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

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

Since this is a vast area. and pool sizes of the substance in question. Current research . on the other. Many compounds contained in the diet have been proposed as having anticarcinogenic properties. Particular attention is directed at identifying dietary factors that modulate environmental and lifestyle factors related to cancer risk. which to a large extent reflects the kinetics of metabolism and transport. some of them mainly reflecting recent dietary intake and others indicating more the individual’s long-term nutritional status. 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. 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. food components and food preparation methods involved. are considered in detail. 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. 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. and bioactive compounds. vitamins and selenium on one hand. Markers differ considerably in their characteristics. that of reviewing the mechanisms of action of some of the most promising anticarcinogenic compounds. The ECNIS project aims at addressing important problems in this field and identifying knowledge gaps that exist. Nutritional biomarkers are essential for studies of the links between dietary intake and the risk of cancer. only certain selected topics. Also. which is a very complex field indeed in view of the large number of cancer diseases. The important links found between use of a substance as a biomarker and its mechanisms of action led to a further aim. pathogenetic mechanisms. as outlined below.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. These compounds are found in foods of many different types. Biomarkers are a key tool for assessing nutritional status and dietary exposure to specific substances. the growth conditions and other factors. especially those of relevance for the partners of the NoE ECNIS and its contacts. although plantbased foods appear to be the richest source. particular progress has been made here recently. The degree to which these compounds are present in plant tissue can vary markedly and be dependent on the plant variety.

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

There is also a need of clarifying the mechanistic role of selenium in the etiology of cancer. whereas there are 13 studies that have reported a null effect. epigallo- . colon). 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. In these studies. 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. Experimental studies have shown that selenium compounds affect cell growth. 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. release of cytochrome c and the activation of caspases. Like other types of dietary chemopreventive agents. methylated selenocompounds having been found to have particularly strong chemoprotective effects. cyclin-dependent kinases and inhibitors. The blocking activities include antioxidant effects and the modulation of drugmetabolising enzymes and multidrug resistant genes. 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. The optimal type and dose of selenium supplements needs to also be defined.Executive summary 7 of it on oxidative DNA damage. mainly through intrinsic pathways involving members of the Bcl family. Both organic and inorganic forms of selenium have been evaluated. Flavonoids can also induce cell cycle arrest by modulating key components of cell cycle regulation. DNA repair and signal transduction. 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. Several large human intervention studies have indicated that selenium supplementation can prevent the development of several different forms of cancer (prostate. The relationship of low selenium status and selenium supplementation to cancer disease is another important segment of focus in the diet-cancer field. primarily glutathione peroxidase. It is also important to determine whether there is an increased risk of certain forms of cancer after selenium supplementation. The suppressing activities include the inhibition of signalling pathways responsible for cell proliferation and survival. Further studies are underway. lung. including cyclins. mitochondrial membrane depolarisation. and the induction of apoptosis. Most studies have involved healthy individuals. the cell cycle. It is important to explore the possibility of using other selenoproteins as biomarkers too. Some recent mechanistic findings on the flavonoids apigenin. use has been made of several biomarkers such as the concentration of total selenium in different body fluids as well as different selenoproteins. flavonoids exhibit a wide range of potentially beneficial activities in terms of cancer prevention.

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

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. This can well contribute to cancer prevention. In some systems. 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 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. evidence from human studies still being limited. The strongest evidence for the anticancer effects these can have comes from animal studies. combinations of green tea flavonoids are more active than single compounds of this sort. The development of ideas regarding this has also been stimulated by findings that dietary supplements of only a single compound may have negative effects. one can regard the use of lactic cultures for human cancer suppression as interesting. it appears that many combinations of complementary modes of action may be involved. promising and as deserving of closer scrutiny.Executive summary 9 being activated. 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. Many healthful diet effects are attributed to probiotic bacteria. growth arrest or apoptosis is to occur are determined. whereas at higher doses cell cycle arrest and apoptosis occur. An understanding of the mechanisms involved is a necessary basis for the development and validation of new biomarkers of nutritional status. 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. The synergistic effects of dietary phytochemicals need to be investigated further. It is presently unclear how these different types of responses are coordinated by the cell and how matters of whether adaptation. Even with the above reservations and the limited number of human studies available in mind. More work needs to be done to identify the specific strains and strain characteristics responsible for particular antitumour effects and the mediating mechanisms involved. Identification of the mechanisms that control such outcomes would be very useful. and green tea flavonoids can also show increased activity when present together with other phytochemicals. 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. As has been indicated here. Although our understanding of the molecular mechanisms behind the observed combinational effects is still limited. Several possible mechanisms may explain how lactic acid bacteria can protect against tumour development in the colon. 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. Much hope is attached in this connection to the powerful techniques available today within the field of nutritional genomics.

.

Lund University and Department of Clinical Nutrition.1. Introductory overview and future prospects Björn Åkesson Biomedical Nutrition. Various properties of biomarkers are summarised in Table 1. This is a very complex field in view of the large number of different types of cancer. A further aim is to examine and explore the mechanisms responsible for the action for different promising anticarcinogenic compounds. A brief overview of food components that appear to have an anticarcinogenic effect is provided in Appendix 1. The ECNIS project aims at addressing important problems in this field and identifying various knowledge gaps. 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. Pure and Applied Biochemistry. 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.g. Lund. their physiological basis and the use of biomarkers in studies of different types.1.1. Introduction 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. Table 1.2. the pathogenetic mechanisms involved. that role that various food components and food preparation methods play in connection with this. Sweden Dietary assessment methodology and the development and validation of biomarkers of nutritional status are key scientific fields within nutritional science. 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. . Some major issues regarding nutritional biomarkers Markers of dietary intake vs.1. Lund University Hospital. A number of textbooks and reviews are available within the field of nutritional biomarkers [see e. 1–6] dealing with the basic theories and methodology.

