]

Estimates from the author’s lab. This may change with new faster LC techniques.

Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A, C and E
27

Biomarkers of vitamin A related effects in the eye

VAD leads to dryness of the conjunctiva of the eye and moderate deficiency leads to decreased night vision due to interruption of light-sensitive chemical processes in the eye. Permanent blindness may ensue in severe cases. The WHO has compared the sensitivity of different methods for determining VAD (as modified in Table 2.2.). Biological effects on the eye are still the only reliable means of detecting moderate to severe VAD, whereas the biochemical detection of retinol in blood samples is needed to identify mild cases and to be able to intervene at an early stage [12]. As already indicated, simple yet sensitive assays to determine the presence of this condition are thus still in demand. Histological markers that are employed include corneal cytology of the eye by direct visual inspection and by sampling a specimen of the conjunctiva for staining and microscopy. Functional markers include dark adaptation and the ability to see contrasts. The direct visual tests include staining with rose Bengal to visualize dry areas or to detect inflammation, but tests of this sort have been shown to be less reliable [13]. The standard today is the conjunctival impression cytology test which makes use of a vacuum pump to lift a small portion of the epithelium from the inferior temporal conjunctiva onto a filter paper disc, fix it in glacial acetic acid and stain it with periodic acid Schiff and haematoxylin for histological examination [14,15].
Table 2.2. Biological indicators of subclinical VAD*

Indicator (cutoff) Functional tests Night blindness (age-specific) Biochemical markers Serum retinol (≤ 0.70 Ķmol/l) Breast-milk retinal (≤ 1.05 Ķmol/l) Histological markers Abnormal conjunctival impression cytology

Prevalence cutoffs for defining a public health problem and assessing its level of importance mild > 0 to < 1% ≥ 2 to < 10% < 10% < 20% moderate ≥ 1% to < 5% ≥ 10% to < 20% ≥ 10 to < 25% ≥ 20% to < 40% severe

≥ 5% ≥ 20% ≥ 25% ≥ 40%

* Common biological indicators of subclinical VAD in children 6–71 mo of age. A public health problem is considered to exist when the prevalence criteria of at least two of the above indicators of VA status are met (adapted from [1,12].

The dark adaptation tests measure the time needed to adapt to a defined level of limited illumination. A fast adaptation procedure involving the ability to discriminate between red and blue objects for field and for screening studies has been described [16]. When the eye shifts from cone-mediated day vision to rod-mediated night vision, the Purkinje shift occurs, the retinal peak light sensitivity shifting from red towards blue, blue objects being perceived then as lighter shades of grey than for red objects. Patients have to be taught use of the test, which must be repeated afterwards several times. In some studies the test results have been found to be more closely related to vitamin A intake by dietary assessment than to the plasma retinol level [13].

Lars O. Dragsted
28

Biomarkers of oxidative stress after supplementation with vitamin A

There seem to be no studies reporting on markers of oxidative stress or oxidative status following intervention with use of increased doses of vitamin A. Neither retinol nor beta-carotene supplements to blood samples in vitro have been found able to affect a number of markers for antioxidant stability of the plasma and erythrocytes [17], which indicates that this vitamin may have a limited capacity to act as an antioxidant in vivo.
Conclusions

Serum retinol is still the most reliable indicator of vitamin A deficiency. HPLC methods are the most sensitive, but they are not useful for large screenings or for field studies in poor areas of the world where deficiency is a common problem. Although simpler tests using fluorescence, as well as immunological techniques possessing good accuracy and high precision exist, there is still a need of methodology which is simpler, faster and cheaper yet and requiring no complicated sample treatment or use of complex equipment. Vitamin A appears to have only limited capacity as a direct antioxidant in vivo.

Vitamin C
Biomarkers of vitamin C in body fluids

Vitamin C (ascorbate and dihydroascorbate, Figure 2.2.) levels have been determined in plasma, serum, dialysates and other body fluids by colorimetric and fluorimetric techniques, by enzymatically based assays and by HPLC with or without post-column derivatisation. Since ascorbic acid is easily oxidised to dehydroascorbate, which can subsequently be degraded to diketogulonic acid, initial treatment by stabilising acids such as metaphosphoric and perchloric acids has to be performed quickly after isolation of a sample for analysis. The effects of the anticoagulants used during sample collection has been compared in one study, heparin being found to result in only a minimal loss, EDTA in contrast giving rise to a significant loss of vitamin C within a 30 min period. Also, oxalate and citrate were found to be less efficient in stabilizing ascorbate than other anticoagulants were [18]. Storage time of the sample and storage conditions are important factors determining the stability of vitamin C. In one early study, the concentrations of ascorbate and dehydroascorbate were found to be unaffected in samples stored in the laboratory at a temperature of 12°C for up to 6 hours [19], but in other studies considerable time- and temperature-dependent losses were found already from the first hour of storage onwards even after optimization of the collection conditions [18]. In another study the storage time and temperature were found to have no effect on loss of vitamin C during 2–14 day storage at either –25°C or –75°C, but a 3.5% loss due to freezing was observed [20]. In a third study, pre-treatment with metaphosphoric acid was compared with treatment by dithiotreitol, a commonly used laboratory antioxidant. The latter performed slightly better than the former since no loss of vitamin C was evident after storage at –80°C for 6 years, whereas the standard procedure involving the addition of metaphosphoric acid led to a small but significant mean loss of < 1% per year [21].

allowing high sample handling rates to be achieved through automation [25]. possibly reflecting higher leakage of haem in this group. with ascorbate and dihydroascorbate leading to the formation of a chromophore or a fluorophore. and about 1. together constituting vitamin C. A method for the simultaneous detection of ascorbate and uric acid by means of capillary zone electrophoresis (CZE) has also been described [29]. Dehydroascorbate is not readily determined by use of this approach. 2. which can be photometrically measured by use of manual or automated equipment. but a few of them use assay blanks produced by adding ascorbate oxidase to the sample. Most of these methods are quite unspecific [24]. since treatment with dithiotreitol is known to reduce dehydroascorbate to ascorbate. Some of these methodologies are very fast. Postcolumn derivatization can be used to reduce dihydroascorbate so that it can be determined by use of an electrochemical detector. around 0. erythorbate. Structures of ascorbate. Recovery is better than 98% with .8% under the same conditions in the case of smokers [23].Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A.27–28]. the stereoisomer of ascorbate.2. C and E 29 However. the ascorbyl radical and dihydroascorbate. this procedure cannot be recommended if both compounds are to be determined separately. The normal range of plasma concentrations of dehydroascorbate is controversial and the observation of this compound in plasma may be a result of metalcatalysed oxidation following acidification [22]. There was a significant increase in the concentration of dehydroascorbate over time at low total vitamin C concentrations [23]. If dehydroascorbate is physiologically present. The HPLC methods for detecting plasma ascorbate electrochemically give results similar to those using UV detection [26]. Fig. Results in determining plasma ascorbate in this way correlate well with those obtained by use of chromatographic methods [18]. The colorimetric and fluorometric methods are generally based on redox-reactions. can be determined simultaneously [25. its true concentration seems to be very low. creating greater sensitivity with retention of speed.1% of the total plasma vitamin C in non-smokers when the sample has been handled carefully.

In interlaboratory comparisons. following HPLC separation. The interday coefficient of variation (CV) for TBARS with use of HPLC is in the order of 10–20 %. ex vivo LDL oxidation. The method is based on the liberation of aldehydes from amino groups by acid or alkaline hydrolysis followed by a colour reaction involving use of thiobarbituric acid. whereas the intralaboratory variation was about 2 µmol/l. since it may partially measure aldehydes deriving from endogenous metabolism. to detect malondialdehyde. plasma lipid hydroperoxides. which led to relatively larger relative errors being registered at low concentrations [21]. depending on the method employed for hydrolysis. Dragsted 30 use of the HPLC and CZE methods and good linearity is obtained even at low ascorbate concentrations. antioxidant capacity markers. In a European study of laboratories carrying out population surveillance. These flaws have caused some journals to generally regard the method as being invalid as a lipid oxidation biomarker [32]. The most commonly used marker is determination of TBARS. irrespective of the concentration.1 mg/l 0. and since there is no generally accepted assay procedure [31]. and plasma or urinary isoprostanes.Lars O. In another study an interlaboratory difference of about 15% was observed.39] Vitamin C and lipid oxidation markers Many different biomarkers of radical mediated lipid oxidation exist but for the purpose of this review the more commonly used assays appear adequate for comparing studies in this area. Table 2. The performance of different methods for the measurement of vitamin C in plasma is summarized in Table 2. quite disparate results have been obtained with use of these techniques. The product can be determined spectrophotometrically. Typical performance of different methods for determination of plasma vitamin C Test method Reversed phase HPLC Capillary electrophoresis Automated colorimetry ND — not determined.3.5 mg/l 3 mg/l Speed (samples/day] 40–50 40–50 500 Reference [36] [37] [38. The assays employed to this end here are the following: plasma thiobarbituric acid reactive compounds (TBARS). either directly or online. .3. a 13–20% interlaboratory variation was found using plasma samples in the ‘normal’ range of 36–94 µmol/l in a second round after corrections had been instituted at several laboratories [30].3% < 5% CV% DHAA interday < 6% ND ND Limit of detection 0. but as already mentioned this relatively simple method has an odd tendency of sometimes giving spurious results and of varying from one analytical series to another. also within the same laboratory. with or without calibration. Only randomised study designs are included in this review. CV% AA interday < 4% 1. This marker is highly controversial since it is variable both within and between laboratories.

In a larger study involving 56 smokers. In a third study of this sort. A faster method applicable directly to a plasma or serum sample overcomes this problem by using a peroxide-sensitive fluorescent probe with high affinity for LDL [43]. In a smaller parallel study of vitamin C supplementation (1 g/d) versus placebo. Oxidative stress in 10 volunteers as determined by increased plasma MDA induced by infusion of free fatty acids was also found to be unaffected by high-dosage vitamin C infusion [39]. The method is disputed because the outcome depends on the antioxidants present in the LDL and these may be lost during LDL isolation. as compared with placebo treatment. giving a combination of vitamin C (272 mg/d) and vitamin E (800 IU/d) compared to placebo was found to not affect plasma MDA in 77 smokers treated for 90 d [38]. 30 mg beta-carotene and 100 µg organic selenium. Overall it appears that the intake of vitamin C does not consistently affect plasma MDA but that significant increases may be observed following the infusion of large. C and E 31 In a randomised 2-months intervention study of 59 healthy smoking males MDA was found to increase significantly following daily doses of 250 mg ascorbate combined with 200 IU vitamin E. no effect was observed at 12 or at 36 months in the vitamin C group in terms of susceptibility of isolated LDL or VLDL to oxidation ex vivo. randomised double-blind crossover study the effect of vitamin C supplementation (six weeks.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. acute doses. in which isolated LDL is exposed to copper chloride or to a semistable radical such as AAPH. given in a normal formulation. or a placebo in a parallel design. vitamin C plus 182 mg/d dl-α-tocopherol. 182 mg/d dl-α-tocopherol alone. Infusion of large doses of vitamin C (5g) in combination with EDTA treatment resulted initially in a marked increase in plasma MDA but in an overall decrease in this parameter after 16 repeated sessions [40]. No differences between groups in plasma malondialdehyde concentrations were observed either before or after supplementation [34]. In addition. the oxidative formation of conjugated dienes being followed spectrophotometrically at 234 nm [42]. 250 mg/day) was determined in 20 subjects each showing normal (67 µmol/l) or below average (32 µmol/l) plasma vitamin C concentration at baseline. In another counterbalanced design. in which 19 smokers participated for 4 weeks following a 2-week . by a decrease in plasma MDA concentration [41]. In a group of 48 middle-aged male and female participants in a 36-month intervention study receiving 500mg/d of either vitamin C. The interday CV for this latter assay is less than 10%. Oxidative stress induced by Zn deficiency was found to respond to 250 mg/d vitamin C. In another. No effect of either treatment on plasma MDA was observed [35]. the intake of 500 mg/d of vitamin C was found not to affect MDA as determined by HPLC [37]. In a study comparing 8 smoking women with 8 controls during a 14-day period in which 1 g ascorbate was administered daily plasma TBARS was found to not be affected [36]. but MDA to be unaffected by a slow-release formulation as compared with placebo treatment [33]. given for 3 months. Another controversial marker used by many laboratories is the ex vivo LDL oxidation assay. no change in whole plasma ex vivo oxidation was observed in this group [44]. The lag-time to oxidation and/or the slope of the oxidation curve are used as end points. 25 males were exposed to vitamin C (500 or 1000 mg/d) and/or to exercise.

the ferric reducing ability of plasma (FRAP) assay and the trolox equivalent antioxidant capacity (TEAC) assay which also correlated with ex vivo LDL oxidation [58. The infusion of 1000 mg ascorbate for a 1 h period in ten healthy volunteers was found to result in an increased antioxidant capacity as measured repeatedly by two different methods during both the infusion period and the hour following this [60]. Supplementation by 1 g/d ascorbate for 4 weeks in 11 healthy volunteers failed to change the plasma LDL oxidation lag-time as compared with 9 controls [51]. Dragsted 32 ascorbate depletion period (≤ 30 mg/d). Overall there seems to be no consistent effect of vitamin C supplementation on ex vivo LDL oxidation kinetics. there was a significant increase in ex vivo LDL oxidation lagtime in the vitamin-supplemented group [45]. In a study with 30 coronary artery disease patients there was no evidence after 6 months that random assignment to vitamin C (1 g/d) together with vitamin E (800 IU/d). In a subsequent study of 30 young smokers. only a limited response of this sort was observed with use of the FRAP assay during the 24-hour period following a single dose of 500 mg ascorbate with or without 400 IU vitamin E [62].Lars O. especially for whole plasma. the size of the CVs depending on the exact wavelength used for determining the absorbance. In another group. no such increase was observed despite changes in plasma ascorbate [50]. At the same time. These methods generally have interday CVs of less than 10% for repeated measurements of the same sample. decreased LDL oxidation or antibodies to oxidised LDL [47]. 60 mg or 6 g of vitamin C was administered daily during 14 day-periods separated . no effect was observed after 8 weeks supplementation by 1 g/d of vitamin C [46]. They are all ex vivo oxidation systems composed of some oxidising system and some relatively simple marker of plasma oxidation. There are important differences between the methods which call for caution when they are interpreted [56]. A borderline effect on LDL oxidation was observed in a similar study with only 18 participants after a shorter period of 12 weeks [48]. However. No effect on LDL oxidation lag-times of 500 mg/d vitamin C supplementation for 4 weeks as compared with placebo was observed in 30 type II diabetics [49]. the availability of a photometer with exact filters or one equipped with a narrow grid being required.59]. When applied to plasma samples some authors accordingly report poor correlation between methods [57] whereas others observed a high degree of correlation between some of the most commonly applied methods. Marked increases in antioxidant capacity in the plasma of elderly women during the 4 h period after a dose of 1250 mg ascorbate was given were also observed using three different antioxidant capacity measures [61]. or placebo. receiving only 60 mg vitamin C plus iron per day. an increase in ex vivo LDL oxidation lag-time during a 12 week-period was observed in 20 volunteers receiving 260 mg vitamin C in combination with 14 mg iron/d. usually one leading to a change in visual or UV absorption of the test matrix [52–55]. The oxidizability of LDL is inherently an antioxidant capacity assay for this particular compartment. A variety of antioxidant capacity markers exist for other blood compartments. In a study without a control group. in a cross-over intervention study involving 48 non-smoking men and women. in which 0 mg.

Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A, C and E
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by 6 weeks wash-out periods, the plasma antioxidant capacity of these persons was found to be significantly affected [63]. In a 14 d parallel study of 16 women who smoked, half of them treated with 1 g ascorbate daily and half of them given placebo, no effect on the antioxidant capacity of the plasma could be shown [36]. Neither was the plasma TEAC found to be affected by a 150 mg/d vitamin C supplement in a 2-week study involving 18 volunteers [64]. No effect of antioxidants on TEAC could be observed either in 39 lupus erythromatosis patients randomised to being given 500 mg vitamin C together with 800 IU vitamin E a day or a placebo for a 12-week period [65]. In a group of 48 middle-aged male and female participants in a 36-month intervention study in which 500 mg/d of vitamin C, vitamin C plus 182 mg/d dl-α-tocopherol, 182 mg/d dl-α-tocopherol alone, or placebo were given daily in a parallel design, no effect in either of the vitamin C groups on the plasma antioxidant capacity as determined by the TRAP assay was observed at either 12 or 36 months [44]. At the same time, the antioxidant capacity in 6 moderately trained males was found to be significantly affected, following 2.5 h of strenuous exercise stress, by the intake of a drink containing vitamin C (0.15%, approx. 200 mg ascorbate) [66]. In contrast, FRAP was not found to be affected by 1h of hard exercise following the daily administration of 600 mg vitamin C in combination with a range of other micronutrients for a 7-day period [67]. In these various studies, the effect of vitamin C on antioxidant capacity measures could not always be explained simply by an increase in the plasma ascorbate concentration. It appears that, although vitamin C may increase the antioxidant capacity in normal, healthy individuals, the effect is more evident short- term and may be partially be due to indirect actions of unknown character. Although plasma lipid hydroperoxides can be determined individually by HPLC together with electrochemical detection [68,69] or collectively by means of hydroperoxide-sensitive photometric assays [70–72], in most published papers the determination of ‘lipid hydroperoxides’ is a euphemism for TBARS. The effect of ascorbate supplementation on the plasma lipid hydroperoxide concentration in humans under normal conditions has not been frequently reported. The formation of lipid hydroperoxides in LDL was not found to be affected by a 150 mg/d vitamin C supplement in a 2-week randomised study involving 18 volunteers [64]. The effect of an ascorbate supplement in reducing the plasma hydroperoxide level following various conditions of oxidative stress showed a significant effect on this marker. A 30% reduction in exerciseinduced total lipid hydroperoxides was observed after acute supplementation of vitamin C [73]. Also vitamin C supplementation in conjunction with biweekly apheresis for 6 months in dyslipidemic and uremic patients markedly increased the efficiency of such treatment in reducing the plasma phosphatidylcholine hydroperoxide level as determined by HPLC [74]. Thus, Vitamin C seems to affect lipid hydroperoxide formation in some oxidative stress conditions. The least disputed lipid oxidation methods are the isoprostane assays. The determination of plasma isoprostanes can be performed by gas chromatography/mass spectrometry (GC-MS) using electron capture negative ionization (ECNI) or negative ion

Lars O. Dragsted
34

chemical ionization detection (NCI). The compounds need to be extracted from the sample and derivatized. The extraction has not always been straightforward and has involved multiple chromatographic steps, including thin layer chromatography, and has led to a poor overall recovery [75]. Improvements have included a combination of solidphase extraction cartridges and HPLC [76,77], two sequential solid-phase extractions [78] and most recently a single anion exchange cartridge [79]. The derivatization of the compounds is most often done by use of pentafluorobenzyl bromide followed by a silylation step employing bis-(trimethylsilyl) trifluoroacetamide [79]. The overall extraction efficiency is now around 70%, the interday analytical CV% varying from less than 1% to 5–8%, depending on extraction procedures. Since the interindividual variation is much larger, this method has considerable power for detecting the factors causing biological variation. An immunological method for the determination of 8-isoprostane F2α having 5–15% analytical variation also exists [80]. The formation of isoprostanes in plasma was found to not be affected by 150 mg/d vitamin C supplementation in a 2-week study involving 18 volunteers [64]. Using the chromatographic method on plasma samples, no change was observed after a daily dose of 272 mg vitamin C in combination with vitamin E (31 mg/d) and folate (400 µg/d) for a 90 day period in elderly male smokers and non-smokers [38]. A doseresponse study of hospitalised women in which vitamin C (30–2500 mg/d) was administered to them for 186 days following a short ascorbate-depletion period, gave no evidence of change in the levels of isoprostanes in the urine or plasma [81]. In a study of 30 coronary artery disease patients limited evidence was obtained after 6 months that random assignment to vitamin C (1 g/d) together with vitamin E (800 IU/d) versus placebo led to a decrease in plasma isoprostanes [47]. No studies of the effect of ascorbate supplementation on oxidative stress-induced isoprostanes have been reported. The performance of plasma lipid oxidation markers and the effects on them of vitamin C being administered is summarized in Tables 2.4. and 2.5. Overall, the effect of vitamin C on lipid oxidation markers seems limited to a possible effect in certain oxidative stress conditions.
Table 2.4. Performance of plasma lipid oxidation markers

Test method TBARS (HPLC) LDL ex vivo oxidation Antioxidant capacity Lipid peroxides 8-Isoprostane F2α EIA Isoprostanes GC-MS
a

Analytical CV% 15—20 5—10 <10 <10 5—15 <1—6

Normal variation CV% 25 20 20—25 60—65 20—50 50

Speeda (samples/day) 50 10/50 >100 20 50—100 50

Reference [83] [42,43] [55] [72] [80] [78,79]

Estimates in the author’s laboratory with use of standard semiautomated equipment.

Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A, C and E
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Table 2.5. Summary of effects of vitamin C on plasma lipid oxidation markers

Treatment Vitamin C Stress + vitamin C
NR — not reported.

MDA — —

LDL oxidation ex vivo — —

Antioxidant capacity +/— +/—

Lipid hydroperoxides — +

Isoprostanes — NR

Vitamin C and protein oxidation

Proteins have no natural carbonyl groups, so such groups are introduced into proteins by oxidative mechanisms, including the oxidative deamination of the e-amino group in lysine and the oxidation of carbons next to the secondary amine functions in proline and arginine. Other assays for oxidatively modified amino acid residues in proteins include the measurement of preformed hydroxytyrosine, dityrosine, and sulphoxides (e.g. methionine sulphoxide), and the loss of protein sulfhydryls. Oxidised proteins can denature, the denatured proteins being quickly chaperoned towards proteolytic degradation in vivo, but the oxidation may also be insufficient to denature the protein so that protein carbonyls are allowed to accumulate to some extent. The steady-state concentration of protein carbonyls and other oxidative modifications in the plasma or in other biological specimens may thus reflect the accumulated oxidative stress in the compartment in question during the lifetime of the protein, thereby providing a potentially valuable biomarker for low-level oxidative stress. Several approaches to developing biomarkers for protein oxidation have been taken, but only few of them have been applied to studying the effects of micronutrient supplementation. The simplest assay is based on the reaction of carbonyl groups with primary amines to form semi-stable Schiff-bases. The reaction with 2,4-dinitrophenylhydrazine (DNPH) is commonly used for the photometric determination of carbonyls. In its simplest form, it involves the sample reacting with DNPH, precipitation and a washing procedure to remove the unspecifically bound DNPH. After washing, the protein is solubilised to determine the level of specific binding photometrically. This method has the advantage of being fast and of low technical demands. The disadvantages include difficulties in removing the unspecifically bound DNPH, leading to high and variable background readings, a variable loss of protein during washing, and a risk of introducing additional oxidative modification during the procedure, leading to binding of the excess DNPH. The first two disadvantages can be partly overcome by isolating a specific protein fraction prior to precipitation, which leads to a much greater loss of the unspecifically bound DNPH. It also introduces the additional advantage of selecting the specific protein for study, since oxidation can vary between proteins due to differences in the redox microenvironment surrounding the proteins. Another solution is to use an immunoassay procedure involving specific antibodies to the DNPH-modified protein, thus avoiding the unspecifically bound DNPH and extensive washing.

vegetables or nutrient supplements was found in that study on the levels of total plasma carbonyls or immunoglobulin carbonyls by the antibody approach in conjunction with Western blotting [85]. In a very small study. including 150 mg/d vitamin C. The method has been used to study the effect of supplementation by 500 mg/d of vitamin C versus placebo for 30 days in 16 healthy volunteers. The decrease was confined to individuals with a suboptimal intake of vitamin C. the effect of taking 272 mg/d of vitamin C supplement in conjunction with vitamin E (800 IU/d) and folate (400 µg/d) for 90 d was assessed in 39 smokers and 38 non-smokers who habitually had a low intake of fruit and vegetables. Since vitamin C is a cofactor for the lysine oxidases that cross-bind cartilage in the body. showing a rapid depletion of plasma ascorbate and a concomitant decrease in AAS. Another line of evidence comes from studies of dietary fruit and vegetable depletion. unpublished data). None of the volunteers had suboptimal vitamin C levels before intervention. whereas no change in the total plasma sulfhydryls was detected [85]. No effect of the supplement on protein carbonyls was observed [38]. the fluoresceinamine adduct could be detected by HPLC through the use of UV. Using the same method for protein carbonyls. A third method employed for assessment of protein oxidation takes advantage of the reaction of protein aldehydes with fluorescein and reduction of the product by cyanoborohydride to form a stable adduct. No effect of fruit.Lars O. the observed effect of vitamin C on this specific marker may reflect enzyme leakage from the cartilage rather than prooxidative stress. Use of this approach was found in one study to produce a significant time-dependent decrease in AAS during a 24-day period of fruit and vegetable depletion in the diet. A marginal increase in plasma protein AAS was observed (Dragsted. The main disadvantage is the longer time required for analysis and the higher technical demands. involving seven divers. the influence of taking 500 mg/d versus 1 g/d of vitamin C for 2 weeks prior to 30 min of hard exercise was evaluated in 12 males using a counterbalance design. . no effect on the levels of plasma protein carbonyls as a results of diving apnea or of a 1 g/d supplement of vitamin C for a week preceeding a diving experiment was observed [84]. Following protein isolation by a rapid sizeexclusion chromatographic step and complete acid hydrolysis. This procedure has the advantage of being specific for the products of lysine (2-aminoadipic semialdehyde (AAS)) or for proline and arginine (γ-glutamyl semialdehyde) oxidation and of avoiding any further oxidation during workup of the sample. whereas no change was observed after supplementation of the known nutrients from the fruits and vegetables. indicating that the depletion of ascorbate might have an antioxidant effect. fluorescence or APCI-MS detection [86]. Dragsted 36 To assess oxidative stress by use of the simplest version of this assay. The level of protein carbonyls in the immunoglobulins was found to decrease after both 10 and 15 weeks of supplementation. Vitamin C was found to decrease the exercise-induced increase in protein carbonyls in a dose-dependent manner [83]. A more advanced approach with an isolated protein fraction was taken in a study of the effect of a 400 mg/d vitamin C supplement given to healthy volunteers for a 15-week period.

Conclusions Total plasma vitamin C can readily be determined by automated colorimetric assays or HPLC.3. There are no studies currently available on the relationship between lipid or protein oxidation markers. Most of the currently available markers for assessing lipid or protein oxidation in humans have been employed in human intervention studies to assess the effects of vitamin C supplementation. No evidence for the antioxidant effect of vitamin C in the dose range of 60–2500 mg/d in connection with mild ascorbate depletion was obtained using the best lipid oxidation marker available. Vitamin E Biomarkers for vitamin E in body fluids Vitamin E is a collective term for alpha-. beta-. HPLC has the advantage of having lower detection limits. The handling and storage of plasma for vitamin C determination has a strong effect on accuracy. whereas the colorimetric assays have much higher throughput.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. gamma. The reference for the international unit (IU) is 1 mg of all-rac-α-tocopheryl acetate. since in a study comparing the delivery of RRR-α-tocopherol and all-rac-α-tocopherol to lipoproteins in humans following 8 weeks of supplementation by 1600 mg/d of either product. The vitamin E status in rats and possibly in other rodents can be determined as a function of all the active isomers. 1 IU corresponding to 1. and possibly possessing higher accuracy. This is still controversial.and δ-tocopherols or of the tocotrienols are thought to have any vitamin E activity in humans as opposed to the rat. C and E 37 Overall there appears to be certain but limited evidence for suboptimal vitamin C intake leading to an increase in protein oxidation in the plasma immunoglobulins. but in humans vitamin E status is preferably determined as the plasma. including the susceptibility of erythrocytes . γ. of which RRR-α-tocopherol has the highest vitamin E activity.49 mg RRR-α-tocopherol. serum or erythrocyte concentration of d-α-tocopherol. permitting simultaneous determination of ascorbate and dehydroascorbate. There is only limited evidence that plasma ascorbate is a functional antioxidant in the body as assessed by means of these markers and very limited evidence that high dosages of vitamin C may decrease oxidative stress. however. Evidence for the prevention of stress-induced carbonyl formation by increasing the intake of vitamin C is controversial and needs further confirmation. Since only the four R-forms of α-tocopherol (d-α-tocopherol) are recognised by the human vitamin E transporter in the body. that of the formation of plasma isoprostanes.and delta-tocopherols as well as the tocotrienols. Clear short-term pro-oxidant effects on lipid oxidation were observed following high-dose vitamin C infusion into the bloodstream. Various functional tests have also been suggested. none of the four S-forms of α-tocopherol and none of β-. no difference in the total vitamin E content of LDL was observed [87]. The relative vitamin E activity of the various tocopherols and tocotrienols in the rat together with their structures are shown in Figure 2. vitamin C and chronic disease.

Redrawn from [100].3. dl-α-tocopherol equivalents). 2. .Lars O. and deletion of this transporter leads to neurological symptoms [92]. it seems more appropriate to evaluate the relationship of vitamin E supplementation to currently used lipid oxidation markers. Also. Fig. since these functional tests may be lipid oxidation markers. Structures of tocopherols and tocotrienols and their relative potency as vitamin E in the rat (TE. Dragsted 38 to lysis following an in vitro challenge with hydrogen peroxide [88–90]. or the exhalation of breath pentane [91]. In one study no correlation was found between results of the erythrocyte membrane stability test and plasma or erythrocyte vitamin E concentrations [90]. Vitamin E has a physiological transporter in the human that only binds d-α-tocopherol.

C and E 39 Vitamin E and markers of lipid oxidation This review concerns only randomised. In another study. In another study. ex vivo LDL oxidation. 16 young and 16 older male subjects were first given eccentric exercises for 45 min and were then placed on 1000 IU/d of vitamin E supplementation for 12 weeks before being given a second round of exercises. controlled intervention study. given in a normal formulation. In a randomised 2-months intervention study of 59 healthy smoking males. of a group of 14 runners assigned to either a placebo or a 1200 IU/d dose of vitamin E starting 4 weeks before and continuing during 6 days of increased running training. In a study of 49 HIV patients randomised to supplementation with a placebo or with 800 IU/d vitamin E and 1000 mg/d vitamin C for a 3-month period. no effect on exerciseinduced oxidative stress could be shown by serum MDA [109]. a reduction in plasma MDA occurred in the group assigned vitamin supplements [104]. a significant decrease in plasma MDA was found in the vitamin-supplemented group as determined by HPLC [101]. In a subsequent study of 24 diabetic children by use of the same design. but to be unaffected by a slow-release formulation as compared with placebo treatment [33]. or isoprostanes (see desciption in the section on vitamin C above). 56 patients with congestive heart failure were given 335 mg/d of the natural d-α-tocopherol for a 12-week period and no effect on the plasma MDA level was detected [105]. In a three parallel groups of 16 middle-aged male and female participants. . In a study of 24 diabetic patients assigned to take a capsule each day containing 100 IU vitamin E or a placebo for a period of three months. those assigned to treatment with vitamin E (1200 IU/d starting 7 days before the exercises began) were found to not differ from the placebo group in the levels of plasma MDA [107]. No change in plasma MDA either within or between the two groups could be shown [110]. who were given either 500 mg/d of vitamin C together with 182 mg/d dl-α-tocopherol. antioxidant capacity markers. 30 mg beta-carotene and 100 µg organic selenium. there was a significant decrease in erythrocyte MDA in the vitamin-supplemented group [102]. In a trial of lipid oxidation induced in 18 untrained men by three sessions of resistance exercises. controlled studies in which the effects of supplementation by vitamin E was investigated on peripheral blood samples using plasma or serum thiobarbituric acid reactive compounds (TBARS). lipid hydroperoxides. no effect on the plasma MDA was detected [38]. a decrease in serum MDA was found in the group given vitamin E [108]. In a trial involving 80 men showing an increase in plasma lipid oxidation from exposure to either olive oil or menhaden oil. In 49 diabetic patients given either 504 mg/d d-α-tocopherol or a placebo for a 6-month period a decrease in ex vivo induced TBARS in the erythrocyte membranes was found for the supplemented group [103]. giving a 900 IU dose of vitamin E was found to be no better than a placebo in decreasing MDA [106]. In a cross-over intervention study involving 4-week periods of either taking 500 or 1000 IU of vitamin E together with 500 or 1000 mg of vitamin C or taking placebo. MDA was found to increase significantly following daily doses of 200 mg vitamin E combined with 250 mg ascorbate.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. In a study of the effects on 77 smokers of taking vitamins C (272 mg/d) and E (800 IU/d) or a placebo for 90 d. In a double-blind.

vitamin E given at doses higher than 200 IU/d was found to affect the kinetics of ex vivo oxidation of LDL [113]. In a double-blind controlled intervention study of 56 patients with congestive heart failure. In a study of 49 HIV patients randomised for supplementation with a placebo or with 800 IU/d of vitamin E and 1000 mg/d of vitamin C over a 3-month period. In a group of 45 randomised healthy males and females. In a trial involving 80 men who had an increase in plasma lipid oxidation following exposure to olive oil or to menhaden oil. or LDL-hydroperoxides induced ex vivo [87]. Non-induced lipoprotein oxidation ex vivo was found to be delayed in the group given the vitamin supplement. there was found to be a reduction in plasma lipid peroxides and in breath pentane exhalation in the group to which a vitamin supplement was assigned [106]. a dosage of 900 IU vitamin E was found to equal placebo in reducing the level of lipid hydroperoxides in the plasma or the exhalation of breath pentane [112].Lars O. In a dose-response study of 40 healthy men assigned doses of 60–1200 IU of vitamin E for an 8-week period. no change in the group receiving the supplement was detected in the antioxidant capacity of the erythrocytes as determined on the basis of the glutathione concentration and the glutathione peroxidase activity [103]. . this together with 182 mg/d dl-α-tocopherol. the treatment with vitamin E was found to significantly increase the level of plasma F2-isoprostanes as well as of several markers of inflammation. the effects of giving mixed supplements of 200 mg/d vitamin E. In 49 diabetic patients given 504 mg/d d-α-tocopherol or a placebo for 6 months. In a blinded intervention study of 33 triathletes participating in a world championship. LDL-dienes. and the strength of this effect was correlated with the level of plasma α-tocopherol [114]. or placebo. In a study without a parallel control group. 900 mg/d vitamin C and 18 mg/d beta-carotene for 6 months were compared with those of a placebo. In a study comparing the delivery of RRR-α-tocopherol and all-rac-α-tocopherol to lipoproteins in humans following 8 weeks of supplementation with 1600 mg/d of either product. including IL-6 [115]. In a group of 48 middle-aged male and female participants in a 36-month intervention study in which either 500 mg/d of vitamin C. a significant increase in the susceptibility of isolated LDL or VLDL to oxidation ex vivo was observed at 12 and at 36 months in the group given only vitamin E and in group given the combined dosage. 182 mg/d dl-α-tocopherol alone. who were given 800 IU/d vitamin E or a placebo for 2 months prior to the race. Dragsted 40 182 mg/d dl-α-tocopherol alone. no difference was observed in LDL-MDA. treatment with 335 mg/d of the natural d-α-tocopherol for 12 weeks was found to have no effect on the level of activity of plasma glutathione peroxidase [115]. no effect on plasma antioxidant capacity was observed at 12 or at 36 months [44]. a 1000 IU/d vitamin E supplement for a 10 d period was found to reduce the exhalation of breath pentane [91]. A significant change in these groups in whole plasma ex vivo oxidation at 36 weeks was likewise observed [44]. In a double-blind controlled intervention study of 56 patients with congestive heart failure. treatment with 335 mg/d of natural d-α-tocopherol for 12 weeks was found to have no effect on the level of breath pentane exhalation [111]. or a placebo in a parallel design.

In another double-blind placebo-controlled trial with a crossover design. simvastatin together with 600 mg/d vitamin E or a placebo for 2 months. 10 of them were given 500 mg/d and 10 of them 1000 mg/d vitamin E for a period of 3 weeks. C and E 41 In another study. irrespective of the treatment [124]. In a study of the combined effects of giving either vitamin C (500 mg/d) together with d-α-tocopherol (400 mg/d). In a dose-response study. 800. In 43 hypercholesterolemic patients randomised to taking simvastatin. 300. 16 young and 16 older male subjects were given 45 min of eccentric exercise and were then given a 1000 IU/d supplement of vitamin E for a 12-week period before being given a second round of exercises. no effect of either vitamin treatment was observed. 600 or 1200 IU/d of vitamin E for 3 weeks. the 9 patients receiving standard medication together with 600 mg/d vitamin E for a 30-day period showed lower urinary excretion of F2-isoprostanes than 5 controls given only standard medication [123]. In a study without a control group. Running as such was found to increase the plasma isoprostane level. This was not observed for the young men. vitamin C (500 mg/d) together with d-α-tocopherol (290 mg/d) and d-γ-tocopherol (130 mg/d). but this was not changed by the vitamin supplements. Following vitamin E supplementation a significant lowering of the plasma isoprostane level both before and 24 h after the exercise was found for the older men. although the treatment by g-tocopherol was found to affect the exercise-induced increase in plasma and muscle heat shock protein (HSP72) and also the expression of HSP72 in muscle [118]. 21 ultramarathon runners were given either a combination of 300 mg/d d-α-tocopherol and 1000 mg/d vitamin C. isoprostane excretion was found to be decreased at the end of a 2-month period in a group of 15 healthy individuals receiving 400 IU/d of vitamin E [125]. which may have caused the difference. In a third study of the effects of vitamin E on oxidative damage induced by exercise. In a double-blind controlled intervention study of 56 congestive heart failure patients. The inflammatory markers were also affected by running. no effect on the plasma F2-isoprostane level of taking 1000 IU/d of vitamin E was found for 20 patients with endothelial dysfunction [125]. the rest being given a placebo. particularly in the males. No significant change in the plasma F2-isoprostane level could be detected by GC-MS at any time point. In a group of 33 sclerosis patients. No other significant differences were observed between the two groups [116].Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. groups of 5 healthy individuals received either 0. 200. or a placebo during a 28 d period on the plasma isoprostane concentrations induced by exercise. or a placebo during the 6 weeks prior to the race. the adding of vitamin E was found to have no further effect on the urinary excretion of F2-isoprostanes [122]. 1200 or 2000 IU/d of vitamin E for a period of 8 weeks. No effect on urinary excretion of F2-isoprostanes was observed [121]. followed by 8 weeks of washout. The study design could not control for period effects. no effect on the excretion of F2-isoprostane could be observed for any of these interventions [119]. and the vitamin supplement was found to prevent this [117]. treatment with 335 mg/d of natural d-α-tocopherol for a period of 12 weeks was found to have no effect on the plasma F2-isoprostane level [111]. In a small non-blind study of cirrhotic patients. 400. In an intervention study of 46 healthy smokers given 0. .

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In order to produce mutations.3]). which can be produced both in the biochemical utilization of oxygen and as a by-product of O2 metabolism in the mitochondria.Vitamins and selenium: Antioxidant vitamins and cancer risk 51 2. though not generally classed as carcinogenic agents. Nicolaus Copernicus University.8-dihydroguanine (8-oxoGua). but the large numbers of oxidative DNA lesions observed in many tumours strongly suggests that such damage is frequently a cause of cancer [5]. so-called reactive oxygen species (ROS). leads to GC→TA transversions [4]. has led to the proposal that they be used as intermediate markers . corresponding to about 2000 oxidative modifications of guanine per cell daily.2. The presence of 8-oxoGua in the cells can thus result in point mutations. increasing attention has been directed at oxidative DNA damage in relation to cancer. Lesions such as 8-oxodG are used as biomarkers of oxidative stress. can damage cellular molecules of different types — in particular proteins. partly those generated in processing. Not only is DNA mutation a crucial step in carcinogenesis. Cook2. unless repaired prior to DNA replication. oxidative DNA damage needs to occur at a sufficiently high frequency to exceed the cell’s capacity for DNA repair. Poland The role of oxidative DNA damage in the development of cancer Cancer is a disease of the genes. Denmark University of Leicester. has been documented in aging cells and tissues. Food and beverages frequently contain carcinogens. One of the most widely studied lesions of this sort is 8-oxo-7.and purine-derived lesions being formed in the DNA (as reviewed in [1]). together with their mutagenicity in mammalian cells. lipids and nucleic acids. It is of interest to note in this context that a urinary excretion study showed the average 8-oxoGua and 8-oxodG (7-hydro-8-oxo-2’-deoxyguanosine) excretion in the urine of healthy subjects to be some 2. The hydroxyl radicals that ROS creates result in a large number of pyrimidine. An increase in somatic mutations. that metabolites of atmospheric oxygen. Marcus S. Rafa∏ Ró˝alski3. nevertheless. ROS. promotion and malignant conversion (progression) stages of carcinogenesis. Since the cumulative risk of cancer increases at a rate corresponding to the fourth power of age and is associated with accumulated DNA damage. however. Antioxidant vitamins and cancer risk: is oxidative DNA damage a relevant biomarker? Steffen Loft1.5 nmol per kg per day. When mutations accumulate. are human carcinogens. this can be due to the individual’s overall lifetime exposure to endogenous and exogenous agents that damage the DNA. Peter Møller1. Oxidative mechanisms have been shown to have a potential role in the initiation. The presence of 8-oxoGua residues in DNA. This. Ryszard Oliƒski3 1 2 3 University of Copenhagen. Some of these modified DNA bases have considerable potential for damaging the integrity of the genome (reviewed in [2. It is quite possible. UK Collegium Medicum in Bydgoszcz. which are implicated in the development of cancer. and partly carcinogens that occur naturally in the products. such as in the cooking of meat.

creates a selection pressure for the malignant phenotype seen in the case of cancer. . 3. Numerous studies have been concerned with the relationship between the level of oxidative DNA damage and cancer (see [5] for a review). there are serious problems in the chromatographic measurement of 8-oxodG. Peter Møller. there are a large number of pathological conditions in which the level of oxidative DNA damage is elevated without any increase in the incidence of carcinogenesis [5].Steffen Loft. together with the increase in DNA damage that occurs. the nuclei of the undifferentiated. demonstrating that an intervention that reduces oxidative DNA damage also reduces the risk of cancer would provide the evidence needed of the value of such biomarkers in a public health and cancer prevention context. proliferating stem cells have to be affected. There is a need in this context of large prospective studies able to demonstrate to what extent an elevated level of oxidative DNA damage indicates an increased risk of developing cancer. or (ii) there being reduced DNA repair [5]. the mere presence of an elevated level of damage in a tumour does not indicate oxidative damage to have led to the tumourigenic changes that have occurred. Rafa∏ Ró˝alski. In order for a mutation to come about. the analytical procedures in current use are unable to quantify the lesion levels found in the most important ‘target’ cells. Marcus S. not only must the DNA of the target cells be affected. Issues of this sort have to be addressed before a causal link between oxidative DNA damage and cancer can be established. Ryszard Oliƒski 52 of a potential disease endpoint such as that of cancer. For DNA mutations to arise through oxidative damage. This. Although studies that indicate this support the hypothesis that oxidative DNA damage can be an important risk factor for carcinogenesis. 2. regarding the question of ‘cause or consequence’. but also the damage must be within a coding region of the DNA. it has been argued that the mere presence of 8oxodG in DNA is unlikely to be a necessary and sufficient condition for tumour formation. This raises a number of different issues: 1.2 lesions per 106 unaltered guanines and that reports of values outside this range should be viewed skeptically [6].3–4. Oxidative DNA damage may be an epiphenomenon in relation to the ongoing pathophysiological process. such as a high level either of metabolism or of cell turnover. connected with the spurious oxidation of DNA during sample preparation. An elevated level may be due to some well-established characteristic of the tumour. On the basis of an extensive investigation by the European Standards Committee on Oxidative DNA Damage (ESCODD) it has been concluded that the true background level of 8-oxodG in mammalian cells is approximately 0. Elevated ROS levels may activate transcription factors and the corresponding genes being permanently activated. Since tissue samples from both tumours and normal cells represent a heterogeneous mixture of differentiated and undifferentiated cells (the former being likely to predominate). Again. the levels of damage having no causal role in carcinogenesis. At the same time. In addition. Cook. Elevated damage levels have been purported to arise as a result either of (i) the tumour having an environment low in antioxidant enzymes and high in ROS generation. Ultimately. although their suitability for this has yet to be verified in prospective studies of cancer risk.

nevertheless. the mode of action of dietary micronutrients is complex and is far from being fully understood. Both experimental and epidemiological data indicate vitamin C to protect against both stomach and oesophageal cancer [8]. oxidants are involved in each case. At the same time. Reliable detection of urinary 8-oxodG excretion became possible at about the same time [11]. although sampling is usually restricted to use of such surrogate tissues as white blood cells (WBC) and urine. to establish directly that the DNA lesion responsible for a carcinogenic process is the lesion present in a tumor many cell generations later. It is very difficult. Although these events may come about by way of different mechanisms.Vitamins and selenium: Antioxidant vitamins and cancer risk 53 Summing up. The literature on biomarkers of oxidative DNA in WBC employed in small-scale intervention studies of antioxidant supplements has been summarized in a series of reviews [9.12]. as the possibility of detecting 7-hydro-8-oxo-2’-deoxyguanosine (8-oxodG) appeared. It is reasonable to assume that agents that decrease oxidative DNA damage should also decrease the subsequent development of cancer. one can say that in light of the data obtained thus far it appears likely that the development of many types of cancer leads to severe oxidative stress. Nevertheless. therefore. the comet assay. These antioxidants are effective free-radical scavengers and should protect the DNA from oxidative damage. however. A plethora of descriptive epidemiological studies have concerned a possible protective effect of a diet rich in fruits and vegetables [7]. At the beginning of the 1990s. C and E or phenolic compounds. supplementation trials would appear to represent the most relevant way of exploring antioxidant effects. respectively. large-scale intervention studies have failed to demonstrate use of antioxidant vitamins to reduce the risk of cancer [9]. Subsequently. but that it is impossible at present to determine to what extent oxidative stress is directly involved in carcinogenesis. and the enzyme-modified version of the comet assay allowing oxidized purines (including 8-oxodG) and pyrimidines to be detected by means of formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII). since full development of the disease in response to exposure to a carcinogen may take 20–40 years. used for the detection of DNA strand breaks (SB). the first of many antioxidant supplementation trials dealing with this lesion in WBC was carried out [10]. . altered gene expression and mutations are necessary elements in the process of carcinogenesis. that DNA damage. One should bear in mind. this soon being followed by the performance of antioxidant trials in which urinary 8-oxodG excretion served as the key biomarker. Dietary antioxidants as inhibitors of oxidative DNA damage and as a factor decreasing cancer risk There is widely believed to be a link between diet and the incidence of cancer. have become by far the most popular assays for use in conjunction with antioxidant intervention trials. Intuitively. One possible mechanism by which the protective effect of fruits and vegetables is exerted could thus be by way of the antioxidative activities of such plant food constituents as vitamins A.

provides an overview of intervention studies involving optimal design in which the effects of antioxidants or antioxidant-rich food supplements on oxidative damage to DNA in WBC or in urine have been investigated.6 g) for 1 mo 116 M (S) 30—65 No effect on the 24-h urinary excretion of 8-oxodG (HPLC) 13 M (NR) 30±3 Lower 24-h urinary excretion of 8-oxodG (HPLC) in the active group of HIV-infected patients receiving zidovudine therapy 184 MF 58±14 No effect on the 24-h urinary excretion of 8-oxodG (ELISA) (NS) 48 M (22S) 30 NR (NR) 45—69 No effect on the 24-h urinary excretion of 8-oxodG (HPLC) 22±1 No effect on the 24-h urinary excretion of 8-oxodG (ELISA) in subjects undergoing cold-weather training at a moderate altitude 7±2 No effect on the excretion of 8-oxodG (ELISA) in spot urine samples 35 25 28 2x2 parallel study of vitamin C (500 mg) and vitamin E (400 IU) for 2 mo 2x2 parallel study of vitamin C (500 mg) and vitamin E 182 mg) for 12 mo Multivitamin tablete for 2 wk 34 33 Multivitamin tabletf for 21 d 39 MF (NR) 37 Multivitamin tabletg for 24 d 40 M (NR) 18—40 No effect on the overnight excretion of 8-oxodG (ELISA) in subjects undergoing cold-weather training at a moderate altitude 59 MF (11S) 50±6 No effect on the excretion of 8-oxodG (ELISA) in spot urine samples 36 Fruit and vegetable capsulesh for 7 wk 29 . Table 2. but an increase in excretion during the washout period 14 30 Vitamin C (500 mg) for 3 wk 30 MF (NS) 24 Six groups receiving combinations of vitaminsd for 2 mo Vitamin C (1 g) and vitamin E (0. 200 IU α-tocopherol. from use of a non-optimum design. however. and 6 mg β-carotene) for 6 mo Carotenoidsc for 3 wk 100 M (50S) 50—59 A decrease in ENDOIII sites (Comet) in WBC after 20 weeks 13 63 MF (S) 42±9 No difference in 8-oxodG in WBC (antibody-based detection) between the supplemented and the placebo group. Peter Møller. Table 2.6.Steffen Loft. Ryszard Oliƒski 54 Many studies suffer. but a postsupplementation decrease in the urinary excretion of 8-oxodG (ELISA) in the active group 17—49 No effect of vitamin C supplementation on 8oxodG (ELISA) in spot urine. 280 mg vitamin E. but a decline in the course of the trial in both groups 32 MF (NS) 32±11 No effect compared with baseline. Marcus S. Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine Supplement given per day Subjectsa Age (yr)b Effect Ref Multivitamin tablet (100 mg vitamin C.6. and 25 mg β-carotene) for 20 wk Multi-vitamin (250 vitamin C. Cook. Rafa∏ Ró˝alski.

Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont.5 g) or placebo (fiber-free bread) for 2 wk Flavonol (quercetin) rich diet for 2 wk in crossover design among type 2 diabetes patients 12 F (NR) 17 10 MF (3S) 60±7 No effect on ENDOIII sites (Comet) in WBC 16 Vegetable/fruit (500 g) in crossover design 22 M (S) 33±11 No effect on ENDOIII sites (Comet) in WBC for 3 wk with 2 wk washout Vegetable/fruit (600 g) or tablets with the same concentration of antioxidants/minerals for 24 days Cruciferous and legume sprouts (113 g) for 2 wk Kiwi fruit (1—3 pieces) for 3 wk 43 MF (NS) 27±6 No effect on ENDOIII and FPG sites (Comet) in WBC.Vitamins and selenium: Antioxidant vitamins and cancer risk 55 Table 2. Supplement given per day Subjectsa Age (yr)b Effect Ref Antioxidant rich diet or tabletsi for 5 wk 55 MF (NR) 71±6 No effect compared with baseline. but not earlier 68 MF (13S) 18—45 A decrease in the 12-h urinary excretion of 8oxodG (HPLC) 27. 45 Two interventions of green tea with 300 ml for 7 d or 32 oz for 7 days Green tea extractk for 3 wk 317 16 M (8S) 20—31 No effect of supplementation on the 24-h urinary excretion of 8-oxodG (HPLC) but a decrease in excretion during the study in all groups 38 F (NR) NR A decrease in the excretion of 8-oxodG (ELISA) in the active group (statistical test not reported) 44 Soya-hypocotyl tea (> 1 l) for 1 mo 42 . but a decline in all the groups 21—45 No effect on FPG sites (Comet) in WBC 19 20 18 MF (NR) 14 MF (NS) 10 MF (NS) 10 M (NS) 15 26—54 A decrease at the ENDOIII and FPG (Comet) sites 22 Brussels sprouts (300 g) for 1 wk NR No effect on the 24-h urinary excretion of 8-oxodG (HPLC) A decrease in the 24-h urinary excretion of 8-oxodG (HPLC) in the active group 40 Brussels sprouts (300 g) for 12 d NR 41 Fruit juice (480 ml)j for 4 d 11 M (NR) 21±1 A decrease in the 12-h urinary excretion of 8-oxodG (ELISA) in the active group 57 MF (6S) 19—52 No effect on ENDOIII and FPG sites (Comet) in WBC 26 Parallel study of blackcurrant juice or anthocyanin drink (475—1000 ml) for 3 wk Green tea or black tea (4 cups) for 1—4 mo 18 120 MF (S) 18—79 A decrease in the excretion of 8-oxodG (ELISA) in spot urine samples in the green tea group after 4 mo. but a post-supplementation decrease in the 24-h urinary excretion of 8-oxodG (ELISA) in the active group NR No effect on ENDOIII sites (Comet) in WBC 32 Rye crisp bread (76.6. No effect on the 24-h urinary excretion of 8-oxodG (HPLC).

and powder or extract of fruits and berries). cranberry and peach.2 mg). Smokers (S) and nonsmokers (NS) are indicated in brackets. 18 IU vitamin E. lycopene (4. Consisted of tablets containing vitamin antioxidants (400 mg vitamin C.2 mg). Marcus S. In two well-designed studies of the effects of administering a combination of antioxidant vitamins.16]. vitamin C (500 mg). 4) 90 mg coenzyme Q10 in oil. α-carotene (1. Age is shown as range or as mean ± standard deviation. 4 mg β-carotene). 3) 500 mg slow-release vitamin C. vitamin C (330 mg). tocopherols (650 IU). Accordingly.14]. Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont. beet. This suggests that the protective effect of a single antioxidant is relatively short. orange. lutein (500 mg). Ryszard Oliƒski 56 Table 2. spinach. there are fewer that reveal protective effects than those that report only negative results [9.5 mg/10 MJ). parsley. possibly because of differences in the respective rates of bioavailability and elimination. The daily dose consisted of 200 mg vitamin C. N-acetyl-1-cysteine (181 mg). The vegetable capsules were made from carrots. and tomato. or cow milk (1 l) for 4 wk 10 M (NS) 20—50 A decrease in ENDOIII sites (Comet) for soy milk Polyphenol-rich olive oils (25 ml) for 40 d 12 M (NS) 20—22 A decrease in the excretion of 8-oxodG (HPLC) in spot urinary samples following supplementation in a dose-dependent manner 21 43 a b c d e f g h i j k Number of subjects indicated as males (M) and females (F). cabbage. and 15 mg β-carotene. Supplement consisting of β-carotene (6 mg). kale.4 mg).44 mg) per day. Rafa∏ Ró˝alski. Tablets containing micronutrients similar to those of 3 fruits and 3 vegetables (containing 107 mg vitamin C and 83 mg vitamin E). being given rye crisp . 2. The effect of a single dose of vitamin C seems to disappear after several hours. vitamin E (400 IU).6. selenium (167 mg).4 mg β-carotene. lutein (4.12]. broccoli. The fruit capsules were made from juiced apple. Containing vitamin A (240 mg). and papika carotenoids (2. Consisted of β-carotene (20050 IU). lycopene (100 mg). Peter Møller. vitamin C (219 mg). respectively [15. Supplement given per day Subjectsa Age (yr)b Effect Ref Soy milk. Also. 5) 30 mg coenzyme Q10 granulate.12]. The six groups received daily supplements of 1) 200 mg vitamin E.7 mg). 2) 500 mg plain-release vitamin C. and pomegranate extract (5 mg). rice milk. whereas tocopherols and carotenoids exert their effects somewhat longer. selenium (100 mg). Among studies in which multiple doses of single antioxidants are given. vitamin E (1. Antioxidant-rich foods Ingestion of a diet rich in flavonols (including quercetin) and of one rich in cruciferous and legume sprouts (113 g/d for 2 wk) was not found to alter the frequency of ENDOIII and of FPG sites.5 mg). Tablets containing β-carotene (12 mg). a protective effect could be shown after only 20 weeks of supplementation but no effect after 10 weeks (or 6 months of supplementation) [13. the question of whether multiple vitamin supplementation provide better protection against oxidative DNA damage than a single dose does cannot be answered unambiguously. 150 mg vitamin E. pineapple. 60 mg vitamin E. or a carotenoid-rich diet. however. Consisted of 1000 mg extract/kg body weight in meat patties (total phenolics 23. β-carotene (399 mg). catechin (13. bixin (11. and zinc (30 mg). Cook.Steffen Loft.4 mg). capsules (containing 90 mg vitamin C. papaya. 6) placebo. Effects of antioxidant supplementation on oxidative DNA damage in WBC Single doses of simple antioxidants have been found to generally reduce the level of oxidative DNA damage temporarily [9.

The other. Five studies carried out in which a reduction in the level of oxidative DNA damage was observed involved providing very different antioxidant-rich foods that were not easy to compare. Effect of antioxidant supplementation on 8-oxodG in urine Measurement of the urinary excretion of 8-oxodG in antioxidant intervention studies is connected with the idea that it decreases following a steady state ingestion of antioxidants due to the rate of generation of oxidative DNA damage in the body being decreased. whereas 13 studies reported null effect. The only study showing consistent effects involving more than one endpoint was a study of the effects of kiwi fruit supplementation (1–3 kiwi fruits/d for 3 wk).23–44]. for example). There is little support for the notion that ingestion of antioxidant-rich foods is associated with lower spontaneous level of oxidative DNA damage in WBC than intake of single antioxidants. or power to detect a 50% change between these studies reporting beneficial effects and those reporting no effects.or FPG-sensitive sites. unintentional intoxication of the gastrointestinal tract (some of those in the active groups complained of nausea. Drinking black currant juice or an anthocyanine drink (475–1000 ml/d for 3 wk) was also not found to have any beneficial effect on ENDOIII. which showed the numbers of ENDOIII and FPG sensitive sites to be reduced [22]. number of subjects. a cross-over study of male smokers. It can be speculated that the subjects suffered a slight. Anthocyanines have low bioavailability and the dose provided here was rather high. was negative with respect to effects on the ENDOIII and FPG sites [20]. assessment of the DNA damage occurring showed the levels of ENDOIII to be reduced [21]. The one investigation. .5 mg/d for 2 wk) as a source of lignans was not found to be associated with any increase in the plasma enterolactone concentration. there in fact being a tendency in the group of subjects drinking black currant juice for the number of FPG sites to increase [18]. For 24 studies of the effects of antioxidant supplementation on the urinary excretion of 8-oxodG in which a controlled design was employed [20. An overall summary of the studies showed that six investigations reported beneficial effect of antioxidant supplementation. The lack of any effect of lignans in the WBC would seem reasonable in view of the low bioavailability of the active substances in rye crisp bread. a placebo-controlled parallel study in which non-smoking subjects of both sexes were given 600 g/d of fruits and vegetables for 24 days. showed no effect on the ENDOIII sites of ingesting 500 g/d of such food for 3 wk [19]. Drinking soy milk (1000 ml/d for 4 wk) as a source of phytoestrogens in the one study increased the plasma levels of genistein and daidzein but not of enterolactone. or to have any effect on the ENDOIII sites [17]. no appreciable difference was present in terms of duration of the intervention period. Two studies concerned the effects of an intervention of providing vegetables and fruits. and their effects in the gastrointestinal tract are easier to comprehend.Vitamins and selenium: Antioxidant vitamins and cancer risk 57 bread (76.

28. parsley.36]. A comparable study involving supplementation of a mixture of carotenoids (daily intake: α-carotene (1. Mixed results concerning the effects of a dietary supplementation of Brussels sprouts (300 g/d) were obtained in two studies [40. Investigations of the effects of natural food products are distributed about equally between studies reporting beneficial and those reporting null effects. orange. and vegetables. β-carotene (6.e.Steffen Loft. papaya. Marcus S. and paprika carotenoids (2.37] or in subjects undergoing cold-weather field training at a moderate altitude [33.4 mg). beet. In four additional studies. but the results were uncertain because of the only small number of subjects tested and of one of the male subjects showing an unexpectedly high urinary 8-oxodG excretion level [40].45]. in which both sexes were included. pineapple. the effect was less clear. The statistically significant difference in the delta values reflected a marked increase in 8-oxodG excretion in the placebo group and a slight decrease in the excretion of it in the group given mixed carotenoids. that involved only male subjects. In the one study a beneficial effect was found for drinking 300 ml/d for a week [31]. bixin (11.32. the Brussels sprouts supplementation was found to lower the urinary excretion of 8-oxodG [41]. whereas in still another study a beneficial effect of the supplementation of high doses of vitamin C (1000 mg/d) and vitamin E (600 mg/d) was found for HIVinfected patients treated with zidovudine [25]. No effect on the urinary excretion of 8-oxodG was found for the ingestion of capsules containing juices and powders of fruit (apple. and peach) and of vegetables (carrot.41]. cranberry.4 mg).7 mg). Three additional studies in this area concerned the effect of drinking green tea [see 27. neither eating 600 g of fruits and vegetables nor consuming corresponding amounts of minerals and vitamins in tablet form was found to be associated with a lower level of the urinary excretion of 8-oxodG than in a placebo group. neither supplementation of vitamin C or vitamin E or a combination of them was found to have any effect in healthy subjects [24. On the other hand. cabbage. tea. Ryszard Oliƒski 58 Four studies of the supplementation of a single carotenoid showed there in each case to be no effect on the urinary excretion of 8-oxodG [23. and tomato) [29]. Peter Møller. there being a tendency for only the male subjects to benefit from ingestion of Brussels sprouts. In the first study. Taking capsules containing extracts of fruits and berries and eating diets rich in carotenoids were both found to lower the excretion of 8-oxodG [32].35]. A number of studies have involved supplying antioxidants in the form of berries. fruits. kale.39]. A positive effect of vegetable juice consumption on 8-oxodG excretion was also observed in subjects enrolled in a soccer summer training camp [26].0 mg). Ingestion of olive oils with a high content of phenolic compounds was found to be associated with a lowering of the urinary excretion of 8-oxodG [43]. lutein (4.34. the difference between results at the end of supplementation and at baseline) but no difference between the two groups at baseline [30].38.31. lycopene (4. spinach. whereas this intervention was found to have a pronounced period effect [20]. Rafa∏ Ró˝alski. Cook.5 mg). whereas in one of the other two studies .2 mg)) revealed a statistically significant difference between the active and the placebo group in the delta values obtained (i.35. broccoli.44. Further studies showed multi-vitamin tablet supplementation to provide no beneficial effect either in normal subjects [20. In the subsequent study.

such as in the case of severe oxidative stress. such as those of regulating changes in gene expression. It is possible.Vitamins and selenium: Antioxidant vitamins and cancer risk 59 no effect of ingesting green tea extract in meat patties for 3 wk was obtained [44]. 3. and 3) the alterations in the urinary concentration could not be assessed due to insufficient information [42]. therefore. It is worthy of note in this context that presumably healthy men may have the hereditary disease idiopathic haemochromathosis. 5. revealed a statistically significant positive effect of drinking green for 4 months [27. Experimental data suggest that supplementation of vitamin C to iron-overload subjects may increase the oxidative DNA damage that occurs [46]. In still a further study. It is possible that the antioxidants themselves may allow clonal expansion to occur and promote tumour growth by protecting initiated cells from excessive oxidant toxicity and apoptosis that would otherwise kill them [50]. Under some circumstances. that the presence of oxidative stress. 2) the putative active constituents (isoflavones) were not measured in the plasma. The absorption of non-haem iron has also been found to be affected by ascorbic acid [49]. adjustment of the data for a number of variables. which might fail to be recognized. antioxidant vitamins may have biological activities. Interestingly. Paradoxically. The question can be raised as to whether antioxidants in the blood and 8-oxodG in the DNA of lymphocytes and leukocytes are representative of the situation in the target tissue. vitamin supplementation of HIV-infected patients showing very low levels of antioxidant vitamins and significantly increased lymphocyte amounts of 8-oxoGua (as well as other base modifications) was found in one study to result in vitamin restoration to levels characteristic for the control subjects. 4. 2. could increase the likelihood of detecting a protective effect. There are a number of possible reasons for the failure of a positive effect of vitamin supplementation to be shown in connection with the cancer risk that oxidative DNA damage creates: 1. a positive correlation has been shown between LIP and the oxidatively modified nucleoside in human lymphocytes [48]. including baseline 8-oxodG levels. drinking soya hypocotyl tea was found to be associated with a lower urinary excretion of 8-oxodG.45]. such that iron catalytic to free-radical reactions (a so-called labile iron pool — LIP) is present in the blood plasma [47]). It is possible that a preventive effect of the vitamins can be only be seen when their basal levels are very low. Indeed. Although the unadjusted data of the third study indicated no beneficial effect of drinking green tea. which leads to iron overload. . the prooxidative properties of certain of the antioxidant vitamins (vitamins C and A) may take effect. although it should be emphasized that the fate of the antioxidants in this investigations was inconclusive since 1) the plasma concentration of carotenoids decreased. that are separate from their direct antioxidant effects [51]. The authors also noted a significant decrease in the levels of the modified bases in patients who were thus treated as compared with those who received only a placebo.

and to other types of DNA damage. which is not the case with use of single antioxidants.Steffen Loft. a major problem is that many of these studies have too few subjects. to obtain significant results. more attention should probably be devoted to alternative chemopreventive mechanisms such as the upregulation of DNA repair systems. which has been only sparsely investigated. yet the use of WBC as a surrogate tissue may not be particularly well suited for detection of this effect. Rafa∏ Ró˝alski. On an experimental basis. At the same time. which means that. The few well-controlled studies that have reported realistic levels of oxidative DNA damage in the WBC of oxidatively stressed subjects lend little support to the notion that such a population benefits more from antioxidant supplementation than a normal study population. no firm conclusions can be reached on the basis of antioxidant intervention studies. most studies encompassing far fewer subjects than this. This can be interpreted as antioxidants having an overall beneficial effect on the body as a whole. Although single studies may possess considerable power due to specific aspects of their design such as use of repeated measurements. Ryszard Oliƒski 60 Comments There are numerous experimental studies published each year on the potential of antioxidants or antioxidant-rich foods for preventing the oxidation of DNA. although there may be a beneficial effect in the first few hours after ingestion. whereas the protective effect of antioxidants may be more easily detected in subjects who are suffering from oxidative stress. There is an obvious need for controlled antioxidant intervention studies encompassing subjects who are oxidatively stressed and in whom oxidative DNA damage is measured by enzymic or chromatographic methods. Cook. which are of pivotal importance for such studies. however. Marcus S. There is a tendency for supplementation with use of antioxidant-rich food to decrease the urinary excretion of 8-oxodG. it is easy to understand the ingestion of antioxidants being associated with lower levels of oxidative DNA damage. Also the chemopreventive effect of antioxidants on non-lymphatic tissue. that most of the studies reported have used supplements of vitamin E. It should also be borne in mind that the majority of studies involve healthy individuals. particularly as regards the need of proper investigative designs and of the validity biomarkers. should be addressed more thoroughly before the idea of antioxidants having clearly beneficial effects is abandoned. At present. The most likely effect ratio in healthy subjects involves a difference of less than 10%. Hopefully. and many of them lack the power of detecting 50% differences between two separate groups. such studies will benefit from the lessons learned from antioxidant intervention studies. WBC studies provide little support for the idea that long-term antioxidant supplementation lowers the basic level of oxidative DNA damage. Peter Møller. It should be noted. . In the future. hundreds of subjects would be needed. many of the studies performed are of only limited value due to methodological problems related to the study design and the assays. These could be either healthy subjects exposed to oxidative stress (such as exhaustive exercise or hyperbaric oxygen treatment) or patients subject to oxidative stress on the basis of some given disease. which is considered to be the least effective form of antioxidant supplementation. such as those involving bulky DNA adducts.