and they may not respond to overexposure of nutrients at all. A large number of confounding factors such as hormones. Also proteins with a transport function as well as other functions are used as biomarkers. and inflammatory and other diseases can influence the levels of different biomarkers [3]. for example zinc and copper to superoxide dismutase activity and selenium to the activity of glutathione peroxidase and other selenoproteins. 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. variations in metabolism or in excretory pathways. such as the analysis of cobalamine and folate in plasma. trace elements are coupled to the levels of enzyme activities. gender and gene polymorphism (as outlined below). plasma. since the erythrocyte cells have a lifetime of about 120 days [3]. In addition. The selection of the type of sampling to carry out depends very much on the components to be studied and the sampling facilities available. 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. interactions between dietary or endogenous components or xenobiotics. The amount of a substance occurring in the erythrocytes. One such example is olive oil discussed in this volume by Sotiroudis and Kyrtopoulos. Biomarkers show temporal variation. different nutrient-dependent physiological tests can be employed [2]. Various diet-related compounds in the plasma also differ considerably in turnover times. the main function of some markers being that of serving as indicators of malnutrition. 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. 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]. whereas others are performed only in a few specialised research laboratories. diurnal variations. are in routine daily use in clinical laboratories. the gut flora. The level of a biomarker often varies as well with age. . urine or hair. Also. [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. such as the retinol-binding protein and ceruloplasmin. often reflects much more the long-term intake if it than the plasma level does.Björn Åkesson 12 Dietary components as biomarkers Many biomarkers involve the measurement of a nutrient or some other dietary component in the blood. 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. Some methods.

12]. 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]. Their use may increase. These matters are outside the scope of the present review. For this reason. Also. An overview of this matter is provided in [7]. 2) help establish causality of food and nutrient associations in epidemiology studies. buccal mucosa. Folate has served as a prototype here for demonstrating how genetic make-up can influence the nutrient status of an individual. 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. 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. A number of statistical methods for the correction and evaluation of such data are available [8]. The assessment of dietary intake in epidemiological studies has been a major use of biochemical markers of nutritional status. it should be borne in mind that there are very few (if any) “ideal” biomarkers that have been validated for studies of different types. although epidemiological findings on the association between the selenium level and cancer are reviewed by Gromadziƒska et al. 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. or for the calibration of the dietary assessment methods [8]. 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. adipose tissue or biopies from other organs have been developed. together with an assessment of dietary intake by a food frequency questionnaire or by some other methods. methods for measuring nutrients or nutrient-dependent variables in cells in the blood (mainly leucocytes). information on the interactions between dietary intake and gene polymorphisms “can serve to 1) define susceptible subpopulations. Some of these samplings require much skill and effort and can only be used in small studies. faeces. Biomarkers in studies of that type have been used either alone. 3) aid in distinguishing causal components of complex dietary mixtures. thus strengthening dietary associations. 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. This topic is only taken up briefly in the present volume. urine. however.Introductory overview and future prospects 13 Although several of the biomarkers mentioned here are widely used. . There are particularly good reasons for using the 25-hydroxy vitamin D as a biomarker in extended studies in the future. as exemplified in the present volume by Linseisen and his colleagues in connection with vitamin D and polyphenols. The two contributions mentioned also illustrate the difficulty in defining valid cut-off points.

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

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.

3. Lund University. but plant-based foods appear to be their richest source. Plant tissues can also vary enormously in the store of such compounds. a list of putative anticarcinogenic food components has been generated (Table 1. This information was collected with use of a PubMed search conducted in 2005. The criteria used for the selection were their relevance as adjudged from their title.2. for example. and avoiding of similar articles by any given author. 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. Lund. Table 1. emphasis on recent reviews. Overview of possible anticarcinogenic food components Per Mercke Biomedical Nutrition. 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. Sweden There is a vast range of dietary compounds proclaimed to have anti-carcinogenic properties as investigated by the scientific community. Different plant families harbor different mixtures of compounds of this sort.2. depending on the plant variety and the growth conditions.). 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.Per Mercke 18 Appendix 1. 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. . These protective compounds are found basically in all types of food.

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

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

sis: potential role of lipid peroxidation. Farmer PB. Owen RW Exocyclic DNA adducts as oxidative stress markers in colon carcinogene. J Cell Biochem 1996. Bartsch et al. cola. Manson MM. 8. J Nutr 2003. Sanderson P. Pannemans D. [2]. Br J Nutr 2004. Martel P. Rafter JJ. [8] Other non-flavonoid phenolics Lignans Olive oil Adlercreutz [31]. Rowland IR. strawberries. Br J Nutr 2002. Greenwald P. 11. Verhagen H. black-berries.25 Suppl:29–36. Branca F. Rafter et al. et al. Ferguson LR. Recent Results Cancer Res 2005. Diet and cancer: assessing the risk.166:257–75. Br J Nutr 2002. Gescher A. 4. Pool-Zobel B. et al. Downes CS. walnuts. cacao Manson [18] Reference Monoterpenes Citrus fruits (peel) Ferguson [3]. Mutat Res 1999. Steward WP. 5. Rechkemmer G. Pool-Zobel B. Turmeric Branca et al. 3. pecans. Cereals. olive oil References (selected from the PubMed search described above) 1. Kelloff et al. Scientific basis of biomarkers and benefits of functional foods for reduction of disease risk: cancer. Powers HJ. Prospects for cancer prevention. Hematol Oncol Clin North Am 2002. PASSCLAIM — dietrelated cancer.133 Suppl 1:3820S–6S. Rafter J. Wiseman [33] Grapes. [16]. Incorporating basic nutrition science into health interventions for cancer prevention. Innovative agents in cancer prevention.86 Suppl 1:S55–S92. Hanley AB. 6.91:315–23.383:915–21. coffee. Govers M. 10.Overview of possible anticarcinogenic food components 21 Table 1. Manson [18]. Nair J. McGlynn AP. 2. Emerging dietrelated surrogate end points for colorectal cancer: UK Food Standards Agency diet and colonic health workshop report. Cancer risk factors for selecting cohorts for large-scale chemoprevention trials. 9. An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected — cont. Br J Nutr 2001. Johnson IT.88 Suppl 1:S73–87. Turini ME.16:811–40.88 Suppl 2:S219–24. [1]. raspberries. Eur J Nutr 2004.428:329–38. Primary prevention: phytoprevention and chemoprevention of colorectal cancer.3. Class of compound Methylxanthines Example of bioactive substance Caffeine Theophylline Theobromine Limonene Perillyl alcohol Geraniol Menthol Carvone Hydroxytyrosol Curcumin Resveratrol Food source Tea. Mathers JC. Biol Chem 2002. Milner JA.43 Suppl 2:II47–84. Gill CI. dietary fat and antioxidants. . 7. Biomarkers in disease and health. DuBois RN. Bartsch H.

Wilson L. Malone WA. Steele VE. Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis.134 Suppl:1640S–5S.4:1–4. . Anderson DE. J Nutr 2004. Isothiocyanates in cancer prevention. Trends Mol Med 2003. A critical assessment of some biomarker approaches linked with dietary intake. Br J Nutr 2001. 15. Poulsen HE. Breast Cancer Res. et al. Follen M. Mayne ST. Mason JB.90:487–502. Urol Clin North Am 2002. Bingham SA. et al. Andersen R. 21.133 Suppl 3:933S–40S. Exp Biol Med (Maywood) 2002. 24. Nutr Cancer 2003. Braesco V. 29.86 Suppl 1:S37–53. Br J Nutr 2003. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. Maiani G. Alink G. Olmedilla B. Kelloff GJ. Vainio H. Cancer prevention — the potential for diet to modulate molecular signalling. 17. Schottenfeld D. Srivastava S. 14. et al. 22. Nutritional and clinical relevance of lutein in human health. 16. Sharifi R. 30.36:655–67. Wagner KH. Granado F. Chen L. Siddiqui IA. Loft S.48:169–88. Phytoestrogens and breast cancer.555:173–90. Mukhtar H. Br J Nutr 2001.47:13–23. J Nutr 2003.130 Suppl:467S–71S. Biomarkers and their use in cervical cancer chemoprevention. 20.Per Mercke 22 12. Woods JA.46:261–73. Mutat Res 2004. Bowen P. 25. Crozier A.86 Suppl 1:S5–35. Biomarkers of nutrient exposure and status in one-carbon (methyl) metabolism. Saleem M. 19. Sharma RA. J Steroid Biochem Mol Biol 2002. Blanco I. 32. 2002. Clin Cancer Res 2004. Vitamin D and prostate cancer. Konety BR. Ghosh L.227:886–93. The antioxidant conundrum in cancer. Crews H. 31. Drug Metab Rev 2004. 13. Stacewicz-Sapuntzakis M. Milner JA. 23. Gamma-tocopherol — an underestimated vitamin? Ann Nutr Metab 2004.63:4295–8. Kamal-Eldin A. Tea beverage in chemoprevention of prostate cancer: a mini-review. O'Brien NM. Andersson C.10:4901–12. Cancer Res 2003. J Nutr 2000. Adlercreutz H. Biological relevance of adduct detection to the chemoprevention of cancer. Elmadfa I. 28. Crowell JA. Maruvada P. Mammographic breast density as a biomarker of effects of isoflavones on the female breast. Plant-derived phenolic antioxidants. Boone CW. 26. Bianchini F. Seifried HE. Holst B. Farmer PB. Crit Rev Oncol Hematol 2003. 18.83:113–8. J Mol Med 1996. Progress in cancer chemoprevention: development of diet-derived chemopreventive agents.74:297–312. Curr Opin Clin Nutr Metab Care 2000. Duncan C.133 Suppl 3:941S–7S. Lubet RA. 27. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. J Nutr 2003. Atkinson C. Getzenberg RH.3:447–51. Biomarkers for cancer diagnosis: implications for nutritional research.29:95–106. A critical evaluation of the application of biomarkers in epidemiological studies on diet and health. Duthie G. Zhang Y. Wild CP. Vlastos AT. Greenwald P. Cancer risk and oxidative DNA damage in man. Manson MM. Adhami VM.9:11–8. McDonald SS.