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In addition. Because of its serum half-life time of about three weeks [3]. One of the major problems in measuring 25-(OH)D lies in the molecule itself. Measurement by means of HPLC is considered by most to be the gold standard here [6. Heidelberg. Vitamin D is metabolised by hepatic 25-hydroxylase into 25-hydroxycholecalciferol (25-(OH)D). the human diet contains vitamin D in the form of vitamin D3 (in food of animal origin) and vitamin D2 (ergocalciferol contained in plant food.8]. the determination of circulating 25-(OH)D is not an easy task [7]. Determination of 25-(OH)D As reflected in the recent scientific literature.25-(OH)2D level does not provide valid information on the individual’s vitamin D status [6]. is synthesized in the skin. Cancer Epidemiology. Extraction from the matrix (serum or plasma) is thus required for most analytical procedures (see Table 2.2]. its thus being more strongly affected by very recent sun exposure or dietary intake of vitamin D [4].25-(OH)2D or calcitriol) occurs primarily in the kidney (1α-hydroxylase). is rather short (24 h in the blood circulation). arising from the irradiation of ergosterol).Jakob Linseisen. Germany Vitamin D3. e. Plasma 1. Measurement of serum 25-hydroxycholecalciferol as marker of vitamin D status Jakob Linseisen and Sascha Abbas DKFZ. is also not regarded as a suitable biomarker of vitamin D status [5]. Such matrix effects can markedly diminish the validity of assays carried out [8]. Since the time of first assays in 1971. There is overall agreement that 25-(OH)D is the appropriate biomarker to measure vitamin D-status in humans. Because of its hydrophobic nature. The biologically most active vitamin D metabolite is 1. In the blood vitamin D and its metabolites are bound to the vitamin D binding protein. 25-(OH)D2 and 25-(OH)D3. which is the major circulating metabolite. Sascha Abbas 64 2.25-(OH)2D whereas 25-(OH)D has only limited biological activity [1. to lipids. it has to be performed by experienced personnel.). 25-(OH)D measurements are quite vulnerable to matrix effects. is converted by UV light from the sun (UVB 290–315 nm) into previtamin D3 which slowly isomerizes to vitamin D3.7. As compared with 25-(OH)D. strong efforts have been made to simplify them for routine use [6]. the half-life time of its precursor. Although such measurement is very accurate.g. 7-dehydrocholesterol. First of all it is a very hydrophobic compound and secondly it exists in two forms. the circulating 1. this metabolite is a good indicator of the vitamin D-stores obtained both from exposure to sunlight and ingestion of vitamin D [2].3. Considerable interest has developed in assays for measuring 25-(OH)D. vitamin D (cholecalciferol). Its precursor. with an even shorter serum half-life time of 4–6 h.25-dihydroxycholecalciferol (1. since its conversion from 25-(OH)D is also tightly regulated. Further hydroxylation to 1. or cholecalciferol.25-(OH)2D. requires expensive equipment (HPLC) and is more time-consuming than other .

Table 2.5 7.4 9 11 5 h.9 14 1 h. Serum. 0 min ELISA.4 20 min The laboratory must have an HPLC unit with a silica column CPB. or plasma Nichols 20 µL Institute Diagnostics HPLC. RIA — radioimmunoassay. requires luminometer Comments (nmol/l) CV (%) CV (%) ≤6 <8 <12 RIA. enzyme immunoassays (EIA) and chemiluminescence assays (CLPBA) are easier to handle. ** Does not include extraction. no yield determination required No radioactive waste. and are thus more frequently used. 100% cross-reactivity with 24. 24.5—300 18 6. HPLC — high-performance liquid chromatography. Radioimmunoassays (RIA). Table 2. Sample type Manufacturer and volume* RIA.7.0 5. ELISA — enzyme-linked immunosorbent assay. as well as being fast. CV — coefficient of variation. There are important differences in the performance of the various detection methods.2 8. DiaSorin Serum or plasma.25 (OH2)D The primary antibody is the vitamin D binding protein from serum Fully automated.plasma nostics or urine 500 µL 8—312 2. 10 min Uses vitamin D binding protein * Represents the initial starting volume of the sample.5 9. IDS Serum or plasma 50 µL Serum or plasma 50 µL None 6—360 ≤5 <6 <9 3 h. counting or microplate reading times.6 11. CPB — competitive protein binding.2 75 min Acetonitrile and C18 cartridge extraction Acetonitrile 15—150 4. Immundiag. Commercially available 25-hydroxyvitamin D assays [6] Range of Test principle. . Biomedica Proprietary extraction reagent Unknown 6. 50 µL Extraction detection (nmol/l) Acetonitrile Sensitivity Intraassay Interassay Assay time** 2 h. 0 min ELISA. 10 min Calibrators and controls in the serum matrix. 0 min ChemilumiSerum nescence. summarizes some of the performance characteristics of most of the assays that are available commercially.7. 24.25-dihydroxyvitamin D.3—250 2. 25 (OH2) D.9 3 h. IDK Serum 500 µL 17. however.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 65 methods. IDS Serum or plasma 50 µL Two-step reagent extraction 4—400 ≤5 5. no yield determination required Calibrators and controls in the serum matrix.

). After spiking of the serum samples with a defined quantity of 25-(OH)D. Binkley et al. lists the methods and the normal range for 25-(OH)D measurement in the laboratories that participated. Fig.8.4. The means varied widely between laboratories from 17. Binkley et al. HPLC — high-performance liquid chromatography. a broad range (17–95%) of the expected concentration was found by the different laboratories [11] (Figure 2. .4.6 ng/ml (p < 0. 2.8.).5. Sascha Abbas 66 Validity of different assays Only few studies have compared the different commercially available assays [9–13]. [11] reported recently not only immense variation between different assays but also variability between different laboratories in using a given assay. Table 2.005) [11]. Table 2.). Mean (SEM) results for the serum 25-(OH)D concentrations for 10 subjects as estimated by six different laboratories (for abbreviations see Table 2. the serum of 10 subjects was sent to six different laboratories.8. In their study. The mean serum 25-(OH)D concentrations differed 2-fold between laboratories (Figure 2. On the basis of their results.1 to 35.Jakob Linseisen. and the proportion of subjects below an arbitrary threshold of insufficiency (32 ng/ml) varied between 17 and 90%. 25-(OH)D assay methodology and the normal range employed in different laboratories [11] Laboratory A B C D E F G H Methodology Acetonitrile extraction followed by in-house RIA DiaSorin (RIA) Acetonitrile extraction followed by DiaSorin (RIA) Chemiluminescent assay Acetonitrile extraction followed by DiaSorin (RIA) Chemiluminescent assay Chemiluminescent assay Ethyl acetate extraction followed by normal phase HPLC Normal range 10—55 ng/ml 10—40 ng/ml 8—38 ng/ml 20—57 ng/ml NA 6—54 ng/ml 10—68 ng/ml NA NA — not applicable. [11] recommended the use of RIA techniques for 25-(OH)D measurement until other methodologies have been developed further. RIA — radioimmunoassay.

Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 67 Fig. This suggests that the contribution of 25-(OH)D2 to the overall 25-(OH)D supply is less important than had been previously thought. However. However. Figure 2.6. According to the latest DEQAS (Vitamin D External Quality Assessment Scheme) report. especially those in the US.7 to 18 ng/ml. There has been discussion regarding the importance of the assays detecting 25-(OH)D2 as well as 25-(OH)D3. the authors of the report state that the validity of the 25-(OH)D results that are obtained will justifiably continue to be questioned. 59% of the participating laboratories were found to have met the target [12.8). the greatest increment being detected by HPLC [11]. Another study compared the DiaSorin RIA assay with the Nichols chemiluminescence assay. shows the latest results of this external quality control project. Hollis also emphasizes the need for validation of the user’s assay system in the laboratory in question regardless of the claims made by the manufacturers [8]. The means varied widely between laboratories from 27. The increment was less than anticipated and varied between laboratories (p < 0.005) from 7. are still treated with 25-(OH)D2 and the monitoring of vitamin D therapy is complicated by the presence of assays that underestimate 25-(OH)D2. . Both assays also underestimated the 25-(OH)D-concentration as compared with the HPLC-method [10]. The HPLC method enables 25-(OH)D2 and 25-(OH)D3 to be quantified whereas there are concerns regarding the detection of 25-(OH)D2 with other assays (Nichols procedure and IDS RIA) [9]. These give the impression that only method 6 exceeded the limit.5.6 ng/ml (p < 0. and the requirement for meeting the target being to get 80% or more of the results obtained be within ± 30% of the All-Laboratory Trimmed Mean (ALTM). Mean (SEM) results for the 25-(OH)D concentrations in the serum samples of 10 subjects spiked with the 25-(OH)D standard. as estimated by five different laboratories (for abbreviations see Table 2. a subsequent study refuted these findings and also concluded that vitamin D2 is less potent and has a shorter duration of action than vitamin D3 [14].4 to 44. however. Patients. Each of the laboratories was given five serum samples quarterly. several authors have called for international standardization of the vitamin D assays that are available and that are affordable for practicing clinicians. In accordance with these findings. 2.05).13]. finding discordant serum 25-(OH)D-values.

18–23]. Accuracy of 25-(OH)D methods used by the different DEQAS participants [13]. around 25–40 nmol/L [1. In a recent publication. IDS RIA. Hollis defined 25-(OH)D levels below 80 nmol/L as being deficient. method 5. Populations at risk include nursing home residents and the elderly. competitive protein-binding assay. race. 2. but there may also be a significant proportion of the population that exhibits asymptomatic subclinical vitamin D insufficiency. due to the serious health risks encountered at levels less than this [7]. especially the home-bound elderly.6. Method 1. and the composition of the population being studied. There is growing evidence that there is not only a specific seasonal decline in serum 25-(OH)D during the winter months. age. as shown by the seasonal variation in vitamin D status that occurs [17. dietary intake. method 2. Recent reports suggest European and North American populations to have a higher degree of insufficiency than had previously been thought [15. Each column shows the mean deviation (% bias) and SE from the target value (ALTM) during the period of 2000–2004. that set the cutoff points at much lower levels. DiaSorin RIA. A summary of studies on vitamin D status in the elderly is given elsewhere [1]. In epidemiologic studies. suggested cut-offs for insufficiency range from 10 to 30 ng/ml (25–80 nmol/l). HPLC.Jakob Linseisen. Apart from the analytical problems. In addition to the high degree of uncertainty that the divergence of the results for different techniques and different laboratories creates. method 3. method 6. season. IDS EIA.16]. Nichols automated chemiluminescence assay. but the level of circulating 25-(OH)D sufficient to maintain good health and avoid clinical symptoms or consequences due to an inadequate vitamin D supply. It is important to note that “normal” is not defined as the median population level. Having an adequate definition of the normal range is essential in routine clinical practice in order to be able to define groups that are at risk for the negative consequences of vitamin D deficiency. . This is in contrast with most of the published studies.18]. epidemiological variability needs to also be taken into account.15. Sascha Abbas 68 Fig. The method means of samples known to contain 25-(OH)D2 were excluded. the serum 25-(OH)D concentrations in different populations vary with latitude. method 4.

J Clin Endocrinol Metab 1971. Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects. Hollis BW. et al.89:3152–7. Issues of methodology. vitamin D3). J Clin Endocrinol Metab 2004. . the measurement variation introduced by different assays and different laboratories. Immunodiagnostic Systems (IDS). Editorial: The determination of circulating 25-hydroxyvitamin D: no easy task. J Nutr 2005. Clin Chem 2000. Competitive binding assay for vitamin D and 25-OH vitamin D. Goldswain PR. Plum L. 10.89–90:611–4. Lemann J.25-(OH)2-vitamin D3 in healthy adults. 3. Potts JT. Wilz DR.135:317–22. Glendenning P. Myles M. Hollis BW. Cowgill CS. Lips P. McGuiness M. Ann Clin Biochem 2003. J Clin Endocrinol Metab 1978. 2. vitamin D metabolites have various important biological effects. Zhou XY. J Clin Endocrinol Metab 2004. DeLuca HF. Epidemiologic studies in particular can contribute knowledge concerning the association between vitamin D insufficiency and the risk of disease. 8. 7. 6. Hansen KE. Binkley N. et al. This makes strong scientific research efforts needed. epidemiologic variation in vitamin D status and its determinants. Caldas AE. Endres D. standardization and metabolite recognition for 25-hydroxyvitamin D when comparing the DiaSorin radioimmunoassay and the Nichols Advantage automated chemiluminescence protein-binding assay in hip fracture cases. 5. References 1. Zerwekh JE. Musk AA. the determination of vitamin D status is still a challenging task. Several problems are important to bear in mind including the different forms of vitamin D (vitamin D2. Ann Clin Biochem 2004. Which circulating level of 25-hydroxyvitamin D is appropriate? J Steroid Biochem Mol Biol 2004. Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D. DeLuca HF. May 2005.46:756–65. Clemens TL. Hollis BW. Taranto M. Comparison of commercially available (125)I-based RIA methods for the determination of circulating 25-hydroxyvitamin D.46:1657–61. Metabolism and excretion of 3H-1. Overview of Vitamin D Measurement and Methodologies. including the risk of cancers at various sites. Lake E.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 69 Conclusions In conclusion.41:272–81. J Clin Endocrinol Metab 1986.40:546–51. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. Belsey R. 11.33:554–7. Krueger D. 9. Smith GA.63:656–60. Nonetheless. The measurement of vitamin D: analytical aspects.89:3149–51. Gray RW. Hart GR. and problems in defining the appropriate threshold for vitamin D sufficiency. Review SeriesVolume 2. 4. Noble JM. Lindsay R.

Gunter E. Jones G.7:327–35. 13. J Nutr 2005. Vitamin D status: effects on parathyroid hormone and 1. Ovesen L. Miller AB. apparently healthy urban European population.71:1577–81. 20. Davison KS. Vitamin D status of middle-aged women at 65–71 degrees N in relation to dietary intake and exposure to ultraviolet radiation. Morris HA. 25-dihydroxyvitamin D in postmenopausal women. MacFarlane GD.89:5387–91. Holick MF. Prevalence and predictors of vitamin D deficiency in five immigrant groups living in Oslo. Arnaud S. Meyer HE. Woitge HW. Proc Nutr Soc 2003. Geographical differences in vitamin D status. 15. 17. Makin HL. Beard MK.7:439–43. Preziosi P. Jakobsen J. Ersfeld DL. Carter R. Osteoporos Int 1997. with particular reference to European countries. Clin Chem 2004. Body JJ. Prevalence of vitamin D insufficiency in an adult normal population. Metab 2004. Jones J. Nordin BC. 23. Binkley N. Chapuy MC. Aksnes L. et al. Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation. et al. Prevalence of vitamin D inadequacy among postmenopausal North American women receiving osteoporosis therapy. Brustad M. Hollis BW. Armas LA. Obstet Gynecol Surv 2005.89–90:467–71. Engelsen O. 19. . Scheidt-Nave C. Horowitz M. 22. Fenske JS.89–90:621–2. Holvik K. Carter GD. Katzer JT. Norway: the Oslo Immigrant Health Study. Need AG.59:57–63.62:813–21. et al. Vitamin D2 is much less effective than vitamin D3 in humans. Sascha Abbas 70 12. J Clin Endocrinol Metab 1998. Seasonal variation of biochemical indexes of bone turnover: results of a population-based study. 21. Lund E. Andersen R. Jones J. Leidig-Bruckner G.83:68–75.60:658–9. Am J Clin Nutr 2000. Khan A. 14. Kissling C.135:332–7. Hanley DA. Hypovitaminosis D in a normal. Galan P. Eur J Clin Nutr 2005.Jakob Linseisen. Berry J. et al. 16. Hercberg S. 18. Maamer M. Sackrison JL. Carter CR. Haug E. Public Health Nutr 2004. J Clin Endocrinol. Grauer A. J Steroid Biochem Mol Biol 2004. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. Alsaker E.50:2195–7. Carter GD. Siris ES. Heaney RP. Meyer K. Vitamin D insufficiency in North America. Brunvand L. J Steroid Biochem Mol Biol 2004.

. Lund University. When ingested by humans or other organisms.4. and in epidemiological studies of the risk of cancer. containing the amino acid selenocysteine (Sec. so-called selenoproteins. some of these compounds being uncharacterized [4–6]. and methods employed in assessing an individual’s selenium nutritional status — both generally. It is uncertain whether they occur naturally in foods to any major extent.). In addition to the forms of selenium mentioned. Pure and Applied Biochemistry.9. and Department of Clinical Nutrition. together with epidemiological findings on relations between the selenium status in the body and risk of cancer are reviewed. plants and microorganisms is bound within proteins. as well as in connection with long-term trials for investigating the disease-preventive potential of selenium supplementation. In the diet. Another selenium-containing amino acid is selenomethionine. several low-molecular-weight selenium compounds have been shown to be present in different foods. In the chapter thereafter (Gromadziƒska et al. the occurrence of different selenoproteins used as biomarkers of selenium status. Lund. selenoproteins are largely found in animal foods. Lund University Hospital.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 71 2. which is synthesized by plants and by yeast. their functions and mechanisms of action. and the use of intervention trials to study the cancerpreventive effects of selenium supplementation. A major part of the selenium in mammals is specifically incorporated into proteins of defined biological function. nearly all of the selenium in animals. The present chapter concerns the various forms of selenium and their metabolism. Selenite and selenate are inorganic forms of selenium used as dietary supplements. These include the forms of selenium present in the body and in the diet. Different forms of selenium With few exceptions. A variety of issues connected with this are reviewed in two of the chapters. Selenium and cancer — selenoproteins as biomarkers in relation to selenium supplementation Björn Åkesson and Katharina Bruzelius Biomedical Nutrition. The replacement of methionine by selenomethionine appears to be random and to be dependent on the relative concentrations of these amino acids [2]. the mechanisms of action involved. several protein-bound forms having been identified. analogous to cysteine in which sulphur is replaced by selenium) (Table 2. but little is known of the role it has here. it is incorporated non-specifically into proteins as an analogue of methionine [1]. Other proteins can also bind selenium as a ligand [3].). Sweden Introduction The relationship between selenium and cancer involves many different factors.

which is turn is the precursor of selenocysteine in selenoproteins and various excreted forms of selenium. Katharina Bruzelius 72 Table 2. Metabolic pathways of selenium. Selenate and selenite are reduced by glutathione to hydrogen selenide. The major selenium metabolite excreted in urine is selenosugar. selenium is removed as dimethylselenide via exha- Fig. Several compounds can be converted into methylselenol (from [8]).7. Selenomethionine and selenocysteine can also be converted to hydrogen selenide. Some major inorganic and organic forms of selenium Organic Selenocysteine (Sec) Selenomethionine HSeCH2CH(NH2)COOH CH3Se(CH2)2CH(NH2)COOH Inorganic Selenite Selenate SeO32– SeO42– Metabolism of selenium Different chemical forms of selenium are involved in metabolic pathways (Figure 2. which is either transformed into selenophosphate for incorporation into selenoproteins (see below) as Sec or is methylated to selenosugar (1-β-methylseleno-N-acetyl-D-galactosamine). selenocysteine and selenite can be converted into the key metabolite hydrogen selenide (H2Se). At toxic doses. Selenomethionine.7. dimethylselenide or trimethylselenonium ions for excretion. . a much lesser amount being excreted as trimethylselenonium ions [7].).9. 2.Björn Åkesson.

are the selenium compounds most effective in cancer prevention [8]. found in embryos and in the olfactory epithelium Unknown Unknown Unkown. involved in many biological pathways Mitocondrial thioredoxin reductase. Selenoproteins A total of 25 selenoprotein genes were discovered in the human genome several years ago by sequence analysis. such as Se-methylselenocysteine and selenomethionine. a membrane protein Involved in protein folding in the ER Unknown. Several studies have suggested that methylated Se derivatives.9]. involved in the elimination of misfolded proteins from the ER Unknown Unknown. mutations in gene associated with muscular diseases Unknown An antioxidant and selenium transport protein Reduces methionine-R-sulfoxides A membrane protein. mainly in the gastrointestinal tract Reduction of phospholipid hydroperoxides Peroxide reduction.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 73 lation.) [1. Several of these compounds can be converted to methylseleninic acid or methylselenol which have anticarcinogenic effects in vitro [8]. involved in many biological pathways Thioredoxin/glutathione reductase found mainly in testes . Selenoproteins in the human and their putative functions Selenoprotein 15 kDa DI1 DI2 DI3 cGSHPx eGSHPx giGSHPx phGSHPx GSHPx 6 SelH SelI SelK SelM SelN SelO SeP SelR SelS SelT SelV SelW SPS2 TrxR 1 TrxR 2 TrxR 3 (TGR) Involved in protein folding in the ER Thyroid hormone control Thyroid hormone control Thyroid hormone control Peroxide reduction in the cytoplasm Function Peroxide reduction in plasma and other extracellular fluids Peroxide reduction. yet the functions of many of the proteins involved are still unknown (Table 2.10. only found in the testes Can reduce peroxides using glutathione as an electron donor Catalyses the formation of selenophosphate Cytoplasmic thioredoxin reductase. The distribution and concentrations of selenoproteins Table 2.10.

). The cytosolic or cellular form of glutathione peroxidase (cGSHPx) was the first selenoprotein identified [10. The other two major groups of known selenoprotein enzymes are the iodothyronine deiodinases.11]. It is found in almost all tissues and is believed to play a part in the body’s antioxidant defence. The next major step is the interpretation of UGA as a signal to insert Sec instead of stopping the translation. Katharina Bruzelius 74 in different tissues are also not well known. one apical loop.15].8. these organisms tending to have homologue proteins containing Cys instead of Sec [13].Björn Åkesson. but the mechanisms involved have not yet been elucidated (Figure 2. Preservation of the SECIS core and of the length of the helix between the first internal loop and the second internal loop or the apical loop are important for the functioning of SECIS [16]. that regulate thyroid hormones. In eukaryotes the Sec codon and the SECIS element are located at some distance from each other in the mRNA.). Several other glutathione peroxidases containing selenocysteine have been found since then. and the thioredoxin reductases. The current conception is that the mRNA and the SBP2-EFsec loops back towards the translation complex [13. Many of the known selenoproteins catalyse redox reactions in which selenium is at the active site. There are no selenoproteins known to be present in yeast or higher plants. catalysing the reduction of oxidised thioredoxin and other substates [1. and the SECIS core. In the first step.8. which has two [15]. Several of the selenoproteins have an homologue protein containing Cys instead of Sec. The SECIS consensus sequence consists of two helices. which is a short sequence of non Watson-Crick paired nucleotides that appear to exist in all selenoprotein mRNA:s.12]. serine is bound to tRNASec and then transformed to selenocysteine [13]. and the recently discovered L30 protein. . This structure is located in the 3’-untranslated region of the mRNA in eukaryotes and immediately after the UGA codon in prokaryotes [14. In addition to the SECIS element.17]. Biosynthesis of selenoproteins Translation of the codon UGA to selenocysteine (Sec) and its insertion into proteins is a complicated process (Figure 2. Except for the SECIS element there are no features in the DNA-sequence that selenoprotein genes are known to have in common. but the catalytic ability of these latter proteins is much lower. additional factors are required for the insertion of Sec in eukaryotes such as the SECIS binding protein 2 (SBP2). All known eukaryotic selenoproteins have one SECIS-element. one internal loop. the Sec-specific elongation factor (EFsec). This takes place when a special stem-loop structure called the selenocysteine insertion sequence (SECIS) is present in the mRNA. selenoproteins also differing markedly in the nucleotide sequence of their SECIS elements. except for SeP.

Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 75 Fig. There is also a tissue selenium hierarchy that controls the retention of selenium in the tissues and organs under selenium-deficient conditions. cGSHPx could not be stabilised by replacing its 3’UTR with those from more stable glutathione peroxidase mRNAs. Individual selenoproteins The glutathione peroxidase family The glutathione peroxidases generally catalyse the reduction of peroxides using mainly glutathione as the electron donor. and hydrolysis of GTP [17]. Hypothesised Sec translation complex in eukaryotes. but not with that from cGSHPx.19]. This hierarchy is particularly noticeable in the glutathione peroxidase family. causing a conformational change which leads to the release of Sec-tRNASec. The role of the SECIS for mRNA stability was studied by combining the coding regions of giGSHPx. Accordingly an overview of the individual selenoproteins is provided. where the order of stability is as follows: giGSHPx ≥ phGSHPx > cGSHPx = eGSHPx. at least there are factors in both the coding and the non-coding mRNA regions that influence its stability [19].8. It was shown that giGSHPx and phGSHPx containing each other’s 3’UTRs would remain stable. This indicates that in the cGSHPx mRNA. The stability of selenoprotein mRNAs is affected by the amount of selenium present in the cell. 2. In rats and mice fed a seleniumdeficient diet. thus contributing to the body’s defence against free radicals. The SECIS binds to SBP2 which binds the Sec-specific elongation factor EFSec and its Sec-tRNASec. . The links between selenium and cancer probably involve different selenoproteins. phGSHPx and cGSHPx with the 3’UTRs (3’-untranslated regions) of each mRNA and then to study the system during selenium deficiency. Five selenium-dependent glutathione peroxidases are known to date [9]. A special feature is that transcripts of some selenoproteins are much more stable than those of others. the brain and the testes retain selenium whereas the selenium concentrations in the liver and the kidneys decrease markedly [20]. there being a selenoprotein hierarchy. After association with the ribosome the SBP2 and the L30 switch places. but it is not yet known how this regulation operates [18.

without any obvious damages to the host organism. thyroid. In model systems with an over-expression of cGSHPx. Putative functions of this enzyme are those of protecting against ingested lipid hydroperoxides and reducing susceptibility to colon cancer [21]. milk. Other tissues such as liver. Under conditions of severe selenium deficiency. This is the only selenoprotein that has been identified in milk thus far. In mouse and rat. pancreatic. Disruption of the phGSHPx gene is lethal embryonically. Although the glutathione concentration in the plasma is very low eGSHPx can also use other electron donors such as the thioredoxin system [30]. amniotic fluid. there also being a number of negative effects such as increased obesity and insulin resistance [22]. is mainly found in the gastrointestinal tract and also in the human liver and in mammary cells and tissue according to certain studies [23. each containing one selenium atom. but knock-out of both cGSHPx and giGSHPx leads to colitis in mice [25]. unlike disruption of cGSHPx or giGSHPx. skeletal muscle. protective adapatations against paraquat and other challenges were shown to take place. This enzyme has been implicated in inflammation and molecular signalling. cGSHPx knock-out mice have been used to explore the effects of complete loss of cGSHPx activity. Gastrointestinal glutathione peroxidase (giGSHPx). lung lavage and the aqueous humour [21. catalyses the reduction of hydrogen peroxide and organic peroxides. giGSHPx knock-out models show no particular changes in phenotype to occur compared with the wild type. The most recently discovered member of the glutathione peroxidase family is glutathione peroxidase 6. Phospholipid hydroperoxide glutathione peroxidase. which is found in most tissues. phGSHPx occurs in mitochondrial. Extracellular glutathione peroxidase. phGSHPx. placental and mammary gland tissue produce this enzyme to a lesser extent.24]. which has led to speculations that this enzyme is a form of selenium storage to some extent [21]. The postulated roles of eGSHPx include control of peroxide transport and of the extracellular ‘peroxide tone’ [18. nonmitochondrial and sperm-nuclei-specific forms produced from the same gene [31] and has an important role in sperm functioning [32]. and its functional significance is unclear. is a tetrameric protein produced mainly in the kidney and then excreted into the extracellular environment [26]. the level of cGSHPx in most tissues decreases considerably. which has thus far only been found in olfactory epithelium and in embryos.27–29]. It differs from the three glutathione peroxidases just mentioned by being a monomer and having a different substrate specificity. It consists of four identical subunits.Björn Åkesson. . Katharina Bruzelius 76 Cellular (or cytosolic) glutathione peroxidase (cGSHPx). catalyses the reduction of phospholipid hydroperoxides and is expressed in a wide range of tissues. which consists of four subunits. This glutathione peroxidase isoenzyme is also found in extracellular fluids such as blood plasma. eGSHPx. the selenocysteine in this enzyme is replaced by cysteine [9]. These mice appear phenotypically normal but when challenged by viruses or oxidising poisons such as paraquat they are more severely affected than mice of the wild type are.21].

can also form complexes with mercury and cadmium [41]. and expression of DI2 and 3 having been found in many tissues. including bovine mammary tissue [34–36]. but is produced primarily in the liver and is secreted then into the plasma.40]. but in mammals they can also reduce other substrates. It can reduce peroxynitrite and phospholipid hydroperoxides [39. There are three main isoforms of thioredoxin reductases. was designated “P” because of its being found in the blood plasma. TrxR 1 and 2 are ubiquitous being located in the cytosol and the mitochondria. such as DNA synthesis and the regulation of apoptosis. Thioredoxin catalyses the reduction of protein disulfides and is involved in a number of vital processes. thus activating or deactivating them. thioredoxin/glutathione reductase (TGR). SeP is expressed in most tissues. DI3). There are indications that it also acts as an antioxidant in the extracellular space. Selenoprotein P is the major form of selenium in the plasma and is involved in selenium transport [37. but not in the thyroid. but a number of splice variants of TrxR 1 and 2 have also been reported. It is localised in the endothelium. DI1 is found in the liver. The iodothyronine deiodinases have high priority in the selenium hierarchy. 2 and 3 (DI1. It can contain from 1 to 17 selenocysteines. SPS2 is a selenoprotein. particularly DI2 and DI3. and can stimulate the survival of nerve cells in culture [42]. also catalyses the formation of selenophosphate [44]. respectively [12]. iodothyronine deiodinase 1. is expressed in small amounts in many tissues but is primarily found in the testis [33].Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 77 The thioredoxin reductase family The thioredoxin reductases (TrxR) catalyse the reduction mainly of thioredoxin. organs that normally are highly prioritised during selenium deficiency.38]. binding to heparin and related carbohydrates [38]. . In some tissues. kidneys and thyroid. The third isoform. their levels remaining virtually unaltered in the case of selenium deficiency. such as vitamin C. Selenoprotein P Selenoprotein P (SeP). for example. The iodothyronine deiodinase family The iodothyronine deiodinase family consists of three enzymes. which is the selenium donor for the formation of selenocysteine from serine bound in tRNASec. unless they are rescued by a high-selenium diet [43]. Truncated isoforms of this protein have also been found. These mice die after weaning of the young. Selenophosphate synthetase 1. this property indicating the existence of a feedback step in the production of selenoproteins. depending on the animal species. SeP knock-out mice exhibit low levels of selenium in the brain and testes. The 15 kDa selenoprotein. the second selenoprotein to be discovered. which is not a selenoprotein. DI1 decreases during selenium deficiency. Additional selenoproteins Selenophosphate synthetase 2 (SPS2) catalyses the formation of selenophosphate. which catalyse the removal of different iodine groups from the thyroid hormones. DI2. Targeted disruption of the TrxR 1 or 2 genes in mouse models is embryonically lethal [22].

the selenium levels in whole blood or the erythrocytes appear to primarily reflect the long-term intake of selenium [53]. SelW occurs mainly in muscle and brain and has been shown to act as an antioxidant. as compared with plasma selenium. A number of other selenoproteins have been discovered in man but information regarding their function is very limited. For instance. This is an important step in the regulation of biological processes and the management of oxidative stress in the cell. SelN is the only selenoprotein in which a mutation of it has been shown to cause a disease [47]. and may thus be suitable as an indicator of short-term changes. Selenoprotein M (SelM) is structurally similar to the 15 kDa protein and is supposedly also involved in protein folding in ER [46]. Although its catalytic function is still unknown. the measurement of selenoproteins can be expected to provide information on specific selenium functions. utilising glutathione to reduce peroxides [51]. also known as VIMP) is a membrane protein in the ER. which also includes non-specifically bound selenium. To date.Björn Åkesson. It has been identified in prokaryotes. Indices of selenium status The most commonly used methods for assessing the selenium status in humans involve analysis of selenium concentrations in the blood or blood fractions. In addition. eukaryotes and archaea [15]. Selenoprotein S (SelS. such as rigid spine muscular dystrophy. Selenoprotein R (SelR) reduces methionine-R-sulfoxides. one that has been associated with the process of eliminating misfolded proteins by transferring them to the cytosol [48] and also with inflammation [49]. The function of selenoprotein W (SelW) has not been elucidated fully but it has been implicated in white muscle disease [50]. In general. In contrast. usually responding rapidly to changes in selenium status or in the dietary intake of selenium [52]. mutations in the gene have been associated with various muscular diseases. nails and urine has been employed. The responses to selenium intake obtained if other blood fractions are analysed can differ. Selenium in the plasma or serum is the best known and most accessible index. and it responds rapidly to changes in selenium status. since above a certain selenium level they tend to reach saturation. since the turnover of erythrocyte selenium is slower. When data from . the determination of selenium in the hair. Selenoproteins as biomarkers The use of selenoproteins as markers of selenium status has only been exploited thus far in a few large epidemiological studies. Selenoprotein N (SelN) is a 70 kDa protein located in the ER. the activity of GSHPx in plasma has been used by a variety of laboratories in selenium supplementation studies. Katharina Bruzelius 78 located in the endoplasmatic reticulum (ER). is believed to be involved in protein folding [45]. Another important conception is that plasma selenoproteins may not be suitable biomarkers under conditions of high selenium status.

43) — — eGSHPx (mg/l) — 352 (306.31) 2.21.73 (0. b GSHPx activity.78 (1.95 (2.74)b — — — 126—205 38 21 16 31 1. 1. 1. 1.83 (0.38 (2.13 (4. glutathione peroxidase and protein-bound selenomethionine are the major selenium fractions contained in plasma [56].47 (1.08. 2.60.41 (1.56.38 (1.20 (1.52) 0.46—1.31—4. 0.65) 3.34. 1.44. Trial I baseline Finland. In the following various results from their use in the authors’ laboratory are summarized (Table 2.66—1.11.51.27) 1.75—2. 0. U/l. Table 2.52 (0.63—1.07) 1.07 (0.55 (0.15) 1.69—5.2 mmol/l [55]. [63]). Selenoprotein P.91 (1.38 (0.16.16) 0. Study European countries Prior to LDL-apheresis After LDL-apheresis Finland.14) 0. mU/mg protein.18 (0.73) Values are means (95% CI). medium and high fish intake.25) Plasma selenium (µmol/l) 1.86 (0. 1.64) 0.14 (1.51) 0.56) 4. 1.47) 1. selenium and eGSHPx in different studies (from ref. 1.0.83) 1. a GSHPx activity.43.83) 0.51 (6.33. 346)a 6. 0.98.86) 0.85) 1.32) 3. respectively. 1.). c Data from subgroups having (top to bottom) low.39.77 (1.83.69. 0.23 (1. Selenoprotein P accounts for at least 40% of the plasma selenium [37].91 (0. 0.69 (0.11.24) 1.10 (1.44) 1.u. Median values (and range) are given. 397)a 302 (259.03 (0. Trial II baseline Cancer cases Controls Elderly subjects (Malmö Food Study) Patients on HPN Latvians with differing fish intakec n 414 13 13 50 45 302 406 SeP (a.69 (0.30—1. 1.) 1. . 4. Immunoassays for measurement of this protein have been developed [57–59].41 (0. 1.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 79 different cross-sectional studies was combined eGSHPx was found to approach a plateau at a plasma selenium concentration of approximately 1 mmol/l [54].6.20—4.22) 0. Plasma concentrations of SeP.12) 0.88) 1.00 (0.92.41. 0. Supplementation by selenate resulted in glutathione peroxidase activity plateauing at a plasma selenium concentration of 1.11.54—1.28.66) 1.21) 1.