Patterson R. Bibl Nutr Dieta 2001.29:115–24. Lifestyle/dietary supplement partial androgen suppression and/or estrogen manipulation. Research strategies and the use of nutrient biomarkers in studies of diet and chronic disease. Quirke P. Kyrtopoulos SA.11:181–8. Viner JL. Kampman E. Who's afraid of n-6 polyunsaturated fatty acids? Methodological considerations for assessing whether they are harmful. Epidemiology and prevention of colorectal cancer. Hollman PC. Moyad MA. Sugar E. Tahir A. 41. 34. Adv Exp Med Biol 2001. 45. observational versus experimental human studies. Bell SM.73:70–8. 37. Eur J Cancer Prev 1997. Methylation and colorectal cancer. Vineis P. Expert Opin Investig Drugs 2000. Hawk ET.20:465–73. 43. Biomarkers. Christie R. 39. J Pathol 2001. McCall MR. Am J Hosp Palliat Care 2003. Moyad MA. Georgiadis P. Public Health Nutr 2002. Sarhill N. 42. Assessment of nutritional status and fluid deficits in advanced cancer. Molecular epidemiological approaches to the study of the genotoxic effects of urban air pollution. Establishing the significance and optimal intake of dietary antioxidants: the biomarker concept. Antioxidant vitamins: evidence from biomarkers in humans.9:1829–40. Int J Vitam Nutr Res 2003. The therapeutic potential of phytoestrogens. 36. Rivlin RS. Nutr Rev 1999. Halliwell B. Future directions. Am J Clin Nutr 1997. Berry EM.492:255–62. . Jubb AM.5:977–84. 38.82:905–41. Carroll PR. 40. Prentice RL. Limburg PJ. Plant foods versus compounds in carcinogenesis. 47. Frei B. Kaaks RJ. Wiseman H. 35. Nutrition and cancer prevention: new insights into the role of phytochemicals. low-dose carcinogenesis and dietary exposures.195:111–34. Mahmoud FA. 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.31:289–300. part I: time to redirect our attention? Urol Clin North Am 2004.57:104–13. Mutat Res 1999. Wang CY. 46. Ocke MC.55:46–67. A novel PSA reducer and preventive/treatment option for prostate cancer? Urol Clin North Am 2002. Lifestyle recommendations to prevent prostate cancer.428:91–8. 44. Nutr Metab Cardiovasc Dis 2001.Overview of possible anticarcinogenic food components 23 33.6:147–51. Arts IC. Surg Clin North Am 2002. Neuhouser M.65 Suppl 4:1240S–5S.

.

1. The possibility of using dried blood spots. menadione and phylloquinones. tocopherols and tocotrienols. the development of fast and simple methods for the determination of vitamin A status has long been given a high priority. The structure of vitamin A and pro-vitamin A carotenoids is shown in Figure 2. the retinol binding protein (RBP) [1. 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. 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. such as pro-vitamin A carotenoids. 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]. has also been demonstrated [1].1. Vitamins and selenium 2. This chapter is concerned with such markers.2. which is advantageous from a collection standpoint. 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. Denmark Since antioxidant vitamins can affect an organism’s capacity for defence against reactive oxygen species.2]. except for markers of DNA damage. 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. a technique that was later superseded by various chromatographic and fluorescent techniques. . although reversed-phase methods able to separate certain of the retinol isomers have been published [8]. Due to worldwide concern for vitamin A deficiency (VAD). Dragsted University of Copenhagen. The direct-phase methods can also typically measure a large number of other lipid-soluble vitamin isomers in the same run. With the advent of high performance liquid chromatography (HPLC). Biomarkers of exposure to and effects of vitamins A.4] or reversed-phase [5–7] liquid chromatography . which are dealt with elsewhere in this volume. xanthophylls. these techniques took over and today retinol can be determined in serum routinely by direct. C and E Lars O.[3. biological markers of the dietary exposure to these vitamins is of importance.

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].

Postcolumn derivatization can be used to reduce dihydroascorbate so that it can be determined by use of an electrochemical detector. Dehydroascorbate is not readily determined by use of this approach. Results in determining plasma ascorbate in this way correlate well with those obtained by use of chromatographic methods [18]. with ascorbate and dihydroascorbate leading to the formation of a chromophore or a fluorophore. A method for the simultaneous detection of ascorbate and uric acid by means of capillary zone electrophoresis (CZE) has also been described [29]. since treatment with dithiotreitol is known to reduce dehydroascorbate to ascorbate. its true concentration seems to be very low. If dehydroascorbate is physiologically present. Fig. around 0. the ascorbyl radical and dihydroascorbate. creating greater sensitivity with retention of speed. 2. can be determined simultaneously [25. but a few of them use assay blanks produced by adding ascorbate oxidase to the sample. There was a significant increase in the concentration of dehydroascorbate over time at low total vitamin C concentrations [23]. which can be photometrically measured by use of manual or automated equipment. The colorimetric and fluorometric methods are generally based on redox-reactions.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. and about 1.2. 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]. Some of these methodologies are very fast. allowing high sample handling rates to be achieved through automation [25]. possibly reflecting higher leakage of haem in this group. Recovery is better than 98% with .8% under the same conditions in the case of smokers [23].27–28]. Most of these methods are quite unspecific [24].1% of the total plasma vitamin C in non-smokers when the sample has been handled carefully. Structures of ascorbate. this procedure cannot be recommended if both compounds are to be determined separately. together constituting vitamin C. the stereoisomer of ascorbate. C and E 29 However. erythorbate. The HPLC methods for detecting plasma ascorbate electrochemically give results similar to those using UV detection [26].

. Table 2. The performance of different methods for the measurement of vitamin C in plasma is summarized in Table 2.Lars O. with or without calibration.3. whereas the intralaboratory variation was about 2 µmol/l. 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. which led to relatively larger relative errors being registered at low concentrations [21]. ex vivo LDL oxidation.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.3. In another study an interlaboratory difference of about 15% was observed. since it may partially measure aldehydes deriving from endogenous metabolism. The interday coefficient of variation (CV) for TBARS with use of HPLC is in the order of 10–20 %. In interlaboratory comparisons. This marker is highly controversial since it is variable both within and between laboratories. In a European study of laboratories carrying out population surveillance. 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. The most commonly used marker is determination of TBARS. The product can be determined spectrophotometrically. following HPLC separation. and since there is no generally accepted assay procedure [31].5 mg/l 3 mg/l Speed (samples/day] 40–50 40–50 500 Reference [36] [37] [38. 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]. These flaws have caused some journals to generally regard the method as being invalid as a lipid oxidation biomarker [32]. The assays employed to this end here are the following: plasma thiobarbituric acid reactive compounds (TBARS). irrespective of the concentration. CV% AA interday < 4% 1. and plasma or urinary isoprostanes. plasma lipid hydroperoxides.1 mg/l 0.3% < 5% CV% DHAA interday < 6% ND ND Limit of detection 0. Dragsted 30 use of the HPLC and CZE methods and good linearity is obtained even at low ascorbate concentrations. also within the same laboratory. depending on the method employed for hydrolysis. Typical performance of different methods for determination of plasma vitamin C Test method Reversed phase HPLC Capillary electrophoresis Automated colorimetry ND — not determined. to detect malondialdehyde. antioxidant capacity markers. quite disparate results have been obtained with use of these techniques. either directly or online. Only randomised study designs are included in this review.

given for 3 months. vitamin C plus 182 mg/d dl-α-tocopherol. 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. No differences between groups in plasma malondialdehyde concentrations were observed either before or after supplementation [34]. 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]. 182 mg/d dl-α-tocopherol alone. as compared with placebo treatment. acute doses. 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. 30 mg beta-carotene and 100 µg organic selenium.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. 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]. the intake of 500 mg/d of vitamin C was found not to affect MDA as determined by HPLC [37]. 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. but MDA to be unaffected by a slow-release formulation as compared with placebo treatment [33]. In addition. No effect of either treatment on plasma MDA was observed [35]. or a placebo in a parallel design. Another controversial marker used by many laboratories is the ex vivo LDL oxidation assay. randomised double-blind crossover study the effect of vitamin C supplementation (six weeks. The interday CV for this latter assay is less than 10%. 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. 25 males were exposed to vitamin C (500 or 1000 mg/d) and/or to exercise. in which 19 smokers participated for 4 weeks following a 2-week . 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. In another. in which isolated LDL is exposed to copper chloride or to a semistable radical such as AAPH. The lag-time to oxidation and/or the slope of the oxidation curve are used as end points. no change in whole plasma ex vivo oxidation was observed in this group [44]. In a third study of this sort. In a smaller parallel study of vitamin C supplementation (1 g/d) versus placebo. given in a normal formulation. Oxidative stress induced by Zn deficiency was found to respond to 250 mg/d vitamin C. In a larger study involving 56 smokers. 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]. In another counterbalanced design. The method is disputed because the outcome depends on the antioxidants present in the LDL and these may be lost during LDL isolation. 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]. the oxidative formation of conjugated dienes being followed spectrophotometrically at 234 nm [42]. 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].

decreased LDL oxidation or antibodies to oxidised LDL [47]. In a study without a control group. A borderline effect on LDL oxidation was observed in a similar study with only 18 participants after a shorter period of 12 weeks [48]. They are all ex vivo oxidation systems composed of some oxidising system and some relatively simple marker of plasma oxidation.59]. 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]. 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. receiving only 60 mg vitamin C plus iron per day. Dragsted 32 ascorbate depletion period (≤ 30 mg/d). 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]. usually one leading to a change in visual or UV absorption of the test matrix [52–55]. no such increase was observed despite changes in plasma ascorbate [50]. 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. in which 0 mg. However. or placebo. Overall there seems to be no consistent effect of vitamin C supplementation on ex vivo LDL oxidation kinetics. 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].Lars O. there was a significant increase in ex vivo LDL oxidation lagtime in the vitamin-supplemented group [45]. the availability of a photometer with exact filters or one equipped with a narrow grid being required. These methods generally have interday CVs of less than 10% for repeated measurements of the same sample. 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]. In another group. especially for whole plasma. A variety of antioxidant capacity markers exist for other blood compartments. in a cross-over intervention study involving 48 non-smoking men and women. 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). The oxidizability of LDL is inherently an antioxidant capacity assay for this particular compartment. 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]. At the same time. 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. no effect was observed after 8 weeks supplementation by 1 g/d of vitamin C [46]. the size of the CVs depending on the exact wavelength used for determining the absorbance. 60 mg or 6 g of vitamin C was administered daily during 14 day-periods separated . There are important differences between the methods which call for caution when they are interpreted [56].

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.

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 fluoresceinamine adduct could be detected by HPLC through the use of UV. The level of protein carbonyls in the immunoglobulins was found to decrease after both 10 and 15 weeks of supplementation. fluorescence or APCI-MS detection [86]. The main disadvantage is the longer time required for analysis and the higher technical demands.Lars O. the observed effect of vitamin C on this specific marker may reflect enzyme leakage from the cartilage rather than prooxidative stress. involving seven divers. including 150 mg/d vitamin C. Vitamin C was found to decrease the exercise-induced increase in protein carbonyls in a dose-dependent manner [83]. Following protein isolation by a rapid sizeexclusion chromatographic step and complete acid hydrolysis. whereas no change in the total plasma sulfhydryls was detected [85]. 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. No effect of the supplement on protein carbonyls was observed [38]. Dragsted 36 To assess oxidative stress by use of the simplest version of this assay. unpublished data). . Another line of evidence comes from studies of dietary fruit and vegetable depletion. 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. 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. indicating that the depletion of ascorbate might have an antioxidant effect. The decrease was confined to individuals with a suboptimal intake of vitamin C. 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. No effect of fruit. whereas no change was observed after supplementation of the known nutrients from the fruits and vegetables. 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]. 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. In a very small study. 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]. A marginal increase in plasma protein AAS was observed (Dragsted. Using the same method for protein carbonyls. None of the volunteers had suboptimal vitamin C levels before intervention. 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.