The scientists there compared the responses to oral supplementation of 200 mg selenium per day in healthy subjects on two occasions. The levels of selenoprotein P and eGSHPx were found to vary markedly for subjects from different regions and with different diseases.68]. Regarding the relationships between SeP. For women. but that at normal selenium status selenoprotein P usually correlated more highly with plasma selenium than eGSHPx did. studies of lead-exposed children in Katowice. significant relationships between milk intake versus selenoprotein P and urinary selenium. with some indication of a plateau. Poland revealed an inverse relationship between the blood lead level and the level of selenoprotein P and of extracellular GSHPx [65]. In the Malmö Food Study [61] the relationship between the intake of different foods and selenium status was investigated. Katharina Bruzelius 80 Selenoprotein P as a biomarker Selenoprotein P levels were measured in healthy adults from 17 European Regions [60]. In the second study. and it is not possible to conclude that lead exposure produces a decrease in the selenoprotein concentration or that a poor selenium status increases susceptibility to high blood lead concentrations. total selenium and selenoprotein P than control subjects did [64]. Considerable attention has been directed at two studies conducted in Finland regarding the use of different forms of selenium [67. Considerable variation between different regions was found. and also between fish intake versus serum and urinary selenium were found.Björn Åkesson. the selenoprotein P values increased after selenium supplementation. In the first study. the selenium status of the subjects thus being higher. A summary of the responses shown by the different selenium indices in the first of these two studies is provided in Figure 2. it was found generally speaking that the selenoprotein P level correlated more highly with the plasma selenium and eGSHPx level at low selenium status than at high selenium status. however.9. a highly significant positive relationship between fish intake and selenium status was obtained as well as an inverse relationship between the plasma selenium and thyroid stimulating hormone levels. In a study of Latvian fisherman [62]. In a recent study of Chinese subjects of low selenium status given different doses of selenite and selenomethionine. Since the direction of causality was uncertain. no significant increase in selenoprotein P levels was observed after selenium supplementation and no differences were found between groups given different forms of selenium [69]. in which the subjects were low in selenium status. Home parenteral nutrition patients with a variety of gastrointestinal diseases showed much lower levels of extracellular GSHPx. eGSHPx and plasma selenium as markers of selenium status. .9. Concerning the association of selenium status and exposure to toxic metals. performed after the introduction of selenium-enriched fertilizers in Finland. There was a close correlation between selenoprotein P and plasma selenium values. plateauing within two weeks [69] (Figure 2. Biomarkers of selenium status in relation to selenium supplementation Many studies on efforts to improve selenium status through selenium supplementation have been performed and a review of different modes of selenium supplementation has been assembled [66].).

Fig. Full expression of selenoprotein P was not achieved at the highest dose of either form (66 µg/d).). in turn found selenium supplementation to reduce total incidence and mortality of cancer in an American study group (see below). particularly among men [75–81]. in view of the many regulatory mechanisms that exist for the incorporation of selenium into selenoproteins and the varying effects of different forms of dietary selenium on indices of the selenium status. β-carotene and α-tocopherol reduced the total cancer mortality and stomach cancer rate in a Chinese population. Interest in the preventive role of selenium supplementation in humans was stimulated markedly by results from two intervention studies.72].87]. Blot and coworkers [73] found that a mixture of selenium.69]). lower prediagnostic plasma selenium was found for cases of cancer than for controls. Biomarkers of selenium status and cancer Experimental studies have shown that the addition of high (0. This suggested that selenoprotein P is a better indicator of selenium nutritional status than glutathione peroxidase is [70]. . Clark and associates [74]. These matters are reviewed in greater detail in the chapter that follows (Gromadziƒska et al. it appears that a more adequate assessment of the selenium status would be obtained through the use of several biomarkers being employed concurrently than through analysis of only total selenium or of a single selenoprotein. In a number of case-control studies. The strongest associations between premorbid plasma selenium levels and risk of cancer were observed for cancer of the respiratory and digestive tracts.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 81 full expression of glutathione peroxidase was achieved with use of 37 µg Se/d in the form of selenomethionine and with 66 µg Se/d in the form of selenite. Percentual changes in selenium status variables after supplementation of Finnish subjects of low selenium status with 200 µg/day of selenium in different forms (from ref [67. whereas in other studies no significant differences in this respect were found between cases and controls [82–86]. The calculated degree of protection was found to differ between cancer sites [81.9. Generally speaking.5–2 ppm) levels of selenium to the diet has a carcinostatic effect in animals treated with carcinogenic chemicals [71. 2.

In addition. and toenail selenium [90–92] were observed in smokers than in non-smokers. 0. whole blood selenium [89. This is probably due to SeP constituting at least 40% of the total selenium in human plasma [37]. Fig. 2. The individual references are cited in [63].85. For increasing quintiles.01).89].3. lower values of plasma selenium [77. plasma selenium has been used as a marker of selenium status. Smokers were found to have significantly lower levels of selenoprotein P than nonsmokers [88]. When cases were divided into subgroups according to cancer site.Björn Åkesson. Smoking and biomarkers of selenium status Several factors other than selenium intake may affect biomarkers of selenium status. normal contour: p ≥ 0. One possible explanation to the lower selenoprotein P level in smokers would be that smoking contributes to chronic low-grade inflammation due to its irritating effect on the respiratory tract and on the vascular endothelial cells. Bold contour: p < 0. in the lowest tertile as compared with the cases in the highest tertile. respectively (p for trend = 0.05. 2. erythrocyte selenium [89]. Katharina Bruzelius 82 Relations between the selenoprotein P level and risk of cancer In most studies of the association between selenium status and risk of cancer. and 0.0. Percentage differences in the prediagnostic selenium and SeP plasma levels between cancer cases and controls (set at 0) for cancer at different sites. .2.6 respectively. In Figure 2. The circles represent selenium and the squares SeP. 2.10. digestive and urinary tract and for cancer of other sites.9.2. The factors contributing to the lower selenium status in smokers are unclear. In other studies.0. More recently the premorbid level of SeP in the plasma of subjects who had developed cancer at different sites was studied in a nested case-control study [88]. The previously reported association of plasma selenium levels with cancer risk can very likely be explained by the corresponding association of SeP levels. the ORs (adjusted for smoking) in tertiles of the SeP level were calculated for the respiratory. the ORs (adjusted for smoking) were 5. the case-control differences of plasma selenium and SeP are compared for major cancer sites in several different study populations.90]. The association between the relative risk of getting cancer and the SeP concentration found was also estimated from quintiles of the SeP level. These were 6.05. 2.0 and 1. 3.4. the SeP levels for cancer of the respiratory tract were found to be significantly lower than in matched healthy control subjects.10.

which could probably in part explain their lower selenium level [90]. The cadmium blood level was found to be negatively associated both with selenium in the blood and with selenium and selenoprotein P in the plasma. α-tocopherol and selenium given for a 5. It has also been shown that selenoprotein P plays a role in the defence against peroxynitrite [39]. In the Linxian trial involving supplementation of β-carotene. which together with superoxide can produce peroxynitrite [39]. suggests that the selenoprotein P levels are reduced by inflammatory activity. It has been shown that the concentration of selenium in the blood is significantly lower in subjects smoking more than 50 g tobacco per week than in never-smokers. This agrees with findings of Dreher and co-workers [93] indicating that the human selenoprotein P promoter is rendered less active by cytokine treatment. Multiple regression analysis also indicated the blood cadmium to increase significantly with a decrease in the selenoprotein P level. several intervention studies having indicated beneficial effects [73. selenomethionine and glutathione peroxidase can scavenge peroxynitrite. The natural presence of cadmium in tobacco smoke may also contribute to the lower selenium status found in smokers. Smoking may also increase oxidative stress since cigarette smoke is a rich source of reactive nitrogen species. a small but significant reduction in total cancer mortality was obtained (RR = 0. Patients with a history of skin carcinomas who . In another study. Intervention trials on the effect of selenium supplementation on cancer No definite proof of a protective effect of selenium in connection with cancer has been presented as yet in human investigations but there has been increasing interest in the cancer preventive action of selenium supplementation. the blood levels of selenium and cadmium and the plasma levels of selenoprotein P were measured in children from the Katowice industrial area in Poland [65].91). In addition. which could exacerbate the risk of cancer associated with smoking [96]. whereas the concentration of cadmium in the blood is significantly higher in smokers [95]. although this association disappeared when lead was included in the model. Multiple linear regression analysis of the data also suggested a depressive effect of cadmium on the concentration of selenium in the blood. and that these correlations are higher (more significant) in smokers. whereas smoking alone did not serve as a true predictor of this effect. in a meta-analysis of 51 published nutritional surveys of the relationship between smoking status and nutrient intakes. smoking was found to be significantly associated with an unhealthy pattern of nutrient intake.74]. a result that could possibly be explained by the covariance of lead and selenium in the blood [65]. It has been reported that smokers eat less selenium than non-smokers do. both being acute-phase reactants. This may also explain the slightly lower selenoprotein P concentration in smokers. in particular mortality due to stomach cancer [73]. which suggests a repression of selenoprotein P expression during an acute phase reaction.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 83 The finding that selenoprotein P is positively correlated with albumin level and negatively correlated with α1-antitrypsin. As reported by Sies and coworkers [94].25-year period.

Food Chem 1994. 4. Kyriakopoulos A.51:45–9. Bansal MP. concerning the purportedly positive effects of selenium in connection with the prevention of cancer provided certain evidence for permitting a qualified health claim regarding selenium and cancer [103]. Annu Rev Nutr 2001.21:453–73. wheras later results showed selenium supplementation to be ineffective in preventing basal cell carcinoma and that it increased the risk of squamous cell carcinoma and of total nonmelanoma skin cancer [97]. 3. β-carotene. selenomethionine supplementation of subjects with mild to moderate esophageal squamous dysplasia showed there to be a nonsignificant trend toward an increased regression and decreased progression of dysplasia as well as a significant beneficial effect in the subgroup showing mild esophageal squamous dysplasia [100]. Larsen EH. colorectal (RR = 0.16:1403–8. Mukhopadhyay R. Effect of dietary methionine on utilization of tissue selenium from dietary selenomethionine for glutathione peroxidase in the rat. Srikumar TS. 2. Scott J. Occurrence of low-molecular-weight and high-molecular-weight selenium compounds in fish.102]. Recent results of the SUVIMAX study showed supplementation with vitamin C. Behne D.Björn Åkesson. The type of selenium supplements best employed is also in need of further investigations. Mukhopadhyay T. selenonium ions and inorganic selenium by ion exchange HPLC with mass spectrometric detection and its application to yeast and algae. It is apparent from this review that selenium can play an important role in cancer prevention. vitamin E. Åkesson B. but additional studies are needed to determine whether there is also an increased risk of some forms of cancer after selenium supplementation. Fan T. 5.50) and in incidence of lung (RR = 0. . evaluation of health claims by the FDA in the U.37) cancer [74]. In another Chinese investigation. Sunde RA. Hansen M. DNA sequencing of a mouse liver protein that binds selenium: implications for selenium's mechanism of action in cancer prevention. Mammalian selenium-containing proteins.S. Cook RG. Medina D.54). Vahl M. selenium and zinc to reduce the rate of prostate cancer in men having normal levels of prostate-specific antigen in their plasma [99]. Experimental findings also indicate that in some experimental systems selenium can both promote and inhibit cancer [98].118:367–74. Katharina Bruzelius 84 were treated with 200 mg selenium per day showed significant reductions in total cancer mortality (RR = 0. Carcinogenesis 1990.11:2071–3. J Nutr 1988.42) and prostate (RR = 0. Waschulewski IH. Speciation of selenoamino acids. J Anal Atomic Spectrom 2001. In addition. References 1. A summary of results of the first generation of nutritional intervention studies to prevent cancer have been presented [101] and future directions and criteria for evaluating the efficacy of such interventions have been proposed [101. A better understanding of the mechanisms by which selenium interferes with the carcinogenesis process is a necessary focus for future research also for the evaluation of selenium related biomarkers.

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and intervention trials to investigate the effect of selenium supplementation on the incidence of cancer are summarized. and in epidemiological studies of risk of cancer — and the outcome of long-term trials concerned with the disease-preventive potential of selenium supplementation.Vitamins and selenium: Anticancerogenic activity of selenium 91 2. Shapiro [3]. In 1963.05–0. [5] published the alarming finding that feeding rats 0. Researchers of the former Soviet Union [4] feeding Se (as selenite) to rats at a 4.3 ppm level. it is quite possible that the organic selenium compound employed there had carcinogenic properties not linked with the element per se. as well as epidemiological findings regarding the relationship between selenium level and risk of cancer are reviewed. and concern for it as a carcinogenic agent dates to 1943 [2]. Anticancerogenic activity of selenium — molecular mechanisms and epidemiological data Jolanta Gromadziƒska. 11 were found to develop liver tumours.1% bis-(4-acetamine)-phenylselenyl hydroxide. History Historically. Nelson et al.3 ppm Se. the methods used in assessing the nutritional status of selenium — in general. Seifter et al. ¸ódê. [2] observed that liver tumours occurred in rats fed protein-deficient fodder and grains originating from Serich (5–10 ppm) regions. however. Wojciech Wàsowicz Department of Toxicology and Carcinogenesis. induced thyroid adenoma. their use as biomarkers of selenium status. observed the development of different forms of liver tumours. whereas such tumours were found in less than 1% of the animals in the control group. Of 53 animals that survived for 18 months when thus fed. including the forms of selenium present in the diet and in the body. interest in selenium (Se) as a toxic trace element dates back to 1937 [1]. suggested that the pathological changes that occurred reflected regeneration of the liver rather than a carcinogenic process. which suggests that the neoplastic changes that occurred could be attributed to Se. Poland Introduction There are many different aspects of the relationship between selenium and cancer. equivalent to inclusion of 103–207 ppm Se in the diet for a 10-day period. in response to selenium supplementation. Two of the chapters deal with these and related matters. nevertheless. in addition to the occurrence of different selenoproteins. Tscherkers et al. It should be noted. Edyta Reszka. . Nofer Institute of Occupational Medicine. however. [6] observed the development of tumours in male rats fed a diet containing 4. In the previous chapter the forms and metabolism of selenium. In the present chapter the mechanisms behind the role that selenium can play in cancer prevention. their functions and mechanisms of action. that since no studies have been reported of animals administered an analogous compound that did not contain selenium.5.

to 3%. the first tumours developed when the animals were 4 months of age. the Se concentration influenced neither the level of malondialdehyde (a final product of lipid peroxidation) in the breast carcinoma cells nor the level of glutathione peroxidase (GSH-Px) activity there [13]. but they also decreased in number with an increase in the amounts of selenium provided. tumours not only developed later.0 ppm) they first developed at 17 months of age. Another early observation of such an effect was that of Clayton and Bauman [8]. Edyta Reszka.1 to 0. respectively. [15]. These results indicated that supplying Se in larger amounts protects animals from developing cancer or delays . In the groups given extra Se in their drinking water. In both groups the incidence of cancer was found to decrease with increasing level of Se in the diet.5 ppm) reduced the incidence of cancer even more.4-dimethylaminobenzene (3’-MeDAB).5 ppm Se to the diet of rats given a carcinogenic substance led to a decrease in the occurrence of tumours from 80% (in the non-supplemented group) to 10% (in the group receiving Se). Ip [14] also studied the effect of selenium on different stages of cancer development in rats given 5 ppm Se at different time intervals after the administration of 10 mg DMBA.0 ppm Se in their drinking water. Supplying Se was found to decrease the number of tumours induced (from 97 to 46%). Se supplied during only one of these two phases had a much weaker effect [14]. whereas in the group receiving the largest amount of Se in its drinking water (1. At the same time.Jolanta Gromadziƒska. using female mice of the C3H strain.15 ppm Se and the other groups receiving. An interesting study of the anticarcinogenic role of selenium was carried out by Schrauzer et al. These findings were confirmed in a study showing that the number of liver tumours induced in male Spraque-Dawley rats by 3’MeDAB decreased from 92% in rats fed simply a control diet to 46% and 67%.1 to 1. in rats to which either of two different forms of Se were administered [9]. Harr et al. [10] showed that adding 0. when Wassermann et al. In the control group. who reported that in rats a diet enriched with 5 ppm Se decreased the incidence of liver tumours brought on by 3’-methyl-1. Two groups of animals received a diet containing maize oil. a supply of 0. Ip and Sinha [13] investigated the effect of various concentrations of Se on the development of breast cancer induced in rats by 7. Providing selenium at a still higher level (2. a strain characterized by the spontaneous development of breast adenoma in 80% or more of the cases. Wojciech Wàsowicz 92 The anticancerogenic effect of selenium in animals was first reported in 1911. The animals were divided into several groups. [7]) managed to inhibit the development of placental tumours in mice by use of Se compounds. (cf. particularly when it was provided at both the initiation and the promotion stage of chemical carcinogenesis. The maize oil content in the one case was high (25%) and in the other case low (5%). Supplementing a standard diet with administration of sodium selenite was also found to reduce the number of tumours that developed in rats that were given 2-acetyloaminofluorene (2-AAF) as a carcinogenic agent [10–12].12-dimethylbenzantracene (DMBA). The authors concluded that the protective role of selenium was not due to its being able to inhibit lipid peroxidation or to its having any antioxidative function in fat metabolism [13]. a control group receiving a basal diet that contained 0. in addition to this.

that selenium compounds can induce cell death through a mechanism distinct from oxidant toxicity [22. viruses or transplanted tumours [18]. The effects can occur at a systemic. its dose and the agent that induces the development of cancer [16]. cellular or nuclear level. The activity of many cellular targets.30] (Tables 2. proliferation and differentiation of the cells can be affected. in which the metabolism.25]. however. In several studies. It has also been shown. in the majority of cases the reduction being quite significant [19]. Glutathione . can prevent mutations by serving as free radical scavengers [26–28]. Two thirds of these experiments provided evidence for high doses of Se reducing cancer development to a moderate extent (15–35% in relation to controls). and perhaps selenoprotein P and other selenoproteins as well. enhancing immune responses. depends on the cellular GSH/GSSG ratio. and 2. Thus a number of mechanisms for the anticarcinogenic effects of selenium and of many selenocompounds have been proposed. The anticarcinogenic effect of Se has been found to depend on the chemical form in which the element is administered. Some reports suggest that the action of selenium toward cells that have been transformed earlier and toward normal cells differ [32]. Mechanisms responsible for the link between selenium and cancer prevention Experimental studies have thus shown that adding high levels of selenium to the diet of animals treated with carcinogenic chemicals has a carcinostatic effect [20]. including their providing protection against oxidative damage.13. resulting in more efficient carcinogen detoxification [24.Vitamins and selenium: Anticancerogenic activity of selenium 93 the carcinogenic processes. The experiments as a whole strongly suggest that consumption of Se in doses higher than those customarily given in such cases appreciably reduces the development of neoplastic tumours. one should bear in mind that the results of animal studies cannot be directly extrapolated to humans. and inhibiting angiogenesis [29. At the same time.). Cells adequately supplied with Se have been shown to be less sensitive to effects of both endogenous and exogenous carcinogens [17].23]. Regarding the mechanisms involved. the activity of xenobiotic-metabolizing enzymes in vivo has been reported to increase when selenium compounds are given. About 200 experiments have been performed aimed at assessing the effects of Se given to laboratory animals in doses higher than those usually employed in standard diets used to counteract the development of cancer induced by chemicals. since reactive oxygen species can promote apoptosis in vitro [21]. The anticarcinogenic effect of selenium has also been found to reach an optimum if it is given prior to the onset of the disease or in an early phase of its development. The anticarcinogenic effects of Se depend upon the chemical form of the Se-compound involved and the nature and dosage of the carcinogen. altering the metabolism of carcinogens.12. it has been postulated that the chemopreventive effect is related to the toxicity of selenium and the oxidative stress it induces. high levels of selenium compounds in the diet can reduce both the extent to which DNA adducts are formed and the extent to which DNA damage by carcinogens occurs. It is also possible that selenium in the form of glutathione peroxidases. In addition. Experiments in which no effect of Se was observed were rather rare. affecting the cell cycle.

The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis (ref. necrosis and cell survival processes [34]. intercellular communication. [16]) Form of selenium Selenite Selenite Selenodiglutathione Selenite. This suggests that selenoproteins participate in the regulation of intracellular signal transduction [33]. Se and the selenoproteins play a regulatory role in the following processes. BSC. DNA synthesis (in vitro) Cell cycle Apoptosis BSC and ist glutathione conjugates p-XSC. selenite Parameter Growth of Ehrlich ascites tumour cells in mice Growth of L1210 leukemic cells Growth of L1210 leukemic cells Cell growth (in vitro) DNA. p-XSC. Se-methylselenocysteine.12. RNA. PKA . for example: — ROS-activation of protein kinases in the cytoplasm and nucleus. BSC ACF COX-2 PKC. — ROS-activated modification of the thiol and hydroxyl groups in the Cys and Tyr. — Controlling changes in the cell redox potential through inducing activation of the transcription factors and initiating de novo gene expression. protein synthesis Apoptosis Outcome I I I I I E I I E I Block S/G2-M E I I I Block G1 E I I I Selenite Selenite DNA synthesis (in vitro) RNA and protein synthesis Cell death (necrosis SSB) Selenite Selenite Selenite Cell growth Cell cycle p53 AP-1 NF-κB CH3SeCN. — Affecting apoptosis. Table 2. Yet non-protein selenium metabolites may also be important in the regulation of intracellular communication and metabolism. Edyta Reszka. and changes in cell growth. — Regulating the expression of membrane and nuclear receptors responsible for cell maintenance. Selenodiglutathione p-XSC. Wojciech Wàsowicz 94 peroxidases and thioredoxin reductases are involved in ROS scavenging.Jolanta Gromadziƒska.

TK — thymidine kinase. PKA. COX-2 — cyclooxygenase. Table 2. selenium dioxide. [16]) — cont. Inorganic Se compounds such as selenite reduce the number of fibronectin receptors at the cell surface. Form of selenium Selenite p-XSC Selenite. In vitro effects of selenite and methylated selenocompounds [31] Endpoints Cell morphology Membrane damage Cell growth inhibition DNA synthesis inhibition Cell cycle block DNA single strand breaks Cell death Gadd gene induction Selenite Methylselenocyanate or Se-methylselenocysteine Normal No ++ ++ G1 None Apoptosis Early Extensive cytoplasmic vacuolisation. whereas these processes can be inhibited by selenate [34]. cell detachment Yes ++++ ++++ S/G2-M ++++ Necrosis Late Fibronectin is an extracellular component that plays an important role in intercellular communication.13. PKC — protein kinase A and C. NE — no effect.12. W — weak. Selenite activates the protein kinases involved in the pathways of cellular response to stimulation by inflammatory agents. triphenylselenium chloride p-XSC PKC TK Parameter Outcome I I I I Dose dependent E/I I I I/E/NE I Phospholipid/Ca+2 dependent PKC PKC JNK DNA cytosine methyltransferase PKC (in vitro) Cell proliferation and cell cycle biomarkers (in vitro) PKC and isoprostane (in vivo) I — inhibition. ACF — aberrant crypt foci.4-phenylenebis(methylene)selenocyanate. Oxidation of the thiol groups . p-XSC — 1. selenic acid Ebselen P-XSC. BSC p-XSC. NF-κB — nuclear factor κB. Since this is an immediate action. selenite Se-methylselenocysteine Se-methyselenocysteine. The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis (ref.Vitamins and selenium: Anticancerogenic activity of selenium 95 Table 2. E — enhancement. JNK — Jun-N-kinase. AP-1 — activator protein 1. BSC — benzyl selenocyanate. BSC. it cannot come about through activity of the selenoproteins.

such as those of proliferation. In human hepatoma cells. This process in turn results in the activation of AP-1 and of such transductional proteins as ras/rac. such as cytokines. fos. This process is made possible by intracellular reduction in the disulfide bridges (-S-S-). as well as such transcriptional and nuclear factors as NF-κB . Wojciech Wàsowicz 96 in the transductional proteins results in their being modified structurally. I-κB. NF-κB is a crucial target in the pathogenesis of various chronic inflammatory. which modifies the structure of Cys in the regulatory subunit of NF-κB so as to prevent oxidation of the thiol group located within this factor [34]. Oxidative stress induced by chemical or physical factors translocates Trx to the nucleus. In human colon cancer cells.36]. This process has been found to be inhibited in cells cultured in an Se-supplemented medium and to be intensified in the case of Se deficiency [43]. the cell cycle. growth suppression. differentiation and senescence. It regulates the effector genes in the promoter regions and can thus activate or repress gene expression. The redox potential is an important factor in regulating the nuclear factor κB (NF-κB) and the activation of AP-1 [39]. The breakdown of hydroperoxides by GSH-Px and the resulting decrease in the cellular hydroperoxide level inhibit the Se-activated kinase p38 [34]. GSH-Px in particular can act as a suppressor of protein kinases. Se inhibits the expression of one of the activating zinc finger proteins that regulates the activation of the c-myc oncogene [38]. Thioredoxin (Trx) plays a key role in this process. The process the translocation of NF-κB and its binding to DNA is multiphasic and highly complex. The reduced thiol group in Cys is essential for maintaining the activity of numerous transcription factors [45]. GSH-Px and SeP are considered to have a particular role in MAPK inactivation [44]. Edyta Reszka. GSH-Px overexpression inhibits the translocation of another NF-κB subunit and reduces the phosphorylation of I-κB [40–42]. mainly in regions in which DNA binding to nuclear factors occurs [46]. NF-κB activation involves changes in protein conformation and in the binding of numerous genes. TrxR thus affects the redox regulation of a variety of enzymes and receptors. MAPK inactivation inhibits the cellular proliferation occurring in the course of the carcinogenic process.Jolanta Gromadziƒska. intensifying the binding of NF-κB and AP-1 to the DNA. to the promotor regions. the immune response. directly. Reactive oxygen species (ROS) activates the phosphorylation of one of the NF-κB subunits. and in cellular proliferation or differentiation [35]. selenite inactivates the c-myc oncogene and activates the c-fos genes that lead to the growth of normal cells [37]. NF-κB is also an important factor in regulating activation of the genes involved in the control of cell necrosis and apoptosis. as well as the genes involved in apoptosis activation. which can lead to their becoming activated. for example. The activation of individual kinases of the MAPK family triggers the transcription factors involved in changes in chromatine conformation and in the expression of numerous genes of proinflammatory and antioxidative proteins. The phosphorylation of mitogen-activated protein kinases (MAPK) represents one of these phases. The strength of the binding of NF-κB to DNA is influenced by Se. myc and c-Jun N kinase [35. degenerative and neoplastic diseases. and repair processes [39]. Through acting as a protein disulfide reductase. The upor downregulation of the effector genes can modulate many different cell pathways.

ASK1) directly. modulation of tumour angiogenesis. p53. Trx limits DNA synthesis. cellular growth. affect cellular metabolism. Trx inhibits certain kinases of the MAPK family (e. whereas Trx/TrxR activates ribonucleotide reductase. In cells with a high Se level. a key enzyme in the deoxyribose formation needed for DNA synthesis. Se is also known to arrest several kinase pathways important in signal transduction. At the same time. high concentrations of selenides tend to also be generated. etc. Selenite. ref-1. . TrxR also regulates gene expression by affecting the cellular redox status and activating numerous DNA-binding transcriptional factors: NF-κB. but also its metabolites. not only the selenocysteine incorporated into the protein structure. selenodiglutathione. the concentration of Se in the tissues also being found to be higher after the administration of selenomethionine than after selenite supplementation. Effect of different selenium compounds in cancer prevention The chemoprevention of cancer by selenium can involve the action of different selenometabolites. Effects of selenium on apoptosis and necrosis In 1992 it was shown that Se(IV) compounds can induce the necrotic death of a cell by damaging a single DNA strand [53]. reduction in the oxidative DNA damage that occurs. AP-1.55]. diacylglycerol kinase and thymidyl kinase [48]. It has been shown that selenium creates a dose-dependent increase in TrxR activity and in the expression and stability of TrxR mRNA in different cancer-cell lines [39]. [50. When selenides are generated in sizeable amounts. Some low molecular weight Se compounds have been shown to influence the regulation of gene expression.51]. and the glucocorticoid receptors [34. It was also observed that methylated Se derivatives can induce apoptosis [48]. Selenite has been shown to be more effective than selenomethionine in the inhibition of cancer cell growth during chemically induced carcinogenesis [31. The anticancerogenic activities of various Se compounds are summarized in Table 2. It is believed that the role of selenium in the inhibition of carcinogenic processes is associated with oxidative damages caused by the redox cycle of selenides [57]. selenomethionine and Se-methyloselenocysteine can be transformed into selenides. It has been found that both sodium selenite and selenomethionine (SeMet) suppress tumour growth in many animal models involving a dose-dependent response [20. the bioactivation of carcinogens. such as protein kinase A.g.54. The various chemical forms of Se have been shown to differ widely in their anticancer properties.12.. they react with oxygen to produce superoxide and hydrogen peroxide [56].49]. [16]. Ca+2-dependent and -independent kinase C (PKC). but it is likely that one of Se metabolites is essential to this process. From this it can be concluded that a high concentration of Se is not crucial for the inhibition of carcinogenesis.54].Vitamins and selenium: Anticancerogenic activity of selenium 97 and AP1 [47]. It is important to note that different selenocompounds are essential for the growth and differentiation of both normal and neoplastic cells [52].

selenomethionine. Edyta Reszka. DNA strand breaks after selenite treatment being an example. This modulation of the p53 molecule may be a specific mechanism of p53 activation. [49] demonstrated that the Se concentration is a determinant of basal p53 activity. even at rather high concentrations (10–50 µM) [29]. only about 5% of it being found in other metabolites [58]. were found to cause phosphorylation of the serines and threonines contained in the p53 molecule. in the previous chapter). p53 is often mutated and its functional activity suppressed. Regarding the selenocompounds. sodium selenite and methylseleninic acid. that have been investigated most frequently [49]. Methylselenocysteine is one of the most important selenocompounds for chemopreventive activity [32]. Experimental studies have shown that the organic Se compounds induce apoptosis without DNA damage. modulates p53 activity.13. 1. SeMet oxidizes the thiols of the p53 molecules at 275 and 277 cysteine residues. which have been shown in in vitro experiments to affect apoptosis or to arrest the cell cycle (see Figure 2. Selenium compounds can also affect activation of the p53 gene since the main dietary selenium compound. Seo et al. p53-dependent DNA repair being activated within the concentration range of 10–20 µM.4-phenylenebis-(methylene)selenocyanate and benzyl selenocyanate inhibit the activity of DNA cytosine methyltransferase. Two other Se compounds. Fiala et al. The results of in vitro experiments on cells treated with selenite and methylated selenocompounds are summarized in Table 2. suggested that methylated Se derivatives are the most effective selenium compounds for cancer prevention [61]. it is the actions of sodium selenite (Se IV). In normal cells. They suggested that this pathway may be the major mechanism involved in the chemoprevention that selenium compounds provide after the initiation of carcinogenesis. It is twice as active as selenomethionine in suppressing mammary carcinogenesis in rodents [62]. which is not a DNA-damaging agent. Wojciech Wàsowicz 98 When Se is supplied in high concentrations. p53 regulates the activity of genes involved in DNA repair processes. [63] showed that the selenium compounds as selenite. In human cancer cells. an enzyme involved in DNA repair processes. Over 90% of the circulating Se is incorporated into the structure of the selenoproteins. Ip and Ganther [59.Jolanta Gromadziƒska. Inorganic Se compounds added to cell cultures at a concentration of 5–10 µM can induce 8-OHdG lesions or DNA single-strand breaks and cell death by necrosis.60]. responsible for single and double DNA strand breaks. It appears then that the anticarcinogenic action of Se is due to the combined effect of selenium and selenoproteins [33]. in connection with studies of Se metabolism they conducted. Such methylated selenium compounds as methylselenocysteine and selenomethionine show powerful anticarcinogenic activity and also lack some of the toxic effects produced by other Se forms. The metabolic pathways of Se-methylselenocysteine and selenobetaine involve the release of methylselenol or methylseleninic acid derivatives. [31]. any surplus of it is excreted from the body.5–1 µM was found to promote DNA repair processes by increasing the expression of two proteins associated with the p53 . Selenomethionine has been shown to act together with the p53 tumour suppressor protein in affecting the redox status.7. Methylseleninic acid in a range of concentration of 0. however. and of selenomethionine.