that of the formation of plasma isoprostanes. serum or erythrocyte concentration of d-α-tocopherol. including the susceptibility of erythrocytes . gamma. and possibly possessing higher accuracy. however. HPLC has the advantage of having lower detection limits.and delta-tocopherols as well as the tocotrienols. Since only the four R-forms of α-tocopherol (d-α-tocopherol) are recognised by the human vitamin E transporter in the body. 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.3. This is still controversial. Vitamin E Biomarkers for vitamin E in body fluids Vitamin E is a collective term for alpha-. γ. Various functional tests have also been suggested. permitting simultaneous determination of ascorbate and dehydroascorbate. The reference for the international unit (IU) is 1 mg of all-rac-α-tocopheryl acetate. 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. none of the four S-forms of α-tocopherol and none of β-. of which RRR-α-tocopherol has the highest vitamin E activity. Clear short-term pro-oxidant effects on lipid oxidation were observed following high-dose vitamin C infusion into the bloodstream. vitamin C and chronic disease. no difference in the total vitamin E content of LDL was observed [87].and δ-tocopherols or of the tocotrienols are thought to have any vitamin E activity in humans as opposed to the rat. Conclusions Total plasma vitamin C can readily be determined by automated colorimetric assays or HPLC. The relative vitamin E activity of the various tocopherols and tocotrienols in the rat together with their structures are shown in Figure 2. 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. 1 IU corresponding to 1. beta-.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. Evidence for the prevention of stress-induced carbonyl formation by increasing the intake of vitamin C is controversial and needs further confirmation. 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. The vitamin E status in rats and possibly in other rodents can be determined as a function of all the active isomers. The handling and storage of plasma for vitamin C determination has a strong effect on accuracy.49 mg RRR-α-tocopherol. 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. There are no studies currently available on the relationship between lipid or protein oxidation markers. but in humans vitamin E status is preferably determined as the plasma. whereas the colorimetric assays have much higher throughput.

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

In another study. In a subsequent study of 24 diabetic children by use of the same design. 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. In a double-blind. . 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]. 30 mg beta-carotene and 100 µg organic selenium. In a trial involving 80 men showing an increase in plasma lipid oxidation from exposure to either olive oil or menhaden oil. In a trial of lipid oxidation induced in 18 untrained men by three sessions of resistance exercises. In a randomised 2-months intervention study of 59 healthy smoking males. In another study.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. no effect on the plasma MDA was detected [38]. no effect on exerciseinduced oxidative stress could be shown by serum MDA [109]. 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 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. there was a significant decrease in erythrocyte MDA in the vitamin-supplemented group [102]. a reduction in plasma MDA occurred in the group assigned vitamin supplements [104]. 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. antioxidant capacity markers. 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). controlled intervention study. a decrease in serum MDA was found in the group given vitamin E [108]. a significant decrease in plasma MDA was found in the vitamin-supplemented group as determined by HPLC [101]. or isoprostanes (see desciption in the section on vitamin C above). giving a 900 IU dose of vitamin E was found to be no better than a placebo in decreasing MDA [106]. but to be unaffected by a slow-release formulation as compared with placebo treatment [33]. given in a normal formulation. 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. In a three parallel groups of 16 middle-aged male and female participants. MDA was found to increase significantly following daily doses of 200 mg vitamin E combined with 250 mg ascorbate. 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]. C and E 39 Vitamin E and markers of lipid oxidation This review concerns only randomised. 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. ex vivo LDL oxidation. lipid hydroperoxides. who were given either 500 mg/d of vitamin C together with 182 mg/d dl-α-tocopherol. No change in plasma MDA either within or between the two groups could be shown [110]. 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].

. In 49 diabetic patients given 504 mg/d d-α-tocopherol or a placebo for 6 months. 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]. LDL-dienes. 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. or LDL-hydroperoxides induced ex vivo [87]. the effects of giving mixed supplements of 200 mg/d vitamin E. Non-induced lipoprotein oxidation ex vivo was found to be delayed in the group given the vitamin supplement. including IL-6 [115]. a 1000 IU/d vitamin E supplement for a 10 d period was found to reduce the exhalation of breath pentane [91]. who were given 800 IU/d vitamin E or a placebo for 2 months prior to the race. In a trial involving 80 men who had an increase in plasma lipid oxidation following exposure to olive oil or to menhaden oil.Lars O. In a double-blind controlled intervention study of 56 patients with congestive heart failure. In a study without a parallel control group. A significant change in these groups in whole plasma ex vivo oxidation at 36 weeks was likewise observed [44]. 182 mg/d dl-α-tocopherol alone. 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. this together with 182 mg/d dl-α-tocopherol. vitamin E given at doses higher than 200 IU/d was found to affect the kinetics of ex vivo oxidation of LDL [113]. 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]. and the strength of this effect was correlated with the level of plasma α-tocopherol [114]. 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 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. or a placebo in a parallel design. 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]. no difference was observed in LDL-MDA. In a group of 45 randomised healthy males and females. no effect on plasma antioxidant capacity was observed at 12 or at 36 months [44]. the treatment with vitamin E was found to significantly increase the level of plasma F2-isoprostanes as well as of several markers of inflammation. 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. 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]. or placebo. In a dose-response study of 40 healthy men assigned doses of 60–1200 IU of vitamin E for an 8-week period. Dragsted 40 182 mg/d dl-α-tocopherol alone. In a double-blind controlled intervention study of 56 patients with congestive heart failure. In a blinded intervention study of 33 triathletes participating in a world championship.

particularly in the males. 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]. 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 43 hypercholesterolemic patients randomised to taking simvastatin. Running as such was found to increase the plasma isoprostane level. No other significant differences were observed between the two groups [116]. In another double-blind placebo-controlled trial with a crossover design. which may have caused the difference. 10 of them were given 500 mg/d and 10 of them 1000 mg/d vitamin E for a period of 3 weeks. vitamin C (500 mg/d) together with d-α-tocopherol (290 mg/d) and d-γ-tocopherol (130 mg/d). The study design could not control for period effects. In a double-blind controlled intervention study of 56 congestive heart failure patients. 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]. no effect of either vitamin treatment was observed. The inflammatory markers were also affected by running. 600 or 1200 IU/d of vitamin E for 3 weeks. In a dose-response study. 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. or a placebo during the 6 weeks prior to the race.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. 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. groups of 5 healthy individuals received either 0. 1200 or 2000 IU/d of vitamin E for a period of 8 weeks. but this was not changed by the vitamin supplements. 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]. 300. 21 ultramarathon runners were given either a combination of 300 mg/d d-α-tocopherol and 1000 mg/d vitamin C. 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]. No effect on urinary excretion of F2-isoprostanes was observed [121]. the adding of vitamin E was found to have no further effect on the urinary excretion of F2-isoprostanes [122]. the rest being given a placebo. In a small non-blind study of cirrhotic patients. followed by 8 weeks of washout. . In a third study of the effects of vitamin E on oxidative damage induced by exercise. or a placebo during a 28 d period on the plasma isoprostane concentrations induced by exercise. No significant change in the plasma F2-isoprostane level could be detected by GC-MS at any time point. This was not observed for the young men. C and E 41 In another study. In an intervention study of 46 healthy smokers given 0. 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]. 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). 800. In a study without a control group. 400. In a group of 33 sclerosis patients. simvastatin together with 600 mg/d vitamin E or a placebo for 2 months. 200.

and beta-carotenes) in human plasma by isocratic liquid chromatography. chemistry. Vitamin E intervention has not been shown to decrease the oxidation of plasma proteins.654:129–33. There is only limited evidence for a protective effect of vitamin E supplements at levels of 200–2000 IU/d on markers of lipid oxidation. Goodman DS. A new. Futterman S. Cancer Lett 2000. editors. alpha. The retinoids: biology. Bui MH. Simple determination of retinol.131:1626S–30S.Lars O. but this parameter was not assessed in the other study [126. Olson JA. Furr HC. Craft NE. Roberts AB. Conclusions In conclusion.127]. alpha-tocopherol and carotenoids (lutein. 2nd ed. 5. In: Sporn MB. J Nutr 2001. Daneshvar B. these proposed functional tests for vitamin E must be regarded as obsolete. and supposed effects of high levels of vitamin E supplementation either on exerciseinduced lipid oxidation as determined by isoprostanes or on inflammatory markers are controversial. Dragsted 42 Effects on protein oxidation In a study without a control group. Breinholt V. Kalina RE.Appl 1994. In one study. p.14:125–30. rapid fluorometric determination of retinol in serum. 4. No evidence was presented that this effect was related to antioxidation. 2. Swanson D. Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat. little effect of taking 600–800 mg/d of vitamin E for 2–3 months was shown. Invest Ophthalmol 1975. a significant increase in the binding ratio to the zona pellucida of the unfertilized oocyte in a competitive binding assay was observed. 179–209. Other markers related to vitamin E effect Intervention trials involving ingestion of synthetic vitamin E either alone or in combination with vitamin C have been performed to assess their effects on various conditions assumed to be caused by oxidative stress. References 1. Lauridsen ST. the plasma carbonyl content in 15 healthy individuals was found to be unaffected by 400 IU/d of vitamin E for a period of 2 months [125]. Sperm counts and sperm motility are thought to be partially influenced by oxidative stress. Innovative approaches to vitamin A assessment. present day HPLC and GC-MS methodology has high precision and accuracy in the detection of vitamin E analogues in plasma. In two randomised studies. all-translycopene. New York: Raven Press Ltd. 3. J Chromatogr B Biomed. and medicine. Analytical methods. . 1994. Jakobsen J. Since plasma vitamin E levels are not always correlated with the stability of the erythrocyte membrane or with the exhalation of breath pentane.154:201–10. Bawa AB. Vitamin E deficiency is also known to cause semen abnormalities and infertility in rats. each with 30 men.

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

. proliferating stem cells have to be affected. Again. An elevated level may be due to some well-established characteristic of the tumour. Although studies that indicate this support the hypothesis that oxidative DNA damage can be an important risk factor for carcinogenesis. This raises a number of different issues: 1. Numerous studies have been concerned with the relationship between the level of oxidative DNA damage and cancer (see [5] for a review). together with the increase in DNA damage that occurs. the levels of damage having no causal role in carcinogenesis. 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 0.3–4. the analytical procedures in current use are unable to quantify the lesion levels found in the most important ‘target’ cells. creates a selection pressure for the malignant phenotype seen in the case of cancer. Issues of this sort have to be addressed before a causal link between oxidative DNA damage and cancer can be established. 3. This. 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. Marcus S.2 lesions per 106 unaltered guanines and that reports of values outside this range should be viewed skeptically [6]. although their suitability for this has yet to be verified in prospective studies of cancer risk. Ryszard Oliƒski 52 of a potential disease endpoint such as that of cancer. Since tissue samples from both tumours and normal cells represent a heterogeneous mixture of differentiated and undifferentiated cells (the former being likely to predominate). 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]. Cook. Oxidative DNA damage may be an epiphenomenon in relation to the ongoing pathophysiological process. Peter Møller. there are serious problems in the chromatographic measurement of 8-oxodG. Elevated ROS levels may activate transcription factors and the corresponding genes being permanently activated. In addition. 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.Steffen Loft. Rafa∏ Ró˝alski. but also the damage must be within a coding region of the DNA. Ultimately. regarding the question of ‘cause or consequence’. 2. such as a high level either of metabolism or of cell turnover. At the same time. connected with the spurious oxidation of DNA during sample preparation. 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. not only must the DNA of the target cells be affected. it has been argued that the mere presence of 8oxodG in DNA is unlikely to be a necessary and sufficient condition for tumour formation. In order for a mutation to come about. or (ii) there being reduced DNA repair [5]. 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. For DNA mutations to arise through oxidative damage.