Seo et al. It has been found that Se derivatives can activate the p53 gene. which has an essential role in recognizing DNA lesions in the nucleotides and in the excision of damaged nucleotides in mammalian cells [69]. Selenium can also act as an inductor of the enzymes participating in phase II detoxification [48] and it modulates expression of the enzymes involved in phase I detoxification. Studies conducted in cell cultures suggest that selenocompounds may exert their chemopreventive effects via induction of apoptosis and cell growth inhibition in the transformed cells. this preventing DNA methylation from occurring. Reducible selenium compounds (phenylseleninic acid. Enhanced formation of the DNA repair complex in cells treated with selenomethionine has been considered to be a possible mechanism for the inducible capacity for repair of DNA [49. DNA methylation is one of the first steps in the carcinogenesis induced by certain chemicals. Limitations in the DNA repair capacity appear to be a key determinant of predisposition to cancer [49. one of the enzymes involved in DNA repair processes.66]. it was found that the human oral squamous cell carcinoma line (HSC-3) lost >80% of its GSH after treatment by 10 µM selenite or 100 µM selenodioxide for 72 h. and 2-nitrophenylselenocyanate) were found to cause a dose-dependent decrease in the activity of formamidopyrimidine-DNA glycosylase (Fpg). In vitro studies of a mammary cell line exposed to DMBA showed 1. as well as damaging the integrity of the genes of the DNA repair enzymes [67.65]. and to be involved in the pathway connected with the p53-dependent activation of DNA repair processes [64].4-phenylenebis(methylene)selenocyanate and its putative glutathione conjugate to inhibit the expression of various CYP450 isoenzymes and to induce the expression of various enzymes involved in phase II or DMBA detoxication [72]. [67] described the incorporation of Se into the factors involved in DNA repair regulation. selenomethionine protects the cells from DNA damage through the induction of DNA repair. [49] showed that in normal human fibroblasts. but that the induction of apoptosis is not a simple result of the regulation of p53 by selenium [16]. ebselen. There is a need of explaining how triggering of the p53-dependent DNA repair process by two different Se compounds can be achieved when a 10–20-fold higher Se concentration is employed. This decreased GSH concentration in the cells induced apoptosis. Blessing et al.68]. They also affect the integrity of the XPA protein (xeroderma pigmentosum group A protein). The postsynthetic methylation of DNA is catalyzed by the family of S-adenosylmethionine-dependent DNA methyltransferases [71]. selenocystine. . Selenium can also affect DNA methylation. which is a dominant mechanism of the cell death these compounds bring about [70]. The methylation of Se compounds to Se(CH3)2 and Se(CH3) results in a decrease in methyl donation. such as benzo(a)pyrene and arsenic [30]. In investigating the ability of inorganic Se compounds to induce apoptosis. an important epigenetic mechanism that exerts control over gene expression [30].Vitamins and selenium: Anticancerogenic activity of selenium 99 gene. These selenocompounds affect the DNA repair processes through oxidation of the zinc finger structures and the release of zinc from DNA. One possibility is that a small fraction of the SeMet is metabolized to a low-molecular-weight compound [32].

Research over the last decade points to a significant protective role of Se in preventing the development of malignant neoplasms. the inhibition of prostaglandin and immunoglobulin synthesis. however. and impairment of the body’s ability to reject implants and to destroy neoplastic tumours [75. Edyta Reszka.80]. Selenium regulates the immune response by stimulating natural killer cells and activating antigens of various types to destroy the tumour cells [78]. the results of epidemiologic studies . Low Se concentrations in the tissues and in human body fluids also appear to be associated with numerous changes in the immune system. Selenium compounds can activate cytotoxic cells. Wojciech Wàsowicz 100 Effects of selenium on immune functions Certain anticancer properties of the selenocompounds may be associated with their effects on cellular immunity. Prospective cohort studies appear to show in a more distinct manner the possible association found between the prediagnostic selenium concentration level and risk of cancer. CTL activity has been found to be significantly increased in Se (SeIV) supplemented patients with cancer located in the head and neck region who were given standard anticarcinogenic therapy. Cellular membranes of the T lymphocytes are particularly sensitive to Se deficiency.Jolanta Gromadziƒska.74]. Insufficient dietary intake of selenium results in many types of defects of the immune processes. such as in connection with antibody production and specific cell immunity [77]. In a Chinese study. such as the Se level associated with the dietary pattern at the time of sampling in cases and controls. methylselenol). Se can stimulate the expression of IL-2 receptors found on activated T lymphocytes and on NK cells [79]. Low plasma selenium concentrations are thought to be associated with increased morbidity and mortality from cancer.g. To date.76]. but no such relationship was found for lung cancer [83]. Case-control studies tend to reflect the short-term Se concentrations in the organism. due to the large amounts of unsaturated fatty acids present in their structure. such as suppression of the host immune response to bacterial and viral infections. micronutrients and phytochemicals [81. These metabolites that are synthetised in the cell can be involved in the cell’s metabolic pathways and destroy the integrity of tumour cells or induce apoptosis in these cells [51. stimulate the expression of cytokine receptors and the proliferation of lymphocytes [73.82]. including nutrients. The decrease in the number and the activity of cytotoxic T lymphocytes (CTL) is accompanied by the reduced excretion of lymphotoxins and the inhibition of both leukocyte and macrophage migration. a statistically significant increase in morbidity from oesophagus and stomach cancer was noted in a population with low levels of selenium. reduction in the activity of T lymphocytes. An inverse association between selenium and risk of cancer has been reported both in case-control studies and in followup cohort studies. NK cells and macrophages. Selenium and cancer risk — epidemiological results Several studies have shown the development of cancer in humans to be inversely related to the intake of specific dietary components. The anticarcinogenic effect of selenium is also partly based on its ability to produce antitumour metabolites (e.

94 and RR = 0. 95% CI: 0. undertaken by Zhou et al [91].85]. representing a daily Se intake of 55 mg.45–1.00 (95% CI: 0.14. only the findings for the two Finnish populations (RR = 0.86 (95% CI: 0.Vitamins and selenium: Anticancerogenic activity of selenium 101 have been rather inconsistent. but only in populations with a low mean Se level. The total RR values for lung cancer in patients showing high Se levels ranged from 0.80 (95% CI: 0.16). Interestingly.89]. dosedependent protective activity of Se was documented in Finnish and Dutch studies [86.14.22). The protective effect of Se in connection with lung cancer could also be noted in studies examining Se concentration in the toenails.20.58–1. those with high toenail Se levels were found to have a particularly high relative risk of lung cancer (RR: 4. to 0. whereas the value for groups with a low basic level of Se was RR = 0.77–1. the risk of lung cancer at high Se concentrations was found to be RR= 0.). to 1. indicated selenium to have a certain protective effect.74 (95% CI: 0.72 (95% CI: 0. whereas no association between selenium level and risk of lung cancer was found in two studies of non-European populations [88. 11 of which had a prospective design.46 (95% CI: 0.17–0. the protective activity of Se could be clearly discerned when the reference group consisted of subjects showing the lowest level of this trace element.57–0. The significant.30–0. In the control group of non-cancer patients the mean serum Se concentration was 100 ng/ml blood serum. 95% CI: 0.). when the study population was divided up according to Se level. Lung cancer Studies to test the hypothesis that low Se concentrations contribute to the development of lung cancer have been conducted in many countries (Table 2. .60) after adjustments for smoking status were made [90]. These finding confirm the assumption that the Se level in the toenails can be used as a marker of long-term Se concentration [91].09–0. It is notable that of the women investigated within the Nurses Health Study.87) when toenail Se was used as a marker of the concentration of Se in the body. whereas others have obtained null results [84. Some authors have reported there to be an association between cancer risk and Se status.87] in which Se in both serum and toenail samples was analyzed. 95% CI: 0.30) in studies of Se intake based on use of a questionnaire.97) (Table 2.50. A review of epidemiologic studies of lung cancer and of the selenium concentration found in biological material.44) and for the Dutch population (RR = 0. Of the studies considered in the meta-analysis.33. 95% CI: 0. The findings for three major forms of cancer are summarized below.54–34.10) for the serum Se level.61–1. the mean value obtained for groups showing a high basic concentration of Se was RR = 0. In a metaanalysis of 14 epidemiologic studies. In most of the reports.24–0.41.81) revealed a statistically significant protective effect of high Se concentrations.

575 (0.7 (18.76 (0. . Edyta Reszka.166) µg/g) 250 (0.02) 1.41) f 0.129) mg/kg) Japan 11—13 77 (113.897 (0.41–1.308) µg/g) 250 (0.20 (0. [94] NCC Serum 0.14.1 (2.102 Table 2.41–2.61 (0.70–2.07 µg/day) 0.43) P for dose-response trend 0.109) µg/g female) 227 (33.0) µg/l USA 5—14 356 (119.01 0.11 Kromhout et al. 0. [97] cohort Diet Zhou et al.001 0.0 µg/l) 120 (119.47–1. [96] Male Female China The Netherlands CC Diet All China — 25 — 1.3) µg/l)c Finland 8—12 153 (57.2 (2.20) 63 (NA) 290 (36. [86] NCC Serum Ratnasinghe et al.66 (0. [91]) Author All All All M.2) µg/day) 290 (39. [90] NCC Toenail Hartman et al.080) µg/g female)g 0.8 (16.17 NA Knekt et al.006 Hu et al. [95] NCC Toenail Van den Brandt et al.56 (0.12) µg/day 227 (33.20–1.537 (0.88) 0.3 317 (0. g data from 370 individuals.09–0.9) µg/l)d 216 (45 µg/l) 47 (0.110 ppm) Study Se index Gender Population Years of follow up No.547 (0. 112.98 (0.49 NA Comstock et al.3) µg/l F) Male All Male Female USA Male Finland 5—8 3.27–1.5) µg/l) USA 15—18 258 (0.0 (16.46 0.0) µg/day) 0.15) 0.52 0.6) µg/l) 356 (117.6 (15.134) mg/kg) China 4—5 108 (46. [98] CC Diet Jolanta Gromadziƒska.0 (13. e male subjects randomized in the earliest in the trial.17–0.20 (0.19) 0.20 (0.5) µg/l)b 145 (57.08 0.36) 0.550 (0.41 (0. Wojciech Wàsowicz a Nested case-control.126) µg/g male.94) 1.40) 4. low Se) 0. controls (mean Se level) RR (95% CI) (high vs. c data from 91 individuals. [93] NCC Serum Knekt et al.60) 0.529 (0. cases (mean Se level) No.80 (33.537 (0.50 (0.48 >0. [87] cohort Toenail All The Netherlands 3. [89] NCC Serum Kabuto et al. [88] NCC Serum Garland et al.30–0.44)e 0. 2459 (0.206) µg/g male.77–1. d data from 177 individuals. Epidemiological studies of the selenium level in the body and risk of lung cancer (according to Zhuo et al.2 (24.10 (16.65 (0.30 (0.1 (19. f male subjects randomized in the 5th year.33 (0.7) µg/l)b 153 (61.5 47 (0.54–34.108 ppm) 515 (0.811 (0.5 µg/l) Finland 16—19 77 (53. b data from 189 sets.60–2.0) µg/day) 870 (64.81) 0.37–1. [92] NCCa Serum Goodman et al.

although a protective effect of a high Se level (fifth quintile) was found (OR = 0. In a US case-control study of the relation between the risk of colonic malignant or benign tumour and the serum Se level.25–0. A lower Se concentration in the serum was found to be associated with a stronger tendency to be afflicted with a colorectal tumour [109]. in the Physicians’ Health Study. high Se concentration in the toenails was associated with an appreciably lower risk of prostate cancer [101].84) in cohort studies and 0. Also.74 (95% CI: 0.15. defined as the average of the 1st and 4th quintiles or the 1st and 3rd quartiles.85). In the Health Professionals Follow-Up Study. The findings of this review showed that the pooled RR of prostate cancer for a particular Se intake. In addition.61-0.Vitamins and selenium: Anticancerogenic activity of selenium 103 Prostate cancer A majority of prospective and case-control studies show high levels of selenium intake to have a protective role in preventing the development of prostate cancer (Table 2. especially men who had a PSA concentration equal to or higher than 4 ng/ml [106]. the odds ratio (OR) for the development of cancer that was associated with a high level of Se intake was 0.103]. A nested case-cohort study conducted on Japanese-American men (at a > 20-year follow-up) also confirmed the association between a high Se level in the plasma and a decreased risk of prostate cancer (OR = 0. colorectal cancer patients with low Se concentrations were found to have a significantly lower survival time . In the Netherlands Cohort Study.3 years’ follow-up period. 95%CI: 0.72 (95% CI: 0. no protective effect of higher Se levels was found in either of the groups investigated [108]. A systematic review of sixteen studies (11 cohort studies and 5 case-control studies) was conducted recently to investigate the association between selenium level and risk of prostate cancer. however.3–0.38. Colorectal cancer Results of prospective and case-control studies of the risk of colorectal cancer in relation to the selenium level in the body are summarised in Table 2. indicating that the intake of selenium was able to reduce the risk of prostate cancer [107]. to be associated (at a 13-year follow up) with a significantly reduced risk of prostate cancer.).61–1. in which the Se concentration in the toenails served as a measure of long-term Se intake. Several studies have shown that selenium can be of specific help in combatting prostate cancer [99]. A high prediagnostic Se level was found.16. was 0.17–0.9) [105]. the protective effect of high Se levels and high α-tocopherol concentrations in the plasma was only particularly strong when the γ-tocopherol level was high [104].39) in case-control studies. The lack of a protective effect of Se in connection with risk of prostate cancer risk was observed in the men participating in the Carotene and Retinol Efficacy Trial (CARET) [89]. In two large case-control studies of male populations in Great Britain and Canada.96) [100]. 95% CI: 0.5. Similar results were obtained in a 6-year follow-up nested case-control study in which the mean toenail Se concentration in prostate cancer cases and in matching controls were not found to differ significantly. the Se level in the toenails was not found to be associated to any marked degree with the level of risk of prostate cancer [102.49 (95% CI: 0. which involved a 6.

cases (mean Se level) No.40) 1. controls (mean Se level) RR (95% CI) (high vs. [89] NCC Yoshizawa et al.6 (19.60) 0. [100] NCC Helzlsouer et al.8) µg/l)b Study Se indicator Population Years of follow up No.3 540 (0. Epidemiological studies of the selenium level in the body and risk of prostate cancer Author Serum Serum Toenail Toenail USA 1—6 117 (0.99) 0.16 0.82 µg/g) 181 (0.126) µg/g) 249 (131. [105] Case-control Serum Li et al.02 (0. [94] NCCa Goodman et al. b data from 51 sets.73—2.090) µg/g) NCC Van den Brandt et al.4) µg/l)b 46 (58. [106] NCC Plasma 577 (0.17—0.39 (0.79 µg/g) USA 5 181 (0.48—0. .4) µg/l) Finland 8—12 46 (59.78 (0.106 (0.42—2.611 µg/g) 0.02 Nomura et al.3 (14.6) µg/l) 456 (114.69 0.008 0. [103] Case-control Nails Jolanta Gromadziƒska.05 0.018) µg/g) — 249 (128.84) 0.13) 1.104 Table 2.85) P for dose-response trend 0.547 (0. [101] Cohort 1211 (0.6 µg/l) 0.108 (0.5 (0.3 (20.65—1.622 µg/g) 13 586 (0.15.77 µg/g) 233 (0. low Se) 1. Edyta Reszka.10) 0.24 (0.38 (0.69 (0. [104] (2000) Toenail USA (JapaneseAmericans) USA UK — 300 (0. Wojciech Wàsowicz a Nested case-control.18—0.0 µg/l) The Netherlands 6.581 Allen et al.8 (19.00 (0.54—1.530 (0.707 0.12 Knekt et al.3—0.9) 0.96 µg/g) USA 5—14 235 (114.018) µg/g) 300 (0.

5 89 (0. [94] NCC Serum Wallace et al.535 (0.3 Colon 121 1209 USA 3.863 (0. controls (mean Se level) RR (95% CI) (high vs.75) 0.58 (0.42—2.3 (15.0 (18.41—1.575 (0. c data from 59 sets.10 (0. cases (mean Se level) No.65) 29 (64.593 (0.12 0.131 0.3 (17.3 (18.547 (0.50 NA Study Se indicator Gender Population Years of follow up No.106)) Rectal 36 (0.22) 0.3) µg/l)c Finland 8—12 29 (63.16. [94] NCCa Serum Knekt et al.59—4. [111] Van den Brandt Cohort Toenail et al. 105 .2) µg/l)b 0.82 (0.44—1.76 (0.3 Colon 112 1246 (0.43—1.724 0.123) µg/g) The Netherlands 3.0 (18.30) 1.733 (0. Epidemiological studies of the selenium level in the body and risk of and colorectal cancer Author Male Female 1.01 (0.41—2.8) µg/l) Finland 8—12 48 (64.9) All All USA — 25 (138 µg/l) 138 (134 µg/l) USA 1—4 276 (131. [112] NCC Serum Nelson et al.108)) 1. [90] NCC Toenail Van den Brandt Cohort Toenail Vitamins and selenium: Anticancerogenic activity of selenium et al.092) µg/g) Rectal 76 (0.7) µg/l)c 48 (65.9) µg/l)b 1.7) µg/l) 276 (130.00) 0. b data from 32 sets.58) 0.Table 2.186) µg/g) 2.88—4.091)) The Netherlands 3.578 (0.843 (0.18—5.91 (0.45) 0. [111] a Nested case-control.7 (0.5 (19.77 (0.5—5.273 0. low Se) P for dose-response trend Knekt et al. [108] Case Serum -control Female Male (0.04 (0.411) µg/g) Female (0.146) µg/g) 47 (0.643 0.560 (0.92) 0.326 Garland et al.

In a Canadian case-control study. age.118]. especially in males. in a recent study no differences between healthy individuals and individuals with adenomatous colon polyps or colorectal cancer were found in terms of Se level. The potential role of dietary Se in the early prevention of colorectal neoplasms would need to be confirmed. Edyta Reszka. smoking status and study site. prostate or colorectal cancer.Jolanta Gromadziƒska. however. However. a number of epidemiologic studies show a low Se level. Summary Results of epidemiologic and laboratory studies have indicated selenium to have a protective role in counteracting or preventing the development of cancer. a significant inverse association was obtained between toenail Se level and risk of colon cancer (OR = 0. to be associated with an elevated risk of lung and prostate cancer. The majority of studies on relations between selenium level and occurrence of colorectal cancer have yielded null results for both males and females.93) [102]. Colorectal cancer risk was also analyzed in a 41-month follow-up of the Nurses’ Health Study [90]. a pooled analysis of data from three clinical trials of colorectal adenoma was conducted: the Wheat Bran Fiber Trial.57.42. gender. A low level of selenium concentration in the body was shown to be associated with a higher risk of lung. environmental exposure. and the like need to be taken into account. a variety of confounding factors such as geographical location.112].19–0. To gain further insight into the anti-carcinogenic potential of selenium. There is a need of clarifying the role of Se . the Polyp Prevention Trial. although this result was only statistically significant in the case of the Polyp Prevention Study (OR: 0. there was found for each of the three studies to be a lower risk of recurrence of an adenoma in patients with blood selenium levels in the highest quartile than in those in the lowest quartile. would need further verification as well. 95% CI: 0. breast cancer was not found to be influenced by the selenium level [114–116]. there are some reports of a significant inverse relationship between blood Se levels and the prevalence of adenomatous polyps [117.34–0. no significant association being found between risk of cancer and toenail Se status. The selenium level was measured in blood specimens of 1763 trial participants. selenoprotein P (SeP) concentration or glutathione peroxidase activity in the plasma [110]. No inverse association of Se level and cancer risk was found for lung cancer in females. In contrast to prostate cancer. genetic susceptibility. A similar result was obtained in a 3. Two studies in which the serum Se status was measured did not indicate there to be a clear association between serum Se levels and risk of colorectal cancer [94. To sum up. and the preventive role of Se regarding cancer of this type. 95%CI: 0. After adjustment for age. gender. However. possibly due in part to the rather low proportion of women in the study population [94]. However. as found in the Polyp Prevention Study [113].95) [113]. Wojciech Wàsowicz 106 and a lower cumulative cancer-related survival rate than such patients with higher Se concentrations did [109]. and the Polyp Prevention Study.3-year follow-up study of a Dutch cohort in which toenail Se level was used as an indicator [111].

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ageing and cancer. quercetin. These polyphenolic compounds are classified. vegetables. . invasion and metastasis. Amongst their health-promoting properties are antioxidant. with major dietary sources including fruit. Bioactive components in foods 3. xanthohumol (a chalone in hops and beer) and genistein (an isoflavone in soy). who also found that quercetin protected human lymphocyte DNA from bulky adduct formation following treatment with benzo[a]pyrene. Examples include quercetin (a flavonol in vegetables. hesperetin). Also in this study. anti-inflammatory. daidzein).1. Similar findings were reported by Wilms et al [6]. apigenin). anthocyanins (cyanidin. flavanones (myricetin. inhibition of carcinogen uptake into cells and enhanced DNA repair. flavones (luteolin. University of Leicester. Total daily intake can range from 50–800 mg. pelargonidin. apples and onions). tea. altered metabolism of procarcinogens in favour of conjugation and excretion of reactive metabolites. Such chemopreventive agents can be effective at different stages of the carcinogenic process. An early study by Duthie et al [5] reported that quercetin protected human lymphocytes from hydrogen peroxide-induced DNA damage. xanthohumol and the novel flavonol. angiogenesis. which led to a significant increase in antioxidant capacity of plasma. epigallocatechin gallate (EGCG). according to structure. chocolate and soy. and anticancer activities. both by blocking initiation and by suppressing the later stages involving promotion. kaempferol). genistein. epicatechin-3-gallate) and chalones (xanthohumol). catechins (epicatechin. as flavonols (quercetin. UK Introduction Many thousands of different flavonoids are found in plant species. narginin. petunidin). resveratrol. the chalone. tricin. Anticarcinogenic effects of flavonoids Margaret M. Those flavonoids which have been studied in most detail exhibit many properties which could be protective against heart disease.3. progression. Some recent data for the well-studied flavonoids apigenin. antiviral. Many flavonoids possess antioxidant or free radical scavenging potential. anti-allergic. isoflavones (genistein. Several recent reviews have summarised the potential chemopreventive mechanisms for a number of flavonoids [1–4]. are summarised here. Manson Cancer Biomarkers and Prevention Group. Leicester. Blocking mechanisms Possible ways in which initiation of carcinogenesis can be blocked include prevention of reactive oxygen species attack on DNA. volunteers consumed quercetin-rich blueberry/apple juice for 4 weeks. which varies depending on the hydroxylation status of the benzene rings.

in a manner which suggested that they interact at the substrate-binding sites. They have also been shown to influence the multi-drug resistance phenotype acquired by many tumour cells. Suppressing mechanisms Mechanisms which result in suppression. elimination of tumour cells. including hydroxyl and peroxyl radicals. causing G2/M arrest. and induction of apoptosis involving release of cytochrome c from mitochondria. In another recent study [9]. Manson 116 Flavonoids can interact with the aryl hydrocarbon receptor (AhR) as agonists or antagonists. [14]. tricin decreased the number of intestinal adenomas in Apcmin mice by 33%. NF-κB.1. namely inhibition of signalling through the EGFR family. with inhibition of COX-1 and COX-2 activity. while the isoflavones. p27. Xanthohumol possesses several useful properties to block carcinogenesis including modulation of enzymes involved in carcinogen metabolism and detoxification (inhibition of Cyp1A. flavonoids can both up. but not apoptosis [12]. and PI3K. Such interactions influence the expression of drug metabolising enzymes such as cytochromes P450 [7]. During later stages of carcinogenesis additional useful mechanisms include inhibition of angiogenesis.Margaret M. A significant number of flavonoids.and down-regulate key molecules. induction of cell cycle arrest involving a decrease in cyclin D1 and phosphorylation of Rb. The inhibitory effect of other flavonoids on COX-2 expression and activity has been reviewed by O’Leary et al. the flavonols. Tricin. including JNK. cyclin D1. along with inhibition of superoxide anion radical formation and nitric oxide production [10]. cdc2. alone and in combination. induction of quinone reductase activity). include growth inhibition by induction of cell cycle arrest or apoptosis. Xanthohumol was also found to inhibit COX-1 and COX-2 activities. depending on cell type and experimental conditions. AP-1. have been shown to induce G2/M arrest in SW480 and CaCo2 human colon carcinoma cells [11]. However. p21. depending on structure and cell context. and to be antiestrogenic [10]. invasion and metastasis. scavenging of ROS. modulated intracellular drug levels by inhibiting function. accompanied by upregulation of p21 and p27. genistein and EGCG (reviewed in [2]) have a number of effects in common with those detailed here for apigenin and quercetin. activation of caspases 3 and 9 and downregulation of Bcl family members. Quercetin and silymarin were found to inhibit MRP1/4/5-mediated drug transport from intact erythrocytes with high affinity. The latter led to a 34% reduction of PGE2 levels in small intestinal mucosa and blood. p53. Such interactions might influence bioavailability of anti-cancer drugs in vivo and could be considered for combination therapies [8]. a novel flavonol in rice bran. reduced P-glycoprotein expression and function in multi-drug resistant human cervical carcinoma KB-IV cells. and pAkt. genistein and daidzein. Resveratrol. was shown to inhibit the growth of breast tumour cells. without affecting expression. quercetin and kaempferol. In a subsequent study by the same group [13]. A range of tumour suppressing activities is shown for apigenin (Table 3. or even better.).) and quercetin (Table 3. .2.

↓HER2/neu phosphorylation. ↓p70S6K1. ↓cyclin D1/D3 & cdk4. ↑DFF-45 cleavage. ↓PI3K-HER2 docking. ↓NF-κB p50 & p65 [21] Growth inhibition. ↓TNFα activation of NF-κB ↓Bcl2. COX-2. p18.Bioactive components in foods: Anticarcinogenic effects of flavonoids 117 Table 3. p16.↓HIF-1α. Chemopreventive suppressing effects of apigenin Tissue/cell type Jurkat T cells Mechanism ↓chymotrypsin-like activity of 20S and 26S proteosomes. ↓NF-κB-DNA-binding. p27.↓HIF-1α. ↓TNF a-induced NF-κB activation Apoptosis. ↓p70S6K1.9 and cIAP-2 ↓fatty acid synthase activity Apoptosis [16] [17] Growth inhibition and apoptosis ↑p53. ↑cleavage of caspase 3 [18] Apoptosis (p53-dependent) ↓Her2. HT-29 colon cancer cells ↓CK2. ↓VEGF and in nude mice OVCAR-3 and A2780/CP70 ovarian cancer cells ↓Akt. [26] . ↑cleavage of caspases 3. ↑p27 Apoptosis. apoptosis HER2-overexpressing breast tumor cells ↓PI3K & Akt activity. LAN-5. ↓HDM2 Growth and angiogenesis inhibition Angiogenesis inhibition [23] [24] HCT 116 colon carcinoma cells ↑ERK & p38 phosphorylation and activity ↑phosphorylation of Elk & ATF2 [25] HCT 116.7. ↑cleavage of caspase 3. ↑cyt c release. ↓VEGF. DU145 prostate tumour cells Breast and prostate cancer cells NUB-7. ↓Bcl2. ↑p53. SK-N-BE neuroblastoma cells Breast cancer cells ↑ROS. ↑HER2 degradation Apoptosis [22] A549 lung cancer cells in vitro ↓Akt. ↑Bax. PC-3. ↑IκBα . ↓Akt. LNCaP. MMP9. ↑p21. ↑p21. ↓IκBα degradation and phosphorylation. ↓IKKα actvity. cyclin D1. ↑IκBα Apoptosis Effect Reference [15] PWR-1E. ↓ colony formation [19] PC-3 prostate cancer cells ↓p50. ↓CDK2/4/6.8. ↑APAF-1. altered Bax:Bcl2 ratio. ↓p65.1. ↑Bax. VEGF Apoptosis [20] DU145 prostate cancer cells ↓cyclin D1/2 & E. NOS-2. G1 arrest.

↑p21 ↓HSP70 ↓c-myc Growth inhibition. ↓Akt Growth inhibition and apoptosis [30] LNCaP. ↓phosphoAkt Growth inhibition and apoptosis [32] ↓β-catenin/Tcf transcriptional activity ↑cyclin B1. During the carcinogenic process. Manson 118 Table 3. such as the cell cycle inhibitor p16 and the retinoic . epithelial cells migrate to the apex of the crypt. Chemopreventive suppressing effects of quercetin Tissue/cell type HL60 human myeloid leukeamia cells Jurkat T cells Mechanism ↑Bax. ↑phosphoBcl2. ↓Pgp Apoptosis Effect Reference [27] Inhibiting chymotrypsin-like activity of 20S and 26S proteosomes. ↑cleavage of caspase 9 Suppression by 4-fold. apoptosis ↑3-fold [28] MiaPaCa pancreatic tumour cells MCF7 breast tumour cells ↓phosphoFAK Decreased invasion [29] ↑PTEN. both hypermethylation of the promoter regions of tumour suppressor genes and hypomethylation of oncogenes can occur. G2/M arrest Apoptosis [33] [34] [35] One recent report by Fenton and Hord [36] has suggested a novel chemopreventive mechanism for flavonoids. which is often mutated in colon cancer. naringin and hesperidin induced a greater migratory response in APCMin/+ cells compared to those expressing wild type APC. ↓Bcl2.or over-expression. SW480 colon cancer cells SW480 colon cancer cells A549. In normal colon. H1299 human lung carcinoma cells PC3 prostate cancer cells ↓Sp1 interaction with AR. ↑IκBα Apoptosis [15] Breast and prostate cancer cells Colonic aberrant crypt foci ↓fatty acid synthase activity Growth inhibition and apoptosis [17] ↑Bax. Both EGCG [37] and genistein [38] have been shown to reactivate a number of key genes. ↑p53. PC3 prostate tumour cells HT29. resulting in under. These authors reported that apigenin.Margaret M. ↑p27. ↑c-jun Inhibition of androgen receptor activity [31] ↓ErbB2/3. epicatechin. ↑Bax. ↓Bcl2.2. a process involving the APC gene. Such flavonoid-induced migration was dependent on matrix metalloproteinase activity. ↑survivin. ↑phospho cdc2.

involved direct interaction with the enzyme. Phosphorylation of JNK1/2 and p38 was increased by the combination. cyclin dependent kinases and inhibitors. like other types of dietary chemopreventive agents.393:223–31. showed that quercetin and ellagic acid acted synergistically in inducing apoptosis. Eur J Cancer 2005. Manson MM. Surh Y-J. Collins AR. References 1. Their blocking activities include antioxidant effects. Inhibition of survival signalling by dietary polyphenols and indole-3-carbinol. using MOLT-4 human leukaemia cells. Conclusions Flavonoids. Duthie GG. modulation of drug metabolising enzymes and multidrug resistant genes. [39]. suggesting the potential use of ‘flavonoid cocktails’ to reverse multi-drug resistance in treatment of this cancer [40]. Cancer Prevention — the potential for diet to modulate molecular signalling.3:768–80. 4. Mut Res Gen Toxicol Environ Mutagenesis 1997. while quercetin alone only induced p38 phosphorylation. Dodson VL Quercetin and myricetin protect against hydrogen peroxide-induced DNA damage (strand breaks and oxidised pyrimidines) in human lymphocytes. 2.41:1842–53. and induction of apoptosis.Bioactive components in foods: Anticarcinogenic effects of flavonoids 119 acid receptor (RARβ). which. exhibit a wide range of potentially useful activities for cancer prevention. cytochrome c release and activation of caspases. mitochondrial membrane depolarisation. synergistic or antagonistic effects of combinations of more than one chemopreventive agent. Li YW.9:11–8.555:53–64. nor quercetin stability were affected by ellagic acid. For example Mertens-Talcott et al. Nature Rev Cancer 2003. . The mechanism proposed was through inhibition of DNA methyltransferase. in several different cancer cell types. including cyclins. Duthie SJ. 5. 3. in the case of EGCG. Mut Res 2004. Sarkar FH. Trends Mol Med 2003. Combinations of flavonoids were found to have an inhibitory effect on the breast cancer resistance protein (ABCG2). Combined effects There is now accumulating evidence for the additive. Manson MM. Cancer chemoprevention with dietary phytochemicals. They can also induce cell cycle arrest by modulating key components of cell cycle regulation. Neither the generation of ROS. mainly through intrinsic pathways involving Bcl family members. Cell signaling pathways altered by natural chemopreventive agents. Ellagic acid potentiated the inducing effect of quercetin on levels of p21 and phosphorylation of p53 at serine 15. Suppressing activities include inhibition of signalling pathways responsible for cell proliferation and survival.

p53-mediated apoptosis by apigenin in human neuroblastoma. Inhibition of P-glycoprotein function and expression by kaempferol and quercetin. Kao MC. . Al-Fayez M.111:1877–82. Cancer preventive activity of xanthohumol. Williamson G. Wang WQ. Alt A. Mol Cancer Ther 2002. Biochem Pharmacol 2005. The rice bran constituent tricin potently inhibits cyclooxygenase enzymes and interferes with intestinal carcinogenesis in Apcmin mice. J Chemotherapy 2005. 17. Mut Res 2004. Chen D. 8. 11. O’Leary KA. Platton S. 10.272:4725–40. Klimo K. Watson RWG. Morrissey C. Tunstall RG. Verhoeven.17:86–95. Brusselmans K.48:106–14.91:1364–71. 7. Induction of caspase dependent. Neumann I.551:245–54. Irons KA. Pintha K. Hudson EA. 13. Febs Lett 2005. Gupta S. O’Neill A. Mol Cancer Ther 2005. O’Brien NM. 16. Dietary flavonoids as proteosome inhibitors in human leukemia cells. Env Health Persp 2003.Margaret M. Prostate 2005. Dou QP. Modulatory effects of plant phenols on human multi-drug resistance proteins 1. Manson MM. Zhang S. a natural product derived from hop. Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity. Greaves P. Christoffel V. Wilms LC. Nutr Cancer 2004. Heiss E.63:131–42. Greaves P. 19. Brit J Cancer 2004. Barrand MA. Birt DR. Hladky SB.280:5636–45.579:145–52.4:1287–92. Swinnen JV. et al. Individual and interactive effects of apigenin analogs on G2/M cell cycle arrest in human colon carcinoma cell lines. Bao YP. Lavoi JF. Fitzpatrick JM. 12. Shukla S.4:1–11. Suppression of constitutive and tumor necrosis factor alpha-induced nuclear factor (NF)-kappa B activation and induction of apoptosis by apigenin in human prostate carcinoma PC-3 cells: correlation with down-regulation of NF-kappa B-responsive genes. 18. Landis-PiWowar KR. Torkin R. Ambudkar SV. Kuhn DJ. Febs J 2005. Vrolix R.69:1421–32. Steward WP. Cai H. Khantamat O. Kaplan DR. Degradation of Her2/neu by apigenin induces apoptosis through cytochrome c release and caspase 3 activation in HER2/Neu overexpressing breast cancer cells. Hollman PCH. 9. Chen S. Wu CP. Effect of flavonoids and vitamin E on cyclooxygenase-2 (COX-2) transcription. J Biol Chem 2005. Boots AW. Chen MS. Safe SH Flavonoids as aryl hydrocarbon receptor agonists/antagonists: effects of structure and cell-context. Protection by quercitin and quercitin-rich fruit juice against induction of oxidative DNA damage and formation of BPDE-DNA adducts in human lymphocytes. 4 and 5 (ABCC1. Growthinhibitory and cell-cycle arresting properties of the rice bran constituent tricin in human-derived breast cancer cells in vitro and in nude mice in vivo. Way TD. Verschoyle RD. 4 and 5). Daniel KG. Yeger H. Lin JK. Cai H. Qin CH. Mut Res 2005. 20. Kleinjans JCS. et al. 14. Knauft J. Gamal-Eldeen A.582:155–62.10:3169–78.1:959–69. Calcagno SB. Spengler B. VanAlstyne PC. Mol Cancer Ther 2005. Stewart JW. Steward WP et al. Clin Cancer Res 2004. Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells. Gerhauser C. de Pascual-Tereasa S. Limtrakul P. 15. Manson 120 6.

68:635–43.48:182–8. 36.285:G919–28. Mol Carcinogenesis 2004. 32. Knudson A. Flavonoid quercetin. Fenton JI. Liu HF. Calderwood SK.279:4479–89. Kim WK. Neoplasma 2005. Sinden MR. Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells.133:3800S–4S.20:835–49. Waite KA. 26. Modulation of aberrant crypt foci and apoptosis by dietary herbal supplements (quercetin. Nutr Cancer 2004.26:793–801. . Gupta S. Carcinogenesis 2005. J Biol Chem 2004. Moussavi M. Sulikova M. Park S. Farah M. Eng C. Mol Pharmacol 2005. J Nutr Biochem 2005. Van Dross R. curcumin. Cho HJ et al. Apigenin inhibits expression of vascular endo.26:1450–6. The 70 kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer. 22.6-dichloro-ribifuranosylbenzimidazoleand apigenin-induced sensitization of colon cancer cells to TNF-alpha-mediated apoptosis. Fang J. Jung KC. 34. Zhao MJ. Park CH. Anticancer Res 2005. Cao ZX.14:1457–63. Sedlak J. Bang MH. induced apoptosis in human myeloid leukemia cells and their resistant variants. Zazrivcova K. Volate SR. Davenport DM. 33. Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells. Jiang BH. Am J Physiol 2003.Bioactive components in foods: Anticarcinogenic effects of flavonoids 121 21. Pelling JC. 31. Eivemark S. Apigenin induces apoptosis through proteosomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway. Chao JI. Shukla S. Lee LT. J Nutr 2003. Lui LZ. Jones EL. Kang NE. Zhou Q.25:2017–25. Quercetin. Carcinogenesis 2005. 30. Reed E. Lin YS. Kim HK. Gong AY. The chemopreventive bioflavonoid apigenin modulates signal transduction pathways in keratinocyte and colon carcinoma cell lines. Kim ES. Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K and HDM2/p53.328:227–34. Kuo PC. Bodo J. Int J Hyperthermia 2004. Kao MC. 35. silymarin. Salh B. 39:114–24. FASEB J 2005. Fang J. Yuan HQ. thelial growth factor and angiogenesis in human lung cancer cells. Xue Y. 25. Phytoestrogen exposure elevates PTEN levels. Targeting of focal adhesion kinase by flavonoids and small interfering RNAs reduces tumor cell migration ability. Lee MT. Wargovich MJ. 27.279:55875–85. Stevenson MA. Way TD. Young CYF. Lin JK.52:273–9. 23. Quercetin decreases the expression of ErbB2 and ERbB3 proteins in HT-29 human colon cancer cells.19:342–53. Muga SJ. Chang JY. Hahm ER. a potent inhibitor against beta-catenin/Tcf signaling in SW480 colon cancer cells. Hu XW Shi XL. Hord NG. 5. Huang YT. but not apigenin or luteolin. Flavonoids promote cell migration in nontumorigenic colon epithelial cells differing in APC genotype: implications of matrix metalloproteinase activity. Zheng JZ. Parhar K. 29. Human Mol Genetics 2005.16:155–62. ginseng and rutin). Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells. Lee PPH. Biochem Biophys Res Comm 2005. J Biol Chem 2004. 24. Yang CH. Duraj J. 28. Jiang BH.

39. Morris ME. RARβ and MGMT genes by genistein and other isoflavones from soy. Percival SS. Ai N. Christman JK. 38. Romero C. Bomser JA. Combined effects of multiple flavonoids on breast cancer resistance protein (ABCG2)-mediated transport. Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. .135:609–14. Wang Y. Ellagic acid potentiates the effect of quercetin on p21 (waf1/cip1) p53 and MAP-kinases without affecting intracellular generation of reactive oxygen species in vitro. Sun Y. Pharmaceutical Res 2004. Fang MZ. et al. Reversal of hypermethylation and reactivation of p16 ink4a. J Nutr 2005. Sun Y. Chen D. Hou Z.21:1263–73. Talcott ST. Zhang SZ.Margaret M. Yang CS. Yang XN.11:7033–41. Clin Cancer Res 2005. Manson 122 37.63:7563–70. Fang MZ. Lu H. 40. Cancer Res 2003. Mertens-Talcott SU.

Long-term dietary habits are usually explored in epidemiological studies by use of food frequency questionnaires (FFQ). isoflavones. Some of the polyphenols are found in a rather wide variety of foods (kaempferol contained in many vegetables.2. flavones. The validity and reproducibility of nutritional biomarker measurements. Heidelberg. The basic flavonoid structure and various examples of them are given in Figure 3. Bioavailability is determined by different factors. whereas others are limited to only a few kinds of food (such as apigenin found in parsley and celery). flavanones. followed . Flavonoids are classified in several different sub-groups.3 to 43% (based on urinary excretion) of the dose administered.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 123 3. and components for which bioavailability is low. Biomarkers of dietary polyphenol intake for studying diet-cancer relationships Jakob Linseisen and Sabine Rohrmann Division of Clinical Epidemiology. reaching plasma concentrations of 0–4 µmol/l at an intake of 50 mg aglycone equivalents [2]. associations that are accompanied and supported by basic research findings enabling one to identify and understand insofar as possible the steps contained in the causal chain of disease development.1. Polyphenols Polyphenols are provided by plant-derived foods. anthocyanins and proanthocyanins. particularly concerning dietary components provided by only certain kinds of foods. such as various secondary plant products. needs to be demonstrated in advance of their use in epidemiological and etiological studies [1]. flavanols. lignans. for example. The use of biomarkers for such compounds should overcome some of the methodological problems in nutritional epidemiology just referred to. including beverages. from imprecision. the sugar moiety of the compound in question and its further metabolism by the gut microflora. however. German Cancer Research Centre. is the fact that the polyphenols markedly differ from one another in their bioavailability and intestinal metabolism. Even more problematic. The analytical data obtained are objective and more precise. Current evidence from bioavailability studies suggests that the bioavailability varies from about 0. Dietary measurements tend to suffer. the hydroxybenzoic and the hydroxycinnamic acids. Nutritional epidemiology deals with dietary factors that are difficult to assess. measurement error being independent of that contained in the corresponding questionnaire data. Isoflavones and gallic acid are polyphenols that are absorbed to the highest extent. Estimates of dietary intake estimations are hampered by incomplete or missing data in food composition tables. Germany Introduction The primary aim of analytical cancer epidemiology is to detect associations between exposure variables and disease endpoints. however. for example). phenolic acids consist of two main subgroups. these including the flavonols.

is a promising approach. the aglycones being quantified. The plasma concentrations present in free-living (fasting-status) subjects are even lower than the abovementioned range obtained after intervention. The polyphenol concentrations contained in biological specimens are estimated in most studies after hydrolysis of the glycosides involved. their being substrates for methylation. sulfation. For many of the polyphenols.Jakob Linseisen. data on the phenolic acids are very limited. the galloylated tea catechins and the anthocyanins are much less available. Sabine Rohrmann 124 by the catechins. concentrations of them reaching baseline levels within 24 hours [3]. Measurement of the compounds found in urine samples. and glucuronidation. 3. flavanones and quercetin glucosides. their half-life time in the plasma is short. ideally 24-hour samples. The basic structure of flavonoids and the chemical structure of selected polyphenols. The proanthocyanidins. The steady-state levels of the plasma polyphenols should only be achievable through regular intake of the foods in question. although the kinetics involved differ considerable. Flavonoids undergo extensive first-pass phase II metabolism in the intestinal epithelial cells and the liver. . Thus far.1. Fig. due mainly due to the higher concentrations of polyphenols — at least after extraction and enrichment — as compared with plasma samples.

the fluorescence emitted is recorded by means of a plate reader.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 125 Laboratory techniques Two recent reviews provide an overview of the analytical methods used to determine the structure and content of the flavonoids and phenolic acids contained in foods and in food-based matrices [4. Hydrolysis (acidic or enzymatic) is frequently used to simplify the analytical procedure. frequent use is made of enzymic hydrolysis by means of sulfatase and glucuronidase unless a study aims at determining the exact metabolites. and the phenolic acids are genarally quantified by means of GC after derivatisation. although a part of them is excreted unmodified. although the use of LC-MS is becoming increasingly common. or urine samples. Thus far.3. In biological specimens. the respective aglycones and acids being subsequently detected and quantified. mass spectrometric techniques need to be used. Phenolic acids undergo further metabolism and degradation. HPLC has become the method of choice. flavonoids mainly undergo modification by means of phase II enzymes leading to methylated. Although in principle these methods can be applied to human specimens. combined with different detection systems (Table 3. the clean-up procedures for this type of samples may be more complicated than required for many food systems.). new developments include the creation of HPLC-ESI-MS-MS systems and similarly coupled devices. the flavonoids are usually glycosylated and the phenolic acids are ester-bound. Identification of compounds by means of mass fragmentation is used as a gold standard. Thus. sulfated and glucuronidated compounds. with the option of time-resolved measurement. Accordingly. the scientific literature contains no report on use of the metabonomics techniques (NMR. For the purpose of quantification. Separation and detection systems The two major separation techniques for the quantification of polyphenolics are HPLC and GC. Clean-up procedures For human specimens. do require only minor sample-preparation steps.5]. However. The availability of antibodies for isoflavones and lignans has enabled the antibodybased assays with a high degree of sensitivity to be developed [6]. serum. Hydrolysis In foods. a single mass-selective detector often fails to fulfil the requirements for sensitivity. such as plasma. For the structural characterization of compounds. For the flavonoids. . Some techniques such as liquid chromatography-mass spectrometry (LC-MS). LC-MS) to characterise clusters of polyphenolic compounds that can potentially be used as biomarkers. solid phase extraction (SPE)-columns provide the most convenient solution for removing from a sample any matrix compounds that would disturb the analysis.

Principal techniques and detection systems used for the identification and quantification of flavonoids and phenolic acids in human specimens (metabonomics/NMR excluded) Separation HPLC Detection system UV/VIS spectroscopy (Diode array detection) Mass spectrometry Electrochemical Fluorometric Substances class technique Flavonoids.50 Urinary excretion2 (% of intake) Mean(SEM) Range Elimination half-life (h) Mean (SEM) 5.6 6.6) 5.6 (1.0–5.5 (0. Differences between the various compounds and of classes of polyphenol are striking nevertheless.0 7.3 3.8) Range 3.26–4.0) 19. Kinetic data from the experiments in question are summarized in Table 3.4.0–55.3.84 (0. LC — liquid chromatography.3 (0.76–3.8) 8.56 (1.2 Cmax (µmol/l) Mean(SEM) 1. even after the administration of polyphenol preparations or polyphenol-rich food corresponding to 50 mg aglycone equivalents.0) 21.5 15.6) 4.1 6.00 0.0) 3–24.4–8.0) 6.0 3.6 4.4–62.8 (0.6) 4.7) 7.0–9.21) Range 0.4. Phenolic acids Isoflavones. Relatively low plasma concentrations were obtained in each case.7–10. Phenolic acids Phenolic acids Flavonoids.3 (3.88 (0.6 (4.46 (0.3 5. Phenolic acids Flavonoids.9 2.8 4.8–29.0 5. Phenolic acids Flavonoids. Phenolic acids Flavonoids.8–7. [7]) Tmax (h) Mean (SEM) Range Daidzin Daidzein Genistin Genistein Glycitin Hesperidin 6. Sabine Rohrmann 126 Table 3.52) 1.46–4.4–9. Table 3.04 1.0–6. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al.27) 2.26 42.0 27.14 0.1) 5.Jakob Linseisen.5 42.7–9.3) 8.3 0.2 1.1 (0. GC — gas chromatography.25) 1.4–5. lignans GC LC Immunoassays Mass spectrometry Mass spectrometry Time-resolved fluorescence HPLC — high-performance liquid chromatography.5 (0.92 (0.7 7.3 (0.50–2. (according to [7]).87 8.00) 1.21–0.57 (0.9 (12.1 (0.36–3. Most of the studies concerned only one or some few compounds within a given subclass of polyphenols.8) 8.5 (0.9 (1.4 . Bioavailability studies and other short-term interventional studies A report was published vey recently summarizing all scientific studies (n = 97) that has been conducted thus far on the bioavailability of polyphenols [7].38) 0.

0 2.5 17. A review of short-term intervention studies (n = 93) in which polyphenols were administered (either as isolated compounds or in the form of foods or food extracts) to human subjects was published recently [8].45) 0.35 10.1 2.1–61. The authors examined evidence relating to the effects of these polyphenols had on biomarkers used to test various pathophysiologically relevant hypotheses in vivo. The magnitude of the variation in the plasma polyphenol concentrations found between free-living subjects following their usual diet habits was .09) 1.4–39.0 1.7–2.7–4.50 (0.0) 36. AUC — area under the plasma concentration-time curve.26 Range 0. flavanones or isoflavones) as biomarkers of polyphenol intake [9–16].57) 0.2) 1.6 (17.1 1.3–1.02) 0.2) 0.2) 1.008–0. Tmax — time to reach Cmax.09–0.8 2.03 0.0–0.1 (0.5 (0.001–0.03) 4.0 4.5 (1.7) 11.3 0.6 0.30–2.4) 2.5–2.96 (0.5) Range 1.0 1.6–3.2) 10.40 (0.6) 3.0 19.9 (8.20 0.3) 6.5 (0.3) 0.40) 0.20 (0.5–2.07–1.5–2.45–1.4) 2.1 (3.6) 2.17) 2.5–5.26) 0.1) 1.09–1.3–2.10 (0. EGC — epigallocatechin.4 (0.5 0.3–9.4 (0.2) 3.01) 0.7 (1.1) 2. The results of these studies as a whole suggest biomarker measurements of this sort to reflect in an adequate way the degree of short-term intake of the polyphenols that were investigated.03) 2. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al. Rutin (Epi)catechin EGC EGCG Gallic acid Chlorogenic acid Caffeic acid Ferulic acid Anthocyanins Proanthocyanidins 1 2 Cmax (µmol/l) Mean(SEM) 0.8 (3.2 0.2–15.1–30.0 4.1) 1.7) 1.0 (0.03 (0.6–5.8–28.3) 18.9–28.004–5.46 (0.1–55.3 (0.8 (0.5 (0.5 (5.9 4.13–1.00 (0.4 (0.7 (0. [7]) — cont.1 0.9 (2.51–3.52 0. EGCG — epigallocatechin gallate.0 0.02 (0.7 27.1–1.3 (0.0 1. Observational studies: cross-sectional studies and studies related to cancer Various studies have dealt with the suitability of using fasting plasma or urinary concentrations of polyphenols (mainly flavonols.2 1.1 Elimination half-life (h) Mean (SEM) 2.10 0.03–0.5 0.33) 1.12 (0.38 0.4.5 (0.70 37.6 (0. Tmax (h) Mean (SEM) Range Naringin Quercetingluc.1 (0.1 1.3 (0.7 0.1–4.0 0.06) 0.80 0. Usually represent 24-h urine samples.5 0.06 (0.6 0.4) Range 1.3) 1.31–6.3 0. although one study in particular failed to support this conclusion [12].1) 11.50 0.57–4.7–2.03 All the data were converted so as to correspond to a supply of 50 mg aglycone equivalent.1) 1.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 127 Table 3.7 5.70 Urinary excretion2 (% of intake) Mean(SEM) 8.4 0.

1 (94.7 561.0a 58. close to of steady-state plasma concentrations of them can only be achieved if the substance in question is consumed regularly. The correlation coefficients between estimates of the dietary intake of polyphenols of the day before blood sampling took place and fasting plasma concentrations polyphenols were as high as 0.3 (67.7 1348. Table 3.0a 0.5.8 .0 478.0a 0.0a 0.6 339.).0a 19. Sabine Rohrmann 128 found to be rather high (Table 3.3 (42.7 (15.8 82.6) 477.0a 0.2) 1031.5 114.4) 107.1 53.1 6849.Jakob Linseisen. the intra-individual variation also being high [14].5 Mean (SD) Min.7) 277.0a 0.8 (15.8 158.75.9 2641.1 (2.8 26.3 644.8 (77. participating in a cross-sectional study (according to Bolarinwa and Linseisen [16].0a 78.2) 260.4 159.9 372. (nmol/l) Max. and if the bioavailability of the compound is not particularly low.0 742.0a 0.6) 6990.1 (41. Compound Gallocatechin Protocatechuic acid Epigallocatechin Catechin Gentisinic acid Epigallocatechingallate Caffeic acid Vanillic acid Epicatechin Syringic acid p-Coumaric acid Ferulic acid Salicylic acid Ellagic acid Daidzein Quercetin Median 68. a precondition most likely to be fulfilled by compounds such as kaempferol that are widely distributed in plant foods.5) 908.4 2904.9 7103.3 388.5.2 0.6) 2160.1) 520.0a 0.6 460.9) 0.9 4528.3 (153. and that the correlations are much lower when intake calculations are based on data obtained either three or seven days prior to blood sampling.6 2542.0 6103.9) 267.7 2290. Due to the short half-life time of most polyphenols.8 1121.3) 652.0 (3077.6 (275.7 (78. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet.3 1253.1 1357.8) 108.0 0.0a 0.1 (203. 91.7 (48.5) 1304.2 (17.4 104468.6 (254.9) 140.9 563.4 59.0a 48. but it should be emphasized that the validity of such correlations may be limited by a lack of precision in the estimates of dietary polyphenol intake.8 0.4) 10.6 413.0a 0.

0a 0.24 and 0.1 2555. phloretin.9) 31.0a 0. 701(659). To give an example of the polyphenol concentrations found in 24-h urine samples of subjects on a habitual diet. 122.2 639. . respectively.2 Mean (SD) Min.2 (4.1) 37. 1638(1316) µg/24 h. (nmol/l) Max.6). several studies on associations with the risk of cardiovascular diseases and with cancer at different sites. some of which appear very promising. Their work provided the basis for the estimation of dietary flavonol and flavone intake. The urinary excretion of polyphenols in subjects on habitual diets was also shown to be correlated significantly with estimates of the short-term of fruits and vegetables.0 (7.8 5. kaempferol.1 (24. In large-scale epidemiologic (etiological) studies of disease-related effects of dietary polyphenol levels.0a 533.3 (1.4) 9.1 (40. and total flavonoids as being 25(23). are taken up.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 129 Table 3.9) 126. naringenin.74 for plasma isoflavone concentrations.6 36.0a 0.38 [13].7 1356. 76(110). Nielsen and coworkers [13] reported average (SD) concentrations of quercetin.0a 0.7) 545. except as regards the intake of phytoestrogens (isoflavones and lignans).8 (80.3) 0.0a 0.8 Below the detection limit.0a 0. Hertog and colleagues were the first to analyze commonly consumed foods in terms of their flavonol and flavone content by means of HPLC. 50(32).1 27.8) 157.28 and 0.3 14. little use has been made of biomarker measurements. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet. Compound Naringenin Luteolin Genistein Hesperetin Kaempferol Apigenin Isorhamnetin a Median 79.0 108. the dietary intake estimates being based on FFQ data [11]. The urinary polyphenol excretion rates show a high degree of variability. although this has not been investigated tested extensively. In the following. just as was indicated already for the plasma concentrations. the correlation coefficients for selected flavonols and flavanones being between 0. One study reported correlation coefficients of between 0.5 204.7 151.5 (21. participating in a cross-sectional study (according to Bolarinwa and Linseisen [16] — cont.7 388. Plasma and urinary polyphenol concentrations are not expected to reflect long-term or habitual dietary intake. Only few studies concerning the use of biomarkers of polyphenol intake were found to be available (Table 3.5.8 52.

One of the major reasons for the frequent use of biomarkers of phytoestrogen intake in epidemiological studies (see Table 3. Epidemiologic (observational) studies of the risk of cancer in which biomarkers of dietary polyphenol intake (other than that of isoflavones and lignans) were employed Author Dai et al. cases/ controls 250/250 Specimen. [21] Zheng et al. enterolactone Serum. enterolactone Urine. NS — not significant. lignans Plasma. [27] Cohort: nested CCS 97/187 114/219 Serum.6. genistein. equol.Jakob Linseisen.7. Flavonoid Urine. [24] den Tonkelaar et al. tea polyphenols Gastric and esophageal Significant inverse association CCS — case-control study. Table 3. enterolactone Urine. Phytoestrogen Urine. . 2002 [17] Type CCS N. [17] Piller et al. [22] Dai et al. [26] Type CCS CCS CCS CCS CCS CCS Cohort: nested CCS Cohort: nested CCS N. [25] Hulten et al. Sabine Rohrmann 130 Table 3. isoflavones Urine. enabling the attainment of the requirement of sufficient statistical power. isoflavones Urine. isoflavones.7.) may be the ready availability of appropriate immunoassays suitable for measurements on large numbers of samples. Urine. Epidemiological studies of the risk of breast cancer risk in which biomarkers of dietary isoflavone and mammalian lignans intake were employed. [23] Pietinen et al. Author Ingram et al. isoflavones. lignans Higher risk when isoflavone estimates are higher CCS — case-control study. total phenols Breast NS Sun et al. 2002 [19] Cohort 232/772 Urine. citrus flavonoids Cancer site Breast Result NS Zheng et al. cases/ controls 144/144 60/60 18/20 250/250 220/237 194/208 88/268 Specimen. enterolactone for highest and lowest categories Significant positive association Grace et al. [18] Murkies et al. NS — not significant. 1999 [18] CCS 60/60 Urine. enterolactone Result Significant inverse association NS Significant inverse association Significant inverse association Significant inverse association Significant inverse association NS 248/492 Plasma.

in which peak identity cannot be confirmed with sufficient certainty. and reports on the quality of the method in question are usually obtained clearly above the detection limit. such as analytical time and costs). MS-based systems also have their problems in connection with concentrations close to the detection limit. LC-MS.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 131 In view of the usually rather low sample volumes available in epidemiologic studies. and TR-FIA (immunoassay). HPLC-MS-MS.20]. Sensitivity is a major issue with regard to analytical methods for determining polyphenols in biological specimens. except for some few small studies [14]. MS-based systems also have their problems when the concentrations involved are very low. Reproducibility. Accounts of analytical procedures that permit a wide variety of polyphenols to be determined in a single run were published recently [16. the immuno-assays being located at the other end of the scale. In epidemiological studies in particular. . recovery 90–105%). having coefficients of variation close to or above 10% and recovery rates frequently at < 90%. validity and reliability For each well-developed method. the analysis of polyphenol content is restricted in most cases to only a few compounds or classes of compounds. For all these methods. The sensitivity achievable with HPLC-MS. working at the detection limit of a method is always a challenge. However. or use of fluorescence detectors. Detection limits very close to 1 nmol/l of polyphenols have been found for techniques involving HPLC-ECD. Specificity and sensitivity In applying mass-selective detection methods at least to standard mixtures or biological specimens spiked with the compound in question. ECD (electron capture detection). the concentrations were found to be high enough to enable the characteristic mass fragments to be identified. This differs from other methods. no systematic findings on repeated within-subject samples or on losses during sample storage are available. confirmation of their results by use of MS-based techniques is necessary. the degree of sensitivity a method provides can be decisive for whether it can be used or not (along with other factors. however. in which the sample volumes available are usually very small. such as HPLC-UV/VIS. As already mentioned. The higher values here are those for the MS-based techniques (coefficient of variation < 5–7%. To the best of our knowledge. where the characteristic fragments may be absent. satisfactory figures concerning the analytical precision and accuracy are available. Antibody-based assays are also subject to failures in specificity due to cross-reactions with other matrix compounds. GC-MS and HPLC with use of fluorometric detection is slightly lower.

Manach C. and diets high or low in fruit and vegetables. Scalbert A. 2. Review of 93 intervention studies. van Staveren WA. Davey G. naringenin and quercetin in human subjects following their habitual diets. Erlund I.11:459–66. Am J Clin Nutr 2004. Blake A. Nielsen SE. 15. Scalbert A. Manach C. Merken HM. Verkasalo PK. Donovan JL. Stumpf K. Morand C. J Agric Food Chem 2003.41:203–9. Aro A. Free Radic Res 2004. Appleby PN. . Al-Maharik N. 142. Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake. Ritchie MR. Robbins RJ. Fasting plasma concentrations of selected flavonoids as markers of their ordinary dietary intake. 11.38:771–85. Lyon: IARC. Deighton N. Lapcik O. 12. Williamson G.86:415–21.56:891–8. J Agric Food Chem 2000. Soya intake and plasma concentrations of daidzein and genistein: validity of dietary assessment among eighty British women (Oxford arm of the European Prospective Investigation into Cancer and Nutrition). Adlercreutz H. Burns J. Eur J Clin Nutr 2000. Am J Clin Nutr 2005. Phenolic acids in foods: an overview of analytical methodology. Plasma and urinary phyto-oestrogens as biomarkers of intake: validation by duplicate diet analysis. Linseisen J.. Remesy C. Silaste ML. 4. Br J Nutr 2001. Remesy C. Eur J Nutr 2002. Alfthan G. 8. Williamson G. Validated application of a new high-performance liquid chromatographic method for the determination of selected flavonoids and phenolic acids in human plasma using electrochemical detection. Buysman MN. Linseisen J. Am J Clin Nutr 1998. Key TJ. Eur J Clin Nutr 2002. Prediction of dietary flavonol consumption from fasting plasma concentration or urinary excretion. Review of 97 bioavailability studies. Kaaks R. Zock PL.91:447–57.79:727–47. Br J Nutr 2004. editors. Flavonoids in human urine as biomarkers for intake of fruits and vegetables. 5. Hampl R.Jakob Linseisen. In: Toniolo P et al. Measurement of food flavonoids by high-performance liquid chromatography: A review. Crozier A. de Vries JH. Steroids 2000. Biochemical markers of dietary intake.51:2866–87. J Chromatogr B Analyt Technol Biomed Life Sci 2005. Kleemola P.48:577–99. I. Bioavailability and bioefficacy of polyphenols in humans. Manach C. Polyphenols: food sources and bioavailability.81 Suppl 1:243S–55S.54:143–9. Lean ME. II. 10. Cancer Epidemiol Biomarkers Prev 2002. Sinha R. Hollman PC. 1997. 13. IARC Scientific Publications No. Riboli E. 3. Cummings JH. Application of Biomarkers in Cancer Epidemiology.65:339–48. Rantala M. 6. Bolarinwa A. Mutanen M. et al. 7. Plasma concentrations of the flavonoids hesperetin. 16. Uehara M. Pharmacokinetics and metabolism of dietary flavonoids in humans. Allen NE. Meyboom S. Manach C. Beecher GR.68:60–5. Wolfram G. Kesaniemi YA. 14. Freese R. Radtke J. 9. Sabine Rohrmann 132 References 1. Wang GJ. et al. Jimenez L.81 Suppl 1:230S–42S. Kelly IE. Time-resolved fluoroimmunoassay of plasma daidzein and genistein. Noroozi M. Bioavailability and bioefficacy of polyphenols in humans. Morand C. Morton MS.823:143–51. Am J Clin Nutr 2005.

Cancer Epidemiol Biomarkers Prev 2001. Arts CJ. Gao YT. Burger HG. Chang-Claude J.22:241–3. Phytoestrogens and breast cancer in postmenopausal women: a case control study. Urinary excretion of isoflavonoids and the risk of breast cancer.10:223–8. High-throughput profiling of dietary polyphenols and their metabolites by HPLC-ESI-MS-MS in human urine. 23. et al.7:289–96. et al. et al. 18. Sun CL. 21. Murkies A. Urinary tea polyphenols in relation to gastric and esophageal cancers: a prospective study of men in Shanghai. Veer PV. Urinary phytoestrogens and postmenopausal breast cancer risk. Stumpf K. Manach C.13:698–708. 22. Mennen L. Jin F. 26. Ross RK. Shu XO. An incident casereferent study on plasma enterolactone and breast cancer risk. Wahlqvist ML. Linseisen J. Custer LJ. Plasma enterolactone and genistein and the risk of premenopausal breast cancer. Pietinen P. Den Tonkelaar I. Sanders K. Adlercreutz H. Gonthiera MP. Hallmans G. et al. Grace PB. Custer LJ. 19. Lee MJ. Cancer Epidemiol Biomarkers Prev 2002. Piller R. Ingram D. Lenner P. Morand C. Lopez D. Winkvist A. Eur J Cancer Prev 2006. Dalais FS. Lancet 1997. Dai Q. Ito H. Franke AA. Cancer Epidemiol Biomarkers Prev 1999. Menopause 2000. et al.15:225–32. . Jin F. Luben RN. et al. Biofactors 2004. Kataja V. Wen WQ. Mannisto S. Kolybaba M. Cancer Epidemiol Biomarkers Prev 2001. Dai Q. 24. Hebert JR.10:339–44. Phytoestrogen concentrations in serum and spot urine as biomarkers for dietary phytoestrogen intake and their relation to breast cancer risk in European prospective investigation of cancer and nutritionnorfolk. Briganti EM. et al. Remesy C. Uusitupa M. Low YL. Yang CS. Thijssen JH. Taylor JI. Case-control study of phyto-oestrogens and breast cancer. China.11:815–21.8:35–40. Yuan JM. Johansson R. Adlercreutz H. 25. Cancer Epidemiol Biomarkers Prev 2004. Serum enterolactone and risk of breast cancer: a case-control study in eastern Finland. 20. Adlercreutz H. Zheng W. Eur J Nutr 2002.41:168–76. Mulligan AA.23:1497–503.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 133 17. 27. Keinan-Boker L. Carcinogenesis 2002. Shu XO.350:990–4. Healy DL. Botting NP. Hulten K. Urinary excretion of phytoestrogens and risk of breast cancer among Chinese women in Shanghai.

and a peak containing the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol [1–3] (Table 3. two secoiridoids (dialdehydes related to oleuropein and ligstroside but lacking the carboxymethyl group at C4). This indicates olive oil phenolics to maintain their antioxidant activities in vivo. a statistically significant negative correlation has been found between homovanillyl alcohol (HValc. a major metabolite of hydroxytyrosol. tyrosol.2.8.). The occurrence of these constituents also calls for the development of specific nutritional biomarkers that reflect the nutritional status of these dietary constituents with respect to their intake or metabolism and that can provide information useful for nutritional epidemiology regarding the effects of disease processes that can occur [4]. a dose-dependent decrease in the urinary excretion of the F2-isoprostane 8-iso-PGF2α. HValc in urine reflects the in vivo .. Epidemiological studies demonstrate rather conclusively that populations within Europe consuming this diet have a particularly low incidence of a number of common cancers [1. A plethora of minor constituents in olive oil have been identified as being effective agents mitigating against the initiation. It has also been shown that olive oil phenolics are excreted in the urine as glucuronide conjugates and that the urinary free tyrosol concentration is responsive to the dietary intake of virgin olive oil. the aglycone of ligstroside. Soterios A. A brief overview is presented of recent findings concerning the bioavailability of certain minor but important olive oil minor components (polyphenols. Athens. Kyrtopoulos 134 3. are dose-dependently absorbed in humans after the ingestion of realistic doses of virgin olive oil. lignans. Kyrtopoulos Institute of Biological Research and Biotechnology. was observed. Sotiroudis.3. oleuropein. These include tocopherol and carotenoid antioxidants.Theodore G. which represent major antioxidants in olive oil. the triterpene hydrocarbon squalene and the phytosterol β-sitosterol [1–3]. Figure 3.2]. a biomarker of in vivo lipid peroxidation processes. secoiridoids and lignans). Anticarcinogenic compounds of olive oil and related biomarkers Theodore G. hydroxytyrosol. Greece Olive oil is an important ingredient of the Mediterranean diet. a number of simple and bound phenolics (tyrosol. When olive oil samples containing increasing amounts of a phenolic extract of olive oil were administered to human volunteers. Oleuropein and its metabolites tyrosol and hydroxytyrosol. Phenolic compounds HPLC chromatography of the methanol extract of virgin olive oil reveals seven major polyphenol peaks corresponding to hydroxytyrosol. The National Hellenic Research Foundation. Sotiroudis and Soterios A. Thus. squalene and β-sitosterol). together with homovanillic acid — HVA) and isoprostane excretion. the excretion of both HValc and HVA also being significantly correlated with the dose of administered hydroxytyrosol. that have been thoroughly studied. considered as putative nutritional biomarkers in relation to cancer the incidence of cancer. In addition. promotion and progression of multistage carcinogenesis.

After the ingestion of olive oil of low phenolic content plasma glutathione peroxidase activity was found to decrease postprandially.75 [3] 14.42 [2]. a biomarker of the intake of tomato-rich food and hypothesized to be responsible for reducing the risk of various .72 [2] 41.2. It was observed in this respect. (+)-pinoresinol (C). Hydroxytyrosol (A).45 [2].65–4. Table 3. Recent findings suggest that olive oil may also affect the bioavailability of other food bioactive components with a chemopreventive potential. Structures of certain phenolic compounds detected in olive oil. oleuropein aglycone (B). An HPLC method for the simultaneous determination of oleuropein and of its metabolites hydroxytyrosol and tyrosol in human plasma has been developed [13.83–4.8.71 [3] 103–205 [3] 27. 38–65 [3] Fig. 1. that the concentration in human plasma of lycopene. Concentrations of the major phenolic compounds found in virgin olive oil Compounds Tyrosol Hydroxytyrosol Oleuropein aglycone Total secoiridoids Lignans References [2] and [3]. 2. but this was not observed after the intake of olive oils of moderate to high phenolic content [12].Bioactive components in foods: Anticarcinogenic compounds of olive oil and related biomarkers 135 concentration of hydroxytyrosol [5–11].53 [2].14]. mg/kg 27. 3.

proapoptotic and anti-oxidant mechanisms established for them [17. It has been observed that postprandial squalene metabolism is age dependent [23]. their reaching a steady state. very little is known concerning the postprandial metabolism of squalene.000 mg/kg. This triterpene hydrocarbon is found mainly in nonedible shark liver oil. while virgin olive oil is a major source of phytosqualene. suggesting that dietary lignans may be important in the etiology of breast cancer. Nevertheless. that the phase II metabolism of enterolactone and enterodiol already may take place during their uptake in the colon. which is most probably based on its strong inhibitory action on the catalytic activity of beta-hydroxy-beta-methylglutaryl-CoA reductase. and that the content of squalene in the whole plasma and in the lipoprotein fractions (where its ratio to cholesterol is highest in the VLDL and the intermediate density lipoproteins [24]) varies directly with the triglyceride content and is increased in hypertriglyceridemia.18]. Experiments in vitro and animal models suggest squalene to play a tumour-inhibiting role. Recent findings suggest that enterolactone is more rapidly metabolized in human colon epithelial cells and/or excreted by them than enterodiol is. restricted almost entirely to estrogen-receptor alpha negative breast cancer has been found. Kyrtopoulos 136 cancers. anti-angiogenic. Mean residence times and elimination half-lives that have been obtained indicate that enterolignans accumulate in the plasma when consumed 2–3 times a day. Sotiroudis. as compared to the consumption of tomatoes cooked without olive oil [15]. and that the epithelial cells in the colon may be responsible for this metabolism [19]. Nevertheless. whereas the findings of epidemiological studies are controversial [18]. Lignans are plant compounds metabolized in the gut to produce the phytoestrogens enterolactone and enterodiol. was found to improve the antioxidant activity of plasma [16]. a tendency for a lower risk of breast cancer to be associated with higher plasma concentrations of enterolactone. If virgin olive oil were the sole source of dietary fat. increased dramatically after the consumption of tomatoes cooked in olive oil. Plasma enterolignan concentrations can thus be considered to be good biomarkers of dietary lignan exposure and be used to evaluate the effects of lignans [20]. Soterios A. Although animal studies have enhanced our understanding of the possible . but not with sunflower oil. the prostate and the colon. leading to a reduced farnesyl pyrophosphate availability for prenylation of the ras oncogene [26].Theodore G. its content ranging from 800 to 12. A number of in vitro and animal studies support a role for lignan-rich foods and of purified lignans in the modulation of cancer events in the breast. The consumption of tomato products prepared together with olive oil. Phytoestrogens have an anticarcinogenic potential through the anti-estrogenic. particularly in premenopausal women [21]. Squalene It has been suggested that the lower risk of cancers of various types associated with high olive oil consumption (as compared with other human foods) may be due to the presence of squalene (reviewed in [1]). which expands the plasma pool of this metabolically active hydrocarbon [25]. the squalene intake would be more than 200 mg/d [22].

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action of squalene in decreasing carcinogenesis, one should be cautious in extrapolating findings there to humans, both because of possible species differences and because the longterm effects of greater consumption of squalene are unknown. Several factors must be taken into account when examining the evidence for squalene’s inhibition of carcinogenesis factors, such as the effective dose used and exposure time [27]. At present, therefore, its use as a nutritional biomarker is hardly to be considered.

Phytosterols Phytosterols are plant sterols that are structurally similar to cholesterol and that possess anticarcinogenic properties [27]. Together with squalene, they represent markers of cholesterol synthesis and absorption and are transported together with cholesterol in serum lipoproteins [24]. β-Sitosterol, one of the most common phytosterols and the main olive oil sterol [1], together with campesterol are the two predominant phytosterols in the blood. It has been suggested that the high reproducibility and high reliability over time (consistency of the plasma phytosterol level over time) of the plasma measurements of these sterols makes them suitable for clinical and population-based studies of cancer prevention [28]. In recent years, functional foods high in phytosterol-ester content for lowering the cholesterol level have been developed. Although phytosterols act as immune modulators and anticancer agents in vitro [29], the protection (if any) that high concentrations of phytosterol provide against the development of cancer in humans has not been adequately examined, further study of this being needed.

Conclusions Since the phenolic content of the olive oil consumed may account for the postprandial antioxidant activity in vivo after the ingestion olive oils of moderate to high phenolic content, we suggest that these biomolecules, or certain polyphenol metabolites in human plasma and urine, can serve as practical biomarkers for olive oil consumption and as an alternative biomarker for future epidemiological studies in dietary cancer prevention and health promotion.

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and the aromatic glucosinolate gluconasturtiin (Table 3. Claverton Down. United Kingdom Glucosinolates and their association with cancer chemoprevention Epidemiological studies have revealed that regular consumption of cruciferous vegetables. this variable elongation of amino acid side chains entails repetitive additions of methyl groups through a series of transamination. the oxime is metabolised to a thiohydroximate. lung cancer [5].11]. Over 115 naturally occurring glucosinolates have been identified. Bath BA2 7AY. As shown in Figure 3.9. University of Bath. though certain broccoli . cabbage. glucobrassicanapin and progoitrin.4. Anticarcinogenic effects of glucosinolate breakdown products John D. catalysed by members of the cytochrome P450 (CYP) 79 family [19]. cauliflower.2]. Cruciferous vegetables uniquely contain glucosinolates at approximately 20 µmol/g dry mass of vegetable [8. Glucosinolates are substituted β-thioglucoside N-hydroxysulfates.. Michael O. Glucoraphanin has been reported to be abundant in broccoli [9]. gluconapin. which is in turn conjugated with glucuronic acid to form a desulfoglucosinolate before finally being sulfated to yield the glucosinolate [2]. Feeding experiments in animals have also suggested broccoli can protect against liver cancer [7]. Michael O. Kelleher. The types of neoplastic disease in man that these vegetables appear to protect against include colorectal cancer [4]. Hayes. Ian M. Kelleher1 and Ian M. the synthesis of glucosinolates proceeds through the conversion of elongated amino acids to their oxime derivatives. greater health benefit may be obtained from raw as opposed to cooked vegetables [3]. phenylalanine and methionine.17].9]. principally valine. though it can be influenced by environmental factors [16. Eggleston 140 3. and possibly prostate cancer [6].12–15]. is associated with a reduced incidence of cancer [1. phenylalanine. used in their biosynthesis. Hayes1. and it is thought that these phytochemicals are primarily responsible for the putative cancer chemoprevention conferred by eating diets that contain significant quantities of these vegetables [10. Furthermore. tyrosine and tryptophan [2]. isoleucine. swede and turnip. the olefinic glucosinolates sinigrin.John D. University of Dundee. The task of establishing a link between the ingestion of particular glucosinolates and their possible health benefits is not straightforward.3. Subsequently. kale. leucine. Much of the diversity amongst glucosinolates arises from the addition of different sized alkyl groups to the side chain of those amino acids. valine. condensation. The glucosinolate content is primarily under genetic control. Eggleston2 1 2 Biomedical Research Centre. such as broccoli. namely. Each cruciferous vegetable contains a mixture of glucosinolates that varies according to the strain of the plant [8. This endeavour is simplified to some extent by the fact that relatively few glucosinolates are present in the human diet. alanine. methionine. Dundee DD1 9SY Department of Pharmacy and Pharmacology. formed by the plant from any one of eight amino acids. The most common of these are the methylsulfinylalkyl glucosinolates glucoiberin and glucoraphanin. Brussels sprouts. isomerisation and decarboxylation reactions [18].) [9.20]. Ninewells Hospital and Medical School.

Brussels sprouts and cauliflower [9.22]. The R group is derived from the original amino acid and is highly variable. Table 3.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 141 strains also contain substantial amounts of glucoiberin [21].3. The indolyl glucosinolate glucobrassicin is present in Savoy cabbage. cauliflower and kale [9]. The aromatic glucosinolate gluconasturtiin is present in watercress. Sinigrin has been reported to be the predominant glucosinolate in Brussels sprouts. Synthesis of glucosinolates. Trivial names of some glucosinolates with the corresponding side-chain (R) composition Name Sinigrin Gluconapin Glucobrassicin Glucobrassicanapin Progoitrin Glucoiberin Gluconapoleiferin Glucocheirolin Glucoerucin Glucoberteroin R side-chain 2-Propenyl 3-Butenyl 3-Indolylmethyl 4-Pentenyl 2-Hydroxy-3-butenyl 3-Methylsulfinylpropyl 2-Hydroxy-4-pentenyl 3-Methylsulfonylpropyl 4-Methylthiobutyl 5-Methylthiopentyl Fig. and whilst not abundant it can elicit distinct pharmacological effects. cabbage. . 3. gluconapin is also found in high levels in Brussels sprouts [9]. Substantial amounts of progoitrin are present in many cruciferous vegetables [9].9.

myrosinase activity may be present in human colonic microflora. suggesting that glucosinolates can be hydrolysed in the gastrointestinal tract during digestion of food [24. or during chewing whilst eating. Kelleher. Hydrolysis of these phytochemicals is catalysed by myrosinase (β-thioglucoside glucohydrolase. nitriles. In addition. Hayes. Eggleston 142 Production of isothiocyanates. Myrosinase cleaves glucosinolates at the thioglycoside linkage to produce glucose and an unstable aglycone thiohydroximate-O-sulfonate that spontaneously rearranges to yield several breakdown products.25]. Michael O. thiocyanates. EC 3. Epithiospecifier protein (ESP) in the presence of Fe2+ ions interacts with myrosinase to promote the transfer of the sulfur to the alkenyl group from the S-Glucose of the terminally unsaturated glucosinolate. The outcome of the reaction with myrosinase depends on the nature of the aglycone.4B. 3. and 3.4A.John D. during freeze-thawing. Ian M. but rather it appears to be due to certain of their breakdown products. the pH and the presence of ferrous ions (Figure 3. Fig.3.147). an enzyme that is physically segregated from glucosinolates within the intact plant by virtue of the fact that it is sequestered in specialised “myrosin” cells [23]. for example during harvesting. myrosinase is released from the “myrosin” cells and is able to hydrolyse glucosinolates within the damaged plant. Upon wounding of the vegetable. cyano-epithioalkanes and oxazolidine-2-thiones from glucosinolates Evidence suggests that inhibition of carcinogenesis by glucosinolates is not primarily attributable to this class of compound.). Hydrolysis of glucosinolates. At high or neutral pH the formation of isothiocyanates is favoured while at low pH the formation of nitriles is favoured. during food preparation. resulting in the formation of an epithioalkane. as well as the reaction temperature.2. .4A.

such as allyl-ITC. 4-pentenyl-ITC and 2-propenyl-ITC (allyl-ITC). In this case. Certain thiocyanates that are formed during a Lossen rearrangement. At low pH. give rise to a cyano-epithioalkane [27]. an arrow shows that allyl thiocyanate. glucobrassicanapin and sinigrin yield 3-methylsulfinylpropyl-ITC. thiocyanates or nitriles [10.4B. The glucosinolates glucoiberin. 4-methylsulfinylbutyl-ITC (sulforaphane). 3-butenyl-ITC.23]. can convert spontaneously to form allyl isothiocyanate [26].g. ESP interacts with myrosinase to promote sulfur transfer from the S-glycosyl unit to the alkenyl chain derived from the amino acid part of the aglycone [28]. gluconapin and glucobrassicanapin) may. sinigrin. to form their respective isothiocyanates (ITCs). hydrolysis of glucosinolates with aliphatic or aromatic side chains gives rise primarily to isothiocyanates (ITCs). glucoraphanin. Hydrolysis of sinigrin. Thus. On the right-hand side of the figure. formed from sinigrin. respectively. at pH 4 and in the presence of Fe2+ ions.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 143 ESP — epithiospecifier protein. At neutral pH. with the elimination of sulfate. in the presence of an epithiospecifier protein (ESP) and ferrous ions. Following damage to the plant tissue the glucosinolate sinigrin is hydrolysed by myrosinase resulting in the formation of four distinct compounds. The thiohydroximate-O-sulfates formed from methylsulfinylalkyl. are unstable and can undergo a relatively slow spontaneous conversion to their respective isothiocyanate [26]. Fig. Elemental sulfur is also formed in certain circumstances. olefinic and aromatic glucosinolates undergo a Lossen rearrangement. . the thiohydroximate-O-sulfates formed by myrosinase from glucosinolates with a side chain containing a double bond (e. gluconapin. 3.

Hayes.34]. Progoitrin. The best studied of these is glucobrassicin. ESP and Fe2+ ions to an epithionitrile [32] and in the case of epi-progoitrin ((2S)-hydroxy-3-butenyl glucosinolate). 3. Two cDNAs for ESP have recently been cloned from Arabidopsis and broccoli and the purified proteins characterized following their heterologous expression in E.4-epithiobutane [30. glucobrassicanapin can be hydrolysed to 1-cyano-4. A few glucosinolates produce thiocyanates though the mechanism involved is unclear [23]. At neutral pH. and is diminished by heating [40]. Eggleston 144 myrosinase and ESP convert sinigrin to 1-cyano-2.5.). This reaction may involve ESP. Michael O. .John D. is also converted in the presence of myrosinase.36]. coli [35. as a consequence of spontaneous cyclization. Ian M. this reaction probably proceeds Fig. Gluconapin can similarly be converted by the combined actions of myrosinase and ESP to 1-cyano-3. glucoconringiin and gluconapoleiferin [2]. a (2R)-hydroxy-3-butenyl glucosinolate. Examples of these include progoitrin. the sulfur atom may be lost and a nitrile formed [37–39].5-epithiopentane [31]. Kelleher.5. Upon hydrolysis by myrosinase. Production of indoles from glucosinolates.3-epithiopropane [29]. Production of indoles from glucosinolates The indolyl glucosinolates glucobrassicin and neoglucobrassicin are synthesised by the plant from tryptophan. At neutral pH the hydrolysis of glucobrassican by myrosinase leads to the formation of an unstable isothiocyanate intermediate which degrades to form indole-3-carbinol and a thiocyanate ion. those aglycones from glucosinolates that contain β-hydroxylated sidechains form oxazolidine-2-thiones. but rather gives rise to indole-3-carbinol and a thiocyanate ion (Figure 3. Likewise. hydrolysis of glucobrassicin by myrosinase does not generate an ITC. If the aglycone generated by myrosinase is from a glucosinolate with a side chain lacking a double bond. it can be hydrolysed by myrosinase to crambene (1-cyano-2-hydroxy-3-butene) [33.31].

). when compared with the data about thiocyanates. Structures of indoles produced from glucosinolates — 3. 3. both in vivo and in vitro [45. cyanoepithioalkanes. At acidic pH. and stimulation of apoptosis. indolo [3.3’-Diindolylmethane (DIM). hydrogen sulfide and elemental sulfur [22]. It can also combine with ascorbic acid to form ascorbigen [42] (Figure 3. Induction of gene expression mediated by Nrf2 Isothiocyanates increase the expression of antioxidant and detoxication proteins. A challenge in evaluating the literature arises from the emphasis placed on certain glucosinolate breakdown products and the dearth of data relating to others. it is not surprising that a number of distinct cancer chemopreventive mechanisms have been proposed to account for the putative anti-cancer properties of cruciferous vegetables. hydrolysis of glucobrassicin yields indole-3-acetonitrile. A major problem exists in interpreting experiments utilizing vegetable extracts because of uncertainty about attributing biological effects to specific phytochemicals.46]. cell cycle arrest.44]. indole-3-carbinol condenses to form various compounds including indolo[3.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 145 through a Lossen rearrangement generating an unstable ITC intermediate [22]. In the acidic environment of the stomach. and oxazolidine-2-thiones. whereas activation of cell cycle arrest and apoptosis occurs at higher levels of phytochemical [43. There is a dose-dependency in these responses: generally.3∂-diindolylmethane. induction of cytoprotective genes and inhibition of CYP activity occurs at relatively low concentrations of phytochemical. nitriles. These include induction of antioxidant and detoxifying genes.6. Thus. inhibition of CYP enzyme activity.2-b] carbazole (ICZ) and ascorbigen (ASG).2-b]carbazole and 3. such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Fig. both of which have potent pharmacological effects [41]. Chemopreventive mechanisms stimulated by glucosinolate hydrolysis products In view of the diverse spectrum of chemicals generated from glucosinolates by the actions of myrosinase and ESP. Agents such as ITC that increase GST and NQO1 .6. there is a relative abundance of information about isothiocyanates and indoles.

Examination of nrf2–/– and wild-type mice. are sometimes called mono-functional inducers [47]. 7-methylsulfinylheptyl-ITC. The induction of these two genes by ITCs is mediated by the Nrf2 (Nuclear Factor-Erythroid 2 p45-related factor 2) bZIP (basicregion leucine zipper) transcription factor that is recruited to the ARE as a heterodimer with small Maf protein [51]. Kelleher. glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits. Hayes.7. allyl-ITC.54. GST. 3) Benzyl-ITC. Michael O. Sulforaphane. ferritin. Indeed. suggests AREs are present in the promoters of at least 100 genes [53–55]. 3-methylsulfinylpropyl-ITC. 3. Many of these compounds induce GST P1-1 in the rat liver RL-34 cells [50]. Eggleston 146 enzymes without increasing arylhydrocarbon hydroxylase activity. thioredoxin. It is thought that ITCs possess the ability to induce ARE-driven gene expression because they are thiol-active [59]. Importantly. carboxyl esterase. heme oxygenase 1. catalysed by the phase I drug-metabolising enzyme cytochrome P450 (CYP) 1A1. Through this characteristic. UDP-glucuronosyl transferase.55–58].7. as far as is known. GST and NQO1 in the gastrointestinal tract in an Nrf2-dependent fashion [21]. Ian M. Many of these genes are induced by sulforaphane in vivo in an Nrf2-dependent fashion in the stomach. 2) Phenethyl-ITC. all genes that are induced by ITCs contain an ARE in their promoters and are regulated by Nrf2.). 5) 7-methylsulfinylheptyl-ITC. Structures of isothiocyanates which induce NQO1: 1) Allyl-ITC. 4) 3-methylsulfinylpropyl-ITC. NQO1.49] (Figure 3. The mouse nqo1 gene contains a functional antioxidant response element (ARE) in its 5∂-upstream region [51]. The battery of genes regulated by Nrf2 includes those encoding aldo-keto reductase (AKR). multidrug resistance-associated protein. ITCs modify cysteine residues in the Cullin 3:Rbx1 E3 ubiquitin ligase substrate adaptor Keap1. Fig. 6) 8-methylsulfinyloctyl-ITC. microsomal epoxide hydrolase. induce NQO1 in the mouse Hepa-1c1c7 hepatoma cell line [48. small intestine and liver of rodents [53.John D. metallothionein. leading to its inhibition and inability to serve as a substrate adaptor . 8-methylsulfinyloctyl-ITC. feeding broccoli seed to mice increased the levels of GCLC. as does the rat GSTP1 gene (called GPEI) [52]. benzyl-ITC and phenethyl-ITC. thioredoxin reductase.

rat GSTA2.3∂-diindolylmethane can increase the stability of Nrf2 protein is not known.64]. mouse. indicating that AhR ligands could lead to an increased production of Nrf2 mRNA.2-b]carbazole and 3. allyl-ITC does not increase Nrf2 stability [64] and there may therefore be other factors involved in enzyme induction by these phytochemicals. this seems unlikely [72]. rat UGT1A1. Although indole-3-acetonitrile has not been studied in the Nrf2 knockout mouse. The identity of this metabolite is not known. it is a good inducer of GST enzyme activity in mouse liver and small intestine [68]. indole-3-carbinol or ascorbigen [43. and there is abundant evidence from enzyme assays. Whether indolo[3. Amongst genes for drugmetabolising enzymes.2-b]carbazole and 3. rat ALDH-3. However. as does the mouse BAX gene. and thereby generate reactive oxygen species.3∂-diindolylmethane.69]. and rat UGT1A6 contain an XRE.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 147 required for the ubiquitination of Nrf2 under homeostatic conditions [60–62].67]. western and northern blotting that CYP1A1 is inducible by indolo[3. it was not a particularly effective inducer of NQO1 activity in Hepa-1c1c7 cells suggesting that the relatively high potency of induction observed in vivo is due to bio-transformation of cranbene to a thiol-active metabolite. but not CYP1A1.3∂-diindolylmethane [2]. It has been found that administration of cranbene to Fischer 344 rats causes an elevation in hepatic GST and NQO1 enzyme activities. suggesting it is a mono-functional inducer [65]. rat and human CYP1A1 are prototypic XRE-regulated genes. see [70]). The promoters of other genes including rat and human NQO1. the mouse nrf2 gene promoter also contains an XRE [71]. Induction of gene expression mediated by AhR The major effect that indole-3-carbinol has on gene expression occurs because it can condense in acid conditions to form indolo[3. but unless they are metabolised by CYPs. cranbene was found to be an approximately equally potent inducer in the rat [66]. By comparison with sulforaphane. and it is therefore anticipated that activation of AhR by glucobrassicin-derived indoles will influence significantly the metabolism of xenobiotics (for a review. and this may also contribute to gene induction [46]. The indole-containing glucobrassicin breakdown products can activate gene expression by several mechanisms. Interestingly. Examination of the dose of indole required to double XRE-driven reporter gene expression shows that indolo[3. Surprisingly. .2-b]carbazole is a much more potent inducing agent than 3. This cytochrome has O-deethylase activity towards ethoxyresorufin. Consistent with this view. this may not necessarily lead to an increase in Nrf2 protein levels. Treatment of cells with benzyl-ITC causes a rapid increase in the level of reactive oxygen species. exposure of RL-34 cells or HepG2 cells to sulforaphane causes stabilisation and rapid accumulation of Nrf2 [63. Indole-3-carbinol is a modest inducer of Nrf2-dependent ARE-driven gene expression in the liver and small intestine of mice [52. Both these condensation products induce CYP1A1 genes via the xenobiotic response element (XRE) in their promoter regions because they are ligands for the Ah receptor. a fact that implies it works through Nrf2.3∂-diindolylmethane.2-b]carbazole and 3.

such as growth arrest and DNA damage (GADD) GADD34. the ITCs with highest lipophilicity and low reactivity of their NCS group had the greatest ability to inhibit lung tumorigenesis [82].John D. DNA is tightly coiled around an octamer of histone proteins in a structure known as a nucleosome. It is not however clear whether the genes for ATF3. Inhibition of CYP can also be achieved by sulforaphane [80.2-b]carbazole before exposure to benzo[a]pyrene provides a small measure of protection against DNA damage as measured by the comet assay. 3. and this protection was greater than was achieved by either phytochemical alone [43]. GADD45 and GADD153 [75. Tumorigenesis caused by the carcinogens 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and N-nitrosobis-(2-oxopropyl)amine can be prevented by phenethyl-ITC through a process that involves inhibition of activation of pro-carcinogens by CYP isoenzymes [78. as are the drug metabolising enzymes AKR. such as CYP1B1 and CYP19 [73].81]. c-Jun. Indeed.79]. Eggleston 148 In addition to induction of CYP1A1 by indoles. Ian M. NF-IL6 and the GADD proteins are regulated through XREs and whether the process is mediated by AhR. p21 is induced by indole-3-carbinole [77]. sulfotransferase and UGT1 [74]. c-Jun and NF-IL6 as well as genes involved in cell growth. Most importantly. Michael O. It has been argued that induction of CYP1A1 by indoles is potentially deleterious to the cell because the cytochrome can activate polycyclic aromatic hydrocarbons to ultimate carcinogens. Hayes. GST T1-1. Also. Bonnesen et al [43] have reported that treatment of human colon LS-174 cells with indolo[3. recognition of this possibility has lead to considerable recent interest in the ability of HDAC inhibitors to act as both chemopreventive and chemotherapeutic agents.2-b]carbazole and sulforaphane before exposure to benzo[a]pyrene was found to confer substantial protection against genotoxicity. This function is likely to alter gene expression substantially. It may also have profound implications for cell fate as a change in the balance between histone acetyl transferase (HAT) and HDAC could alter tumourigenesis. Inhibition of carcinogen activation by glucosinolate breakdown products Certain isothiocyanates can block the activation of several carcinogens to their ultimate carcinogenic forms.84]. the basic structural unit of chromatin. prior treatment of the LS-174 cells with both indolo[3. other cytochromes are inducible. Kelleher.3∂-diindolylmethane can induce expression of the transcription factors ATF3. Within the cell. Inhibition of histone deacetylase by isothiocyanates The isothiocyanate sulforaphane has been shown to inhibit histone deacetylase (HDAC) [83. In the case of the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Furthermore.76]. Each of the histone proteins contains an evolutionary conserved amino tail protruding from . This viewpoint is probably an oversimplification and does not take into account the multiple changes in gene expression that indoles affect.

The tail is also subject to many post-translational modifications including acetylation. . Stimulation of cell cycle arrest and apoptosis by isothiocyanates Many of the naturally occurring ITCs can suppress the growth of cultured tumor cells by modulating multiple targets that influence cell cycle arrest. allowing transcription factors access to the regulatory regions of genes. human embryonic kidney cells [83] and human colorectal cancer cells [83]. mammalian HDAC is capable of downregulating p53 function. In human PC-3 prostate cancer cells. removal of acetyl groups causes the tail to wrap tightly around the DNA thereby preventing interaction with the transcription machinery. the majority of the studies into mechanisms by which this class of chemical inhibit cell growth have focussed on sulforaphane and phenethyl-ITC.92]. Sulforaphane has been shown to diminish HDAC activity with a concomitant rise in histone acetylation in prostate cancer cells [84]. resulting in a reduction in its transcriptional activity. The loss of Cdc25C was reported to be due to proteasomal activity. ApcMin/+ mice treated with sulforaphane at either 300 ppm or 600 ppm in their diet have been reported to develop fewer and smaller polyps in their small intestine than ApcMin/+ mice on a control diet. p53 [86] and Bax [84. Inhibition of HDAC may prevent the removal of acetyl moieties from histones thus allowing transcription of the tumour suppressor genes. which can determine the accessibility of the DNA to transcription factors. Equally. For example. apoptosis and differentiation [89]. The addition of an acetyl group to the histone tail results in a conformational change which enables the tail to move away from the DNA. sulforaphane has been found to cause a G2/M phase delay with an increase in apoptotic cell fraction in a timeand dose. However. to affect chemoprevention via its ability to alter chromatin structure is of acute importance to the welfare of the cell. The addition and removal of the acetyl groups is carried out by HAT and HDAC. Relocation of Cdc25C was found to be controlled by its phosphorylation at Ser-216. The link between inhibition of HDAC activity and the resultant increase in transcription of tumour suppressor genes has been reported for p21 [84. mediated through activation of checkpoint kinase 2 (Chk2) [84].94].dependent fashion [87]. tumour suppressor genes are associated with deacetylated histones resulting in the inactivation of these genes.85]. In pre-cancerous and cancerous cells. Conversely. respectively. by deacetylation of histones associated with the p53 gene. treatment with sulforaphane or phenethyl-ITC causes an arrest in G2/M phase of the cell cycle that is associated with a decrease in levels of cyclin B1 and cell division cycle (Cdc) 25B and Cdc25C proteins [91. Thus the ability of sulforaphane. Cell proliferation by ITCs may also be achieved by distrupting cytoskeletal structure and tubulin polymerisation [93.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 149 the nucleosome. this was associated with a higher level of apoptosis and lower proliferation in animals on the ITC containing diet [90]. and was accompanied by its translocation from the nucleus to the cytoplasm [92]. In addition. along with other dietary HDAC inhibitors such as diallyl disulfide [88].85].

the mechanism by which JNK activates caspases remains unclear. which appears to be necessary for cell death [92. Ian M. Treatment with ITCs leads to a loss of mitochondrial membrane potential and release of cytochrome c from mitochondria [98]. caspase-8 plays a major role in apoptosis stimulated by phenethyl-ITC [100].3∂-diindolylmethane rather than indole-3-carbinol as significant quantities of the indole spontaneously condense to the dimer in culture conditions [107]. In the case of CDK6.97].103]. in human leukaemia HL60 cells. treatment with 400 µM indole-3carbinol induces the CDK4/6 inhibitor p15INK4b mRNA and protein causing hypophosphorylation of Rb protein [109]. indole-3-carbinol has been . The use of inhibitors indicated that JNK is essential for phenethyl-ITC to cause cytochrome c release and caspase-3 activation in human HT-29 colon adenocarcinoma cells [105]. This may in part be due to 3. though this may be cell specific. However. which neutralize the antiapoptotic effects of Bcl-2. the pro-apoptotic proteins Bok and Bim EL are also induced by ITCs. In the case of CDK2. Kelleher.92. benzylITC and phenethyl-ITC are more effective at activating caspase-9 than caspase-8 [99]. are increased by phenethyl-ITC and sulforaphane in PC-3 prostate cancer cells. Cell cycle arrest at G1 occurs as a consequence of indole-3-carbinol inhibiting both cyclin-dependent kinase (CDK) 2 and CDK6. addition of GSH subsequent to ITC treatment can block apoptosis [44. The generation of ROS by ITCs is accompanied by depletion of intracellular GSH and is achieved through the rapid export of ITC-glutathione and ITC-cysteinylglycine conjugates via MRP1 and Pgp-1 efflux pumps [96]. The levels of pro-apoptotic proteins Bak and Bax.John D. overexpression of catalase suppresses ITC-initiated programmed cell death.95]. Stimulation of cell cycle arrest and apoptosis by indoles Treatment of human MCF-7 breast cancer cells with 100 µM indole-3-carbinol inhibits proliferation through affecting a G1 cell cycle arrest [106]. in HaCaT keratinocytes. Eggleston 150 Administration of ITCs to cells at growth suppressive concentrations results in the rapid generation of reactive oxygen species (ROS). as does pre-treatment with N-acetylcysteine [92]. Michael O. ERK1/2 [104]. Hayes. Consistent with the view that production of ROS is necessary for apoptosis. Furthermore. though the effect is cell-specific [101]. For example. Furthermore. and this is mediated by extracellular signal-regulated kinases. and this is thought to amplify the effects of Bak and Bax [92]. It therefore appears that cyclin D-CDK6 activity can be inhibited by dual mechanisms. There is evidence that ITCs can activate both the intrinsic and extrinsic caspase cascades.101]. Furthermore. in PC-3 cells sulforaphane can increase Fas protein levels and activate caspase-8 whilst simultaneously targeting mitochondria and activating caspase-9 [92]. expression of the gene is reduced because indole-3-carbinol attenuates recruitment of the Sp1 transcription factor to the CDK6 promoter [108]. ITCs down-regulate the anti-apoptotic proteins Mcl-1 and Bcl-xL . In human bladder cancer UM-UC-3 cells. Various ITCs have been shown to activate c-Jun N-terminal kinase (JNK) [102. By contrast. within 1 h of exposure. Besides increasing the levels of these pro-apoptotic proteins. and this may lead to induction of Apaf-1 [91.

cyano-epithioalkanes and oxazolidine-2-thiones from glucosinolates. If so. such as tamoxifen [77].115]. the reduction in CDK2 activity is accompanied by redistribution of the kinase in the 200 kDa complex from the nucleus to the cytoplasm. In HCT-116 human colon cells. an increase in p53 protein levels. Concluding comments It is becoming clear that glucosinolate breakdown products can influence the initiation and progression of carcinogenesis. In cells that contain wild-type p53. Indoles can affect apoptosis in breast and prostate cancer cells. indole-3-carbinol can induce nonsteroidal antiinflammatory drug-activated gene-1 (NAG-1). These changes result in prevention of the CDK2-mediated G1/S transition [111]. ESP should possibly . nuclear translocation of NF-κB is inhibited by 3. Indole-3-carbinol. was found to inhibit the expression of the androgen receptor in human lymph node carcinoma of prostate (LNCaP) cells as well as the probable downstream target gene prostate specific antigen [112]. It is unclear whether the activity of ESP. Specifically. the Akt/PI3K cell survival pathway appears to be targeted by indole-3-carbinol. at the expense of forming isothiocyanates. Furthermore. They also appear to influence apoptotic responses to chemotherapeutic agents. but not 3. and the larger complex also contains an additional 75 kDa cyclin E immunoreactive protein. induction of p21 [111]. Also. nitriles. suggesting that indole-3-carbinol can influence the nucleocytoplasmic shuttling of the kinase [110]. Thus.3∂-diindolylmethane has been found to result in activation of the ATM signalling pathway. It is possible that down-regulation of this receptor represents an antiproliferative mechanism in prostate cells. Treatment of PC-3 prostate cancer cells with 60 µM indole-3-carbinol inhibits the EGF-induced autophosphorylation of PI3K and Akt [113]. a TGF-β family member associated with pro-apoptotic activities [116].3∂-diindolylmethane through a reduction in phosphorylation of IκBα [114. nitriles. treatment with 300 µM indole-3-carbinol or 30 µM 3. A major impediment to our understanding of the chemopreventative mechanisms stimulated by glucosinolates is that relatively little is known about the biological effects of glucosinolate breakdown products other than isothiocyantes and the indole-containing derivatives. It is unclear whether formation of thiocyanates. there are few data about chemopreventative activities of thiocyanates.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 151 reported to decrease the kinase activity in MCF-7 cells and inhibit phosphorylation of Rb protein [77]. which reduces the formation of isothiocyanates from glucosinolates. cyanoepithioalkanes and oxazolidine-2-thiones. The reduction in CDK2 activity is attributed to a selective alteration in the size of the complex in which it is contained. is undesirable. such as MCF-10A. from an active form within a 90 kDa complex to a lower activity form within a 200 kDa complex [110]. This may also mediate the anti-tumour effects of indoles.3∂-diindolylmethane. the 90 kDa and 200 kDa complexes include forms of cyclin E that differ in size. is undesirable from a cancer chemoprevention perspective.

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5. University of Maastricht. de Kok. Evaluating the effects of phytochemicals that act synergistically Carcinogenesis is an extremely complex multistep process in which numerous molecular mechanisms play an important role. however. both in vitro and in vivo. There is a growing body of evidence that the actions of phytochemicals administered as dietary supplements fail to provide the health benefits that have been observed for diets rich in fruits. which are believed to have cancer-preventive effects. therefore. is that purified phytochemicals do not necessarily have the same beneficial health effect as these compounds do when their source is a food or even a complete diet. may lose their biological activity or fail to behave in the same way as in the complex matrix that the original item of food represents. Maastricht. The Netherlands Introduction The relatively consistent epidemiological finding that the consumption of whole foods of different types such as fruits. One of the key problems of research in this field. The intended positive effects of an increased intake of β-carotene or vitamin C as dietary supplements. de Kok and Simone G. Combined action of different dietary compounds preventive of cancer Theo M. Isolated and purified compounds. numerous bioactive compounds have been isolated and identified. . and their potential health-promoting effects evaluated extensively. and there are even reports of increased occurrence of lung cancer in smokers receiving dietary β-carotene supplements [5. in contrast. This can be illustrated by the effects of increased intake of carotenoids and vitamin C in diets high in fruits and green and yellow vegetables. vegetables. evidence that bioactive compounds act synergistically is reviewed. Table 3. van Breda 160 3. The combinations of phytochemicals that natural foods contain can reduce the risk of cancer by affecting different overlapping and complementary mechanisms. Some studies show no reduced incidence of cancer as a result of taking supplements of vitamin C [3] or β-carotene [4]. the cancer-preventive effects that certain whole foods and diets are shown to have can perhaps better be explained in terms of the chemopreventive properties that interactions between the different dietary ingredients involved create. In addition to characterizing the chemopreventive effects of individual compounds. Cancer-preventive dietary compounds may interfere at a variety of different levels with these processes. are far from certain.Theo M. In the research conducted. and the like. evaluation of the effects of synergistically acting phytochemicals is needed. in contrast.6].2]. In this chapter. summarizes various mechanisms by which phytochemicals can modulate the risk of cancer [1. van Breda Department of Health Risk Analysis and Toxicology. vegetables and whole grains is strongly associated with reduced risk of cancer and of other chronic diseases has led to the hypothesis that particular phytochemicals are responsible for the preventive effects observed.10. Simone G. Although relatively high doses of single bioactive agents may show potent anticarcinogenic effects. whole grains.

Epicatechin was found to promote the occurrence . Synergistic effects of combinations of various polyphenols A number of studies report enhanced chemopreventive effects of mixtures of polyphenols from green tea or other dietary sources. presents a selection of relevant studies concerning such synergistic effects. Proposed mechanisms by which dietary phytochemicals can prevent cancer Antioxidant activity Scavenging of free radicals and reduction of oxidative stress Inhibition of cell proliferation Induction of cell differentiation Inhibition of oncogene expression Induction of tumour-suppressor gene expression Induction of cell-cycle arrest Induction of apoptosis Inhibition of signal transduction pathways Enzyme induction and enhancing detoxification Phase II enzymes Glutathione peroxidase Catalase Superoxide dismutase Enzyme inhibition Phase I enzyme (blocking the activation of carcinogens) Cyclooxygenase-2 Inducible nitric oxide synthase Xanthine oxidase Enhancement of immune functions and surveillance Antiangiogenesis Inhibition of cell adhesion and invasion Inhibition of nitrosation and nitration Prevention of DNA adduct formation or DNA intercalation Regulation of steroid hormone metabolism Regulation of estrogen metabolism Antibacterial and antiviral effects Modified from Liu et al. [2].Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 161 Table 3. another green tea polyphenol. [7] reported that the effects of tritium-labelled epigallocatechin gallate (EGCG) being incorporated into human lung cancer cells were enhanced by epicatechin (EC).11.10. one without a galloyl moiety. Table 3. Suganuma et al.

[19] Zhou et al. [8] EGCG. induced by a tea polyphenol having a galloyl moiety. The study showed that the synergistic effects of two green tea polyphenols could result in the tea as a whole becoming a more efficient anticarcinogenic mixture than if supplementation of EGCG alone had been provided. C and ß-carotene Reduction in α-tocopheryl radicals. EC.Theo M. These effects. Selection of studies of the synergistic effects of mixtures of polyphenols and of combinations of these with other types of phytochemicals Combination of compounds Polyphenol mixtures EGCG and EC. EC. Extracellular production of oxygen species Antagonism of TCDD-induced transcription of human CYP1A1 via interaction with the Ah-receptor Suganuma et al. [22] EGCG. were also enhanced in a dose-dependent way by EC. [21] Liu et al. Table 3. van Breda 162 of EGCG-induced apoptosis and to inhibit not only growth of PC-9 lung tumour cells but also the release of tumour necrosis factor-α. EGC and ECG Modulation of CYP1A1 expression in human hepatocytes Williams et al. tumour weight and metastasis Inhibition of breast tumour cell growth Reduction in serum levels of testosteron and DHT Inhibition of tumour angiogenesis. Zhou et al. reduced release of TNF-α Increased production of caspases-3. Inhibition of tNOX.11] Polyphenols and other phytochemicals Green/black tea and soy (SPC)* Green/black tea and soy (SPC) Inhibition of prostate tumours.11. EGC and ECG or gallic acid and α-tocopherol Reduced oxidative stress in micelles and human LDL . A and ß-carotene Reduced oxidative stress Morré and Morré [16] Reduced formation of lipid Gorelik et al. reduced levels of estrogen receptor-α protein and of serum IGF-I. -8 and -9. Simone G. [18] hydroperoxides and malondialdehydes. [13] Green tea infusions and grape Reduced tumour cell growth or grape skin extracts Polyphenols. vitamin E. reduced co-oxidation of vitamins E. [7] EGCG and EC Inhibition of cell growth and induction of apoptosis in gastric carcinoma cells Horie et al. [12] Zhou et al. enhanced apoptosis. [10. induction of apoptosis. de Kok. trap-ping of lipid peroxyl radicals and regene-ration of vitamin E Zhou et al. sulindac or tamoxifen Synergistic effect Mechanisms involved Reference Inhibition of growth of human lung cancer cells Enhanced cellular uptake of EGCG.

[23.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 163 Table 3. EC alone had virtually no effect on carcinomic cell growth or induction of apoptosis. interpreted mainly in preventive terms No indication of specific synergetic mechanisms * SPC — Soy Phytochemical Concentrate. Enzyme induction usually enhances detoxification. EGCG. peas and unions) Up. whereas EGCG acts as a strict AhR antagonist. catalase was found to block the synergistic effects of EC and EGCG. The induction of CYP1A enzymes by PAH and by dioxins such as TCDD occurs at the transcription level and is mediated by the cytosolic aryl hydrocarbon receptor (AhR) [9]. the activity levels of caspases 3. Various gastric cell lines were shown to differ in their susceptibility to EGCG treatment. suggesting that the reactive oxidative species and production of hydrogen peroxide play a part in the synergistic mechanisms here [8]. Some of the cytochrome P450 genes are expressed constitutively. epigallocatechin (EGC). Selection of studies of the synergistic effects of mixtures of polyphenols and of combinations of these with other types of phytochemicals — cont. [25] Jørgensen et al. carrots. and epicatechin gallate (ECG) — and it was not found to be improved further by the addition of other compounds [11].24] Different types of vegetables (cauliflower. but under some circumstances substrates may be activated to become mutagens. Interestingly. 8 and 9 became elevated. Williams et al. were also found in gastric carcinoma cells [8].and down-regulation of genes involved in carcinogenesis. carcinogens or cytotoxic substances [9]. The authors concluded that the modulation of human CYP1A1 expression by green tea extracts cannot be attributed to the action of a single tea catechin. whereas others are inducible by xenobiotic compounds or by phytochemicals. the modulation of their expression and activity by phytochemicals is a potential mechanism by which the risk of cancer can be reduced. Combination of compounds 12 red wine polyphenols Synergistic effect Increased antioxidant potential Mechanisms involved Regeneration of phenoxy radicals by phenolic compounds Reference De Beer et al. . being due instead to the effects of the complex mixture involved. both on the inhibition of cell growth and the induction of apoptosis. After this combined treatment. but it had a significant synergistic effect on the induction of apoptosis when combined with other catechins. indicating them to be involved in catechin-induced apoptosis.11. The optimal degree of synergy was achieved by a combination of the four major tea catechins — EC. [27] Van Breda et al. Co-treatment of human hepatocytes with TCDD and different tea catechin mixtures inhibited synergistically both TCDD-induced CYP1A-promoter-driven luciferase reporter activity (in the HepG2 cells) and CYP1A1 expression (in the HepG2 cells and the primary human hepatocytes). Since the cytochrome P450 (CYP) enzymes are responsible for the metabolism of many environmental carcinogens. [10] demonstrated that complex green tea extracts exert mixed agonist/antagonist activity on the Ah-receptors. Synergistic effects of green tea catechins.

vitamin C can enhance the activity of polyphenols through a synergistic antioxidant effect. Polyphenols and dietary antioxidant vitamins can also have synergistic inhibitory effects on lipid peroxidation and on the co-oxidation of dietary antioxidants. In an immunedeficient mouse model in which MCF-7 human breast cancer cells were implanted. whereas no activity was detected for extracts of grape seeds. In the gastric fluid. Cells in which NOX activity is blocked.Theo M. increasing at the same time the amounts of vitamin antioxidants reaching the blood system. indicating that the effects were not caused by resveratrol. This was accompanied by inhibition of tumour angiogenesis. though to a lesser extent) [18].11. In further investigations it was shown that bioactive compounds in tea (particularly EGCG [14]) and soy (the soy isoflavone genistein as well as SPC) inhibit growth of prostate cancer and tumour metastasis in vivo [15]. all of these being factors in breast cancer development. A phytochemical soy concentrate (SPC) and green and black tea infusions were employed (Table 3. the latter a biologically more active metabolite of testosterone and a prerequisite for the development of benign prostatic hyperplasia and prostate cancer [12]. A tumour-specific growth protein possessing NADH oxidase activity (tNOX) has emerged as a potential target of the anticancer action of plant polyphenols and flavonoids [16].). which is found predominantly in the seeds. such as by phytochemicals. Zhou et al. Simone G. investigated with use of mice models the potential synergistic effects of a combination of bioactive tea components and soy phytochemicals on androgen-sensitive human prostate tumours and estrogen-dependent human breast carcinoma [12. The strongest synergistic activity was found for ethanol extracts of grape skins. Recently. NOX proteins are located at the cell surface and are responsible for the increase in cell size that occurs following cell division [17]. The authors indicated that the resulting synergistic increase . It was demonstrated with use of simulated stomach fluid that phytochemicals can prevent the build-up of oxidized lipid products (lipid hydroperoxides and malondialdehyde) as well as the destruction of vitamin E and β-carotene (and of vitamin C too.13]. the authors suggested that the antioxidant network in the stomach can decrease the level of hydroperoxides and other cytotoxic compounds. SPC in combination with green tea showed a synergistic inhibition of tumour cell growth. The synergistic inhibition of the progression and mestastasis of prostate tumours by use of the combination of green tea and SPC was found to be associated with effective reduction in the serum levels of both testosterone and dihydrotestosterone. They cease to divide and eventually undergo apoptosis. are unable to enlarge. reduced expression of estrogen-receptor (ER) alpha and reduced serum levels of the insulin-like growth factor (IGF)-I. These results suggest that more effective cancer prevention and cancer therapy could be achieved by use of combinations of different phytochemicals. de Kok. van Breda 164 Synergistic effects of polyphenols and other phytochemicals In two different studies. The modulation of these two different mechanisms may be an explanation of the synergistic effects of the combined phytochemicals [13]. In line with this. Morré and Morré [16] showed there to be an exceptionally strong (10-fold) synergy between grape polyphenols and tea catechins in the inhibition of tNOX.

22]. ECG and gallic acid) can effectively reduce α-tocopheroxy radicals so as to allow α-tocopherol to be regenerated.24]. Taking account of the different mixtures found of the 12 phenolic compounds that were present. the Trolox equivalent antioxidant capacity (TEAC) value and phenolic composition were determined for a large number of pinotage wines [25]. the individual vegetables were able to modulate genes which were not significantly modulated by the mixture. In one study. . EGCG. despite their unhealthy dietary habits and high consumption of alcohol in the form of red wine) and the beneficial effects of Mediterranean and Japanese diets that contain complex combinations of polyphenols and other antioxidants [18]. indicating that combinations of different foods containing different complex mixtures of phytochemicals can also have an antagonistic effect on gene expression. It is particularly the elimination of the pro-oxidant effect of vitamin E (or the so-called tocopherol-mediated peroxidation) that can occur in the absence of other oxidants [20]. These green tea polyphenols were also found to trap the initiating radicals (ROO•) as well as the propagating lipid peroxyl radicals (LOO•). When the contributions of the separate phenolic compounds were calculated. plays a major role in enhancing the antioxidant efficiency of vitamin E.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 165 in the systemic antioxidant effect might also explain the French paradox (the fact that people in France suffer from relatively low incidence of coronary heart disease. Synergistic effects of whole foods and complex mixtures of compounds In addition to the synergistic effects of several individual compounds on biomarkers of cancer prevention. On the other hand. combined with an α-tocopherol regenerating reaction involving coexisting antioxidants. [19] demonstrated clearly that several green tea polyphenols (EC. These combined effects may also explain the synergistic antioxidant effects of the polyphenols in tea and of the α-tocopherol in both the micelles and human low-density lipoprotein that members of this same research group have reported [21. In two recent animal studies on the effects of vegetable consumption on the modulation of gene expression. it was found that most of the genes that were differentially expressed before and after vegetables were consumed. Consumption of the mixture of the vegetables was able to modulate genes which were not significantly modulated by one of the specific vegetables present in the mixture. the changes in gene expression could be interpreted as a cancer preventive effect [23. EGC. The effects of consuming one of four different vegetables on gene expression in the colon and the lungs of female C57B16 mice were found to differ from those of consuming a mixture of all four vegetables at once. the synergistic effects of taking whole foods or complex mixtures of compounds have been reported. By studying the kinetics of the reaction of α-tocopherolyl radicals with green tea polyphenols by means of stopped-flow electron paramagnetic resonance. it was found that only 11 to 24% of the TEAC values could be explained in terms of the sum of the values for the individual compounds. Zhou et al. Other examples of the assessment of synergistic effects in complex mixtures are to be found in studies aimed at unraveling the antioxidant capacity of red wines. and assuming their concentrations to be those typical for red wines. This.

Various combinations of drugs have been proposed for further clinical development based on their synergistic activity as shown in vitro or in animal studies. the effects reported by Suganuma et al.28]. The author excludes a potential role of sulphur dioxide in the regeneration of phenolic compounds from their phenoxyl radicals as it does not contribute to the total antioxidant potential at the concentrations normally present in red wines [26]. Administering multiple agents can increase the efficacy and potency of the chemopreventive measures taken and reduce toxic side-effects. Other synergistic effects In addition to investigating the synergistic effects of various phytochemicals. the regeneration of quercetin from its phenoxyl radical by the presence of (+)-catechin. Conclusions An increasing number of in vitro and in vivo studies indicate that combinations of dietary chemopreventive agents can result in a significant level of activity being achieved at concentrations at which any single agent is virtually inactive. Use of retinoids in combination with such SERMs (selective estrogen receptor modulators) as tamoxifen or raloxifene [29. without a loss in treatment potency. synergistic effects between these and the other wine constituents may have contributed to the TEAC values. EGCG and sulindac were also found to synergistically enhance apoptosis. de Kok. in addition to the synergistic effects between the different phenolic compounds. This implies that. [7] of using EGCG to induce apoptosis in human lung cells in vitro have been synergistically enhanced by use of cancer preventive agents such as sulindac and tamoxifen. however. This suggests that the regeneration of phenolic compounds from phenoxy radicals is a mechanism that can contribute to the synergistic effects observed. [27] demonstrated. This effect has also been evaluated in an animal study in which treatment of rats with a combination of EGCG and sulindac was found to reduce aberrant crypt formation after their being treated with azoxymethane for colon carcinoma [31]. A review of the effects of various combinations of chemotherapeutic and chemopreventive approaches in dealing with cancer has appeared recently [28].Theo M. it appears . The results confirm earlier findings showing a combination of phytochemicals and therapeutic agents to provide more effective therapy for cancer than the therapeutic agents alone. van Breda 166 indicated 16 to 23% of the antioxidant activity to be synergistic. Simone G. In that study. Jørgensen et al. The fact that many of these phytochemicals act synergistically may why some food items or diets show cancer preventive effects that cannot be explained on the basis of their separate bioactive ingredients alone. Although our understanding of the molecular mechanism behind the observed combinatorial effects of these chemopreventive agents is still limited. studying the combined effects of dietary factors and therapeutic compounds may be a promising approach toward optimising pharmacological strategies for the prevention and therapy of cancer [1. Also. its also reducing side effects.30] are examples of this. particularly those of sulindac.

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Joseph Rafter 170 3. which has been the focus of intense research activity in recent years are probiotics — ’living micro-organisms which upon ingestion in certain numbers exert health benefits beyond inherent general nutrition’ [1. acidophilus to eleven volunteers on a fried meat diet known to increase faecal mutagenicity. Probiotics and prebiotics — potential anticarcinogenic food components Joseph Rafter Department of Medical Nutrition. serum cholesterol reduction. e. The evidence from animal studies provides strongest support for anticancer effects and data from human studies (epidemiology and experimental) are limited. Mortality from CRC is second only to that of lung cancer in men and breast cancer in women and has shown little sign of decreasing in the last 20–30 years. epidemiology and human intervention studies.g. The list of healthful effects attributed to probiotic bacteria is extensive [3] and includes: alleviation of lactose intolerance symptoms. Bifidobacterium spp.2]. although there are some on breast [4] and bladder cancer [5]. alleviating constipation. relieving vaginitis to name but a few.or prebiotics alone. with defined gut survival properties and associated biological activities and which can be ingested in fermented milk products or as a supplement. that can modulate gut microflora and its metabolism. Diet makes an important contribution to CRC risk [6] implying that risks of CRC are potentially reducible. resulted in a lower faecal mutagenic activity after 3 days . anticancer effects. The vast majority of studies on the anticancer effects deal with colorectal cancer (CRC). Lactobacillus spp. Probiotics usually refer to highly selected lactic acid bacteria. the supportive evidence is stronger for probiotics than prebiotics (possibly because the latter have only recently come to prominence) and is recently suggestive that synbiotics are more effective than either pro. animal models. This has led to intense interest in factors. that have the potential to improve host health’) [3] and synbiotics (combinations of pro. Evidence for protective effects of pro.and prebiotics on cancer is derived from in vitro studies.6. Evidence from a wide range of sources supports the view that the colonic microflora is involved in the etiology of CRC. prebiotics (’a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon.. Karolinska Institute. Evaluation of anticarcinogenic dietary effects Evidence from human studies Consumption of lactobacilli by healthy volunteers has been shown to reduce the mutagenicity of urine and faeces associated with the ingestion of carcinogens in cooked meat.and prebiotics). Administration of L. Overall. such as probiotics. Huddinge. and Streptococcus spp. Sweden Introduction An example of a functional food.

Presently however the lack of well validated biomarkers for colon cancer limits the relevance of such studies although a wide range of potential biomarkers of risk are under development. adjusted for potential confounding variables. did not provide evidence that intake of dairy products is associated with a decreased risk of colon cancer [16]. In summary. despite the high fat intake. During L. Thus. the urinary mutagenic activity on days 2 and 3 was significantly lower compared to the ordinary fermented milk period. As yet. there are few epidemiological studies addressing the association between fermented dairy products and colorectal cancer. prebiotics or fermented milk consumption are causally related to reduction in cancer risk. acidophilus administration. in the near future. Consumption of large quantities of dairy products such as yoghurt and fermented milk containing Lactobacillus or Bifidobacterium may be related to a lower incidence of colon cancer [9]. In another case control study. the most profitable approach would be to use combinations of pro. In a cohort study in the Netherlands. There will also be. the 1980–1988 follow-up of the Nurses’ Health Study and the 1986–1990 Health Professionals follow-up study. In most cases. In conclusion. It will be important to define dose and time relationships and it would appear at present. the opportunity to exploit genomics and proteomics in investigations of effects of pro/prebiotics on gene expression and post-transcription . two companion American prospective studies. an inverse association was observed for yoghurt [12] and cultured milk consumption [13]. data from human intervention studies are of paramount importance in providing evidence that probiotics. and other dairy products [10. particularly in urine.11]. It can also be mentioned that an inverse relationship has been demonstrated between the frequency of consumption of yoghurt and other fermented milk products and breast cancer in women [4. although a weak non-significant inverse association with colon cancer was observed [17]. an inverse relationship for yoghurt consumption with risk of large colon adenomas in men and women was reported [14]. In two population-based case-control studies of colon cancer. colon cancer incidence was lower than in other countries because of the high consumption of milk. this is an area of high priority for future studies.and prebiotics. casei on the urinary mutagenicity arising from ingestion of fried ground beef in the human. at-risk groups and patients. yoghurt. from animal studies. it will become possible to perform studies in healthy volunteers. Once such markers are available.Bioactive components in foods: Probiotics and prebiotics — potential anticarcinogenic food components 171 compared to faecal mutagenic activity after 3 days consumption of ordinary fermented milk (although not significant) [7]. it would appear that the case control studies indicate protective effects while the prospective studies do not. an increase in the number of faecal lactobacilli corresponded to a lower mutagen excretion. On the other hand. High levels of mutagenicity also appeared in urine on days 2 and 3 of the fried meat and ordinary fermented milk dietary regimen.15]. it was shown that the intake of fermented dairy products was not significantly associated with colorectal cancer risk in an elderly population with a relatively wide variation in dairy product consumption. Hayatsu and Hayatsu [8] also demonstrated a marked suppressing effect of orally administered L. An epidemiological study performed in Finland demonstrated that.

It is hoped that the results of this study will provide much needed information on the cancer protective effects of synbiotics in humans and supply us with additional valuable information on the underlying mechanisms.be).22] or DMH [23. adolescentis culture and its supernatant did not show an inhibitory effect in this study [20]. induced by azoxymethane (AOM) [20]. End points used are the tumours themselves or early lesions. Oral administration of lactic acid bacteria has been shown to effectively reduce DNA damage. L. Bifidobacterium Bb-12 and Raftilose Synergyl in adenoma patients. double blind. that Lactobacillus acidophilus. Among different cell fractions from L. acidophilus also inhibited the formation of ACF. In recent years. in gastric and colonic mucosa in rats. In this study. induced by chemical carcinogens. there have been many studies. Pool-Zobel et al. which clearly demonstrate a protective effect of dietary supplements of lactic acid bacteria against colon tumour development. the peptidoglycan fraction and whole freeze-dried cells were antigenotoxic. http://www. It involves a twelve-week randomised. such as aberrant crypt foci (ACF).e. Although B.Joseph Rafter 172 events in colonic biopsies and to identify human groups responsive to pro/prebiotic intervention. prevented MNNG-induced DNA damage. while heat-treated L. Certain strains of lactic acid bacteria have also been found to prevent putative preneoplastic lesions or tumours induced by carcinogens. confusus. longum or inulin was associated with a decrease in AOM-induced colonic small ACF in rats and combined administration . including colonic mucosal markers. L. rhamnosus designated GG can interfere with the initiation or early promotional stages of DMH-induced intestinal tumourigenesis and that this effect is most pronounced for animals fed a high-fat diet.syncan. placebo controlled trial of a food supplement containing Lactobacillus GG. casei subsp. acidophilus. [18] reported. Evidence from laboratory animal studies There are several good animal models for colon cancer which have proved useful for identifying dietary factors which may protect us against the development of this tumour. Goldin et al. [19] showed that a specific strain of L. Of relevance here is a clinical trial which is presently ongoing i. to examine the effect of a synbiotic preparation on colon cancer risk biomarkers in humans (SYNCAN project. These bacteria were also protective toward 1.24]. Metabolically active L. gasseri. acidophilus cells. as well as an acetone extract of the culture. Streptococcus thermophilus. Overnight cultures of L.2-dimethylhydrazine (DMH)-induced genotoxicity. funded by EU. longum were antigenotoxic toward N’-nitro-N-nitrosoguanidine (MNNG). using the comet assay. acidophilus was not antigenotoxic. ACF are putative preneoplastic lesions from which adenomas and carcinomas may develop. all of the ”state of the art” colon cancer risk biomarkers. feeding of bifidobacteria suppressed the ACF formation induced by AOM [21. are being measured. using these models. Consumption of B. and involving 8 research centres in Europe. faecal water markers and immunological markers. Bifidobacterium breve and B. It will also be particularly important to use data on mechanisms of action to develop hypothesis based intervention studies in humans.

It has been demonstrated that feeding of fermented milk or cultures containing lactic acid bacteria inhibited the growth of tumour cells injected into mice [32. while a possible protective effect of probiotics was observed. colon and mammary tumours.e. longum administered in the diet to rats inhibited liver. The authors concluded that. More recently. and that the additional colonisation of B. The mechanisms by which they act are less clear. reported that a single subcutaneous injection significantly suppressed tumour growth. There is additional direct evidence for anti-tumour activities of lactic acid bacteria obtained in studies using pre-implanted tumour cells in animal models. which are acid resistant in contrast to most bacteria. which do not survive contact with gastric acid. Ingestion of B. ornithine decarboxylase activity and expression of the ras-p21 oncoprotein.Bioactive components in foods: Probiotics and prebiotics — potential anticarcinogenic food components 173 significantly decreased the incidence of large ACF [25]. Recently. but the data presented suggested that they may act through a combination of mechanisms involving an increase in short-chain fatty acid (SCFA) production. Feeding of fermented milk increased the survival rate of rats with chemically induced colon cancer [29]. enriched with oligofructose. lower proliferative activity and a variation in the expression of some enzymes involved in the pathogenesis of colon cancer. used in this study contained Streptococcus faecalis T-110. Reddy and Rivenson [27] reported that lyophilised cultures of B. In addition. [34] using whole peptidoglycan isolated from B. longum also significantly inhibited AOM-induced cell proliferation. mindful of the fact that the composition and metabolic activities of the intestinal flora of experimental animals are significantly different from those of humans [35]. infantis strain ATCC15697. breve reduced the number of ACF with four or more crypts/focus and crypt multiplicity which are reliable predictors of malignancy [26]. we exploited human flora associated (HFA) mice to test the effects of a probiotic mixture on a parameter of relevance for colon carcinogenesis i. The probiotic mixture. Biothree®. the results from a previous report from our laboratory.5-f)quinoline (IQ). demonstrated that human intestinal microflora had different effects than mouse microflora concerning DNA adduct formation after exposure to mutagens [37]. five intralesional injections resulted in 70% tumour regression in the mice. Sekine et al. there was a report on the antitumourigenic activity of the prebiotic inulin. Dietary administration of a lyophilised culture of B. Clostridium butyricum TO-A and Bacillus mesentericus TO-A.33]. DNA adduct formation [36]. induced by the food mutagen 2-amino-3-methyl-3H-imidazo(4. longum resulted in a significant suppression of colon tumour incidence and tumour multiplicity and also reduced tumour volume induced by AOM in rats [30]. the results indicated that the prebiotic decreased AOM-induced carcinogenesis. acidophilus not only suppressed the incidence of DMH-induced colon carcinogenesis but also increased the latency period in rats. in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis in the AOM-induced colon carcinogenesis rat model [31]. In addition. It has been . it has been reported that colonisation of bacteria with an ability to produce genotoxic compounds and high β-glucuronidase activity enhanced progression of ACF induced by DMH in rats. Indeed. Goldin and Gorbach [28] showed that dietary supplements of L.

difficile and C. the results of this study demonstrated that the above probiotic mixture had an effect to significantly decrease the DNA adduct formation in the colonic epithelium induced by the food mutagen 2-amino-9H-pyrido[2.3-dihydroxyazetidine [39. the use of lactic cultures for human cancer suppression is interesting. Vibrio parahaemolyticus. even with the above reservations in mind and mindful of the limited number of human studies available. It is possible that different strains target different mechanisms. faecalis T-110 and C. binding and degrading potential carcinogens. Biothree® is used as a clinical therapy in Japan. bile acid-metabolizing bacteria). production of antitumourigenic or antimutagenic compounds. botulinum) was inhibited in mixed cultures of S. such mechanisms might include: alteration of the metabolic activities of intestinal microflora. Salmonella typhimurium. given by gavage.40]. AAC). An important goal for the future should be carefully designed human clinical trials to corroborate the wealth of experimental studies. Two possible mechanisms may be involved: reduction of direct exposure to AAC and/or induction of DNA repair of the DNA adducts in the colonic epithelium. as mentioned above. effects on physiology of the host.3-b]indole (2-amino-alpha-carboline. butyricum TO-A [38]. butyricum TO-A showed strong symbiosis with each other and the growth of enteropathogens (enterotoxigenic Escherichia coli. Thus. enhancing the host's immune response. mesentericus TO-A stimulated the growth of Bifidobacterium by producing 3. faecalis T-110 and C. holds promise and certainly deserves more scrutiny. diarrhoea and constipation. It has also been reported that B. alteration of physicochemical conditions in the colon.g. Mechanisms by which probiotic bacteria may be inhibiting colon cancer The precise mechanisms by which lactic acid bacteria may inhibit colon cancer are presently unknown. Interestingly. i.e. C. Conclusion Many healthful effects are attributed to the probiotic bacteria and some of these effects have more scientific support than the anticancer effect. However. All of the mechanisms have various degrees of support. more work needs to be done to identify the specific strains and strain characteristics responsible for specific antitumour effects and the mechanisms by which these effects are mediated. quantitative and/or qualitative alterations in the intestinal microflora incriminated in producing putative carcinogen(s) and promoters (e. mainly originating from in vitro and animal experiments and some of them even have some support from human clinical studies. The strongest evidence for anticancer effects of probiotics comes from animal studies and evidence from human studies (epidemiology and experimental) is still limited.Joseph Rafter 174 reported that S. It is effective for the improvement of symptoms caused by abnormal intestinal flora. Also. . there are several possible mechanisms which might explain how lactic acid bacteria might protect against tumour development in the colon. However.

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Topeliuksenkatu 41 a A. Jagielloƒska 13. 11635 Athens.ki. 20135 Milan. schoketb@okk.Annex ECNIS participants Nofer Institute of Occupational Medicine (NIOM) Poland.fi Vrije Universiteit Brussel (VUB) Belgium.pl Genetics Research Institute & Ospedale Policlinico (GRI) Italy. Neuenheimer Feld 280. Âw.org Deutsches Krebsforsforschungszentrum (DKFZ) Germany. SE-171 77 Stockholm. 00250 Helsinki. david. 91-348 ¸ódê. dan.it The National Hellenic Research Foundation (NHRF) Greece.Nair@dkfz. SE-22100 Lund. University Road. 10133 Turin. 1050 Brussels. Paradisgatan 5C.segerback@biosci. taioli@policlinico.de Finnish Institute of Occupational Health (FIOH) Finland.ac. Norregade 10. Gyáli út 2-6.de University of Copenhagen (UC) Denmark. H-1097 Budapest. ecnis@ecnis.mi.matullo@unito. bjorn. 55099 Mainz. SW7 3RP London.it Johannes Gutenberg-Universität Mainz (JOGU) Germany.ac. Collegium Medicum in Bydgoszcz Poland.ac.hu Nicolaus Copernicus University. DK-1017 Copenhagen.uk . mkirschv@vub.umk.gr University of Leicester (ULEIC) United Kingdom.be Lund University (ULUND) Sweden.se Fondazione ISI (ISI) Italy. Viale Settimo Severo 65. Teresy 8. Ari.dk Karolinska Institutet (KI) Sweden. oesch@uni-mainz.antsz.Hirvonen@ttl. LEI 7 RH Leicester.ku.uk National Institute of Environmental Health (NIEH) Hungary. Pleinlaan 2. 69-120 Heidelberg.phillips@icr. Vassileos Constantinou Avenue 48.se Institute of Cancer Research (ICR) United Kingdom. steffen. Saarstrasse 21. coordinator. Old Brompton Road 123. skyrt@eie.lth. J. giuseppe.akesson@tbiokem.loft@farmakol. 85-067 Bydgoszcz. ryszardo@cm. Strada Della Carita 10. pbf1@leicester.

London. 08907 L'Hospitalet de Llobregat. Barcelona. p. DD1 4HN Dundee roland. 69372 Lyon. polyphenols.de Institut Catala d'Oncologia (ICO) Spain.es Utrecht University.uu. Nethergate. lennart. Oude Markt 13. Lurup 4.uk . Heidelberglaan 8.moller@csb. H.nl Biochemical Institute for Environmental Carcinogens Prof.vineis@imperial.se Imperial College London (ICL) United Kingdom. Gran Via S/N Km27. cagonzalez@ico. 22927 Grosshansdorf. 3000 Leuven.nl University of Dundee (UNIVDUN) United Kingdom.scs. Stawki 2.uk International Agency for Research on Cancer (IARC) France. J. f. (NETIX) Poland. Groot Begijnhof 58-59. 3508 TC Utrecht.casteleyn@lin. Universiteitssingel 50. K.fr NETIX Skrzypczyƒski. Stallarholmen.wolf@cancer.pl Leocordia AB (Leocordia AB) Sweden. Krzysztofowicz Sp. 00-193 Warszawa. Exhibition Road.seidel@biu-grimmer.Kromhout@iras.be Maastricht University (UM) The Netherlands. Cours Albert Thomas 150. Dr Gernot Grimmer Foundation (BIU) Germany. mask@netix.vlaanderen. albrecht. boffetta@iarc.ki. B-3000 Leuven.vanschooten@grat. ludwine. Selagarden.Dietary vitamins. U. Leuven R&D Department.ac. selenium and probiotics: biomarkers of exposure and mechanisms of anticarcinogenic action 180 Katholieke Universiteit Leuven Belgium. Institute of Risk Assessment Science (IRAS-UU) The Netherlands.unimaas.org. 6200 MD Maastricht.

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