therefore. large-scale intervention studies have failed to demonstrate use of antioxidant vitamins to reduce the risk of cancer [9]. that DNA damage. this soon being followed by the performance of antioxidant trials in which urinary 8-oxodG excretion served as the key biomarker. A plethora of descriptive epidemiological studies have concerned a possible protective effect of a diet rich in fruits and vegetables [7]. the mode of action of dietary micronutrients is complex and is far from being fully understood. Intuitively. 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. respectively. Both experimental and epidemiological data indicate vitamin C to protect against both stomach and oesophageal cancer [8]. used for the detection of DNA strand breaks (SB). as the possibility of detecting 7-hydro-8-oxo-2’-deoxyguanosine (8-oxodG) appeared. C and E or phenolic compounds. One should bear in mind. At the beginning of the 1990s. Subsequently. Nevertheless. although sampling is usually restricted to use of such surrogate tissues as white blood cells (WBC) and urine. It is very difficult. altered gene expression and mutations are necessary elements in the process of carcinogenesis. nevertheless.12]. the first of many antioxidant supplementation trials dealing with this lesion in WBC was carried out [10]. These antioxidants are effective free-radical scavengers and should protect the DNA from oxidative damage. Although these events may come about by way of different mechanisms. however. supplementation trials would appear to represent the most relevant way of exploring antioxidant effects. 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). It is reasonable to assume that agents that decrease oxidative DNA damage should also decrease the subsequent development of cancer. have become by far the most popular assays for use in conjunction with antioxidant intervention trials. oxidants are involved in each case. . Reliable detection of urinary 8-oxodG excretion became possible at about the same time [11]. the comet assay. 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. 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.Vitamins and selenium: Antioxidant vitamins and cancer risk 53 Summing up. At the same time. since full development of the disease in response to exposure to a carcinogen may take 20–40 years. 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. to establish directly that the DNA lesion responsible for a carcinogenic process is the lesion present in a tumor many cell generations later.

Marcus S.Steffen Loft. Peter Møller. 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.6. Cook. Rafa∏ Ró˝alski. but a decline in the course of the trial in both groups 32 MF (NS) 32±11 No effect compared with baseline.6.6 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 . and 25 mg β-carotene) for 20 wk Multi-vitamin (250 vitamin C. 200 IU α-tocopherol. Table 2. from use of a non-optimum design. Table 2. 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. 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. 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. Ryszard Oliƒski 54 Many studies suffer. 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. 280 mg vitamin E.

Vitamins and selenium: Antioxidant vitamins and cancer risk 55 Table 2. but not earlier 68 MF (13S) 18—45 A decrease in the 12-h urinary excretion of 8oxodG (HPLC) 27.5 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. Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont. 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. 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. 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 . No effect on the 24-h urinary excretion of 8-oxodG (HPLC). 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.6.

60 mg vitamin E. Smokers (S) and nonsmokers (NS) are indicated in brackets. α-carotene (1. 4 mg β-carotene). respectively [15. Containing vitamin A (240 mg). Rafa∏ Ró˝alski. kale. cabbage. and 15 mg β-carotene.16]. Tablets containing β-carotene (12 mg).7 mg). tocopherols (650 IU). papaya. catechin (13. broccoli. rice milk. Consisted of β-carotene (20050 IU).2 mg).5 mg). β-carotene (399 mg).4 mg β-carotene. there are fewer that reveal protective effects than those that report only negative results [9. This suggests that the protective effect of a single antioxidant is relatively short. The vegetable capsules were made from carrots. whereas tocopherols and carotenoids exert their effects somewhat longer. possibly because of differences in the respective rates of bioavailability and elimination. lycopene (4. bixin (11.Steffen Loft. The effect of a single dose of vitamin C seems to disappear after several hours.4 mg). The daily dose consisted of 200 mg vitamin C. 5) 30 mg coenzyme Q10 granulate.12]. 2. vitamin C (330 mg). orange. and pomegranate extract (5 mg). vitamin C (500 mg). and papika carotenoids (2.2 mg). Accordingly. 4) 90 mg coenzyme Q10 in oil.4 mg). Ryszard Oliƒski 56 Table 2. Tablets containing micronutrients similar to those of 3 fruits and 3 vegetables (containing 107 mg vitamin C and 83 mg vitamin E). 2) 500 mg plain-release vitamin C. pineapple. and powder or extract of fruits and berries). Marcus S. capsules (containing 90 mg vitamin C. vitamin E (400 IU). Supplement given per day Subjectsa Age (yr)b Effect Ref Soy milk. or a carotenoid-rich diet. Among studies in which multiple doses of single antioxidants are given. 150 mg vitamin E. Supplement consisting of β-carotene (6 mg). N-acetyl-1-cysteine (181 mg). and tomato. lutein (500 mg). 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). however.14]. Peter Møller. a protective effect could be shown after only 20 weeks of supplementation but no effect after 10 weeks (or 6 months of supplementation) [13. 6) placebo. The six groups received daily supplements of 1) 200 mg vitamin E. Cook. Also. lycopene (100 mg). In two well-designed studies of the effects of administering a combination of antioxidant vitamins. 3) 500 mg slow-release vitamin C.44 mg) per day. Consisted of tablets containing vitamin antioxidants (400 mg vitamin C. 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. spinach.12]. The fruit capsules were made from juiced apple. being given rye crisp . vitamin C (219 mg). 18 IU vitamin E. parsley. Age is shown as range or as mean ± standard deviation. vitamin E (1. beet. cranberry and peach. selenium (167 mg). and zinc (30 mg). Consisted of 1000 mg extract/kg body weight in meat patties (total phenolics 23. the question of whether multiple vitamin supplementation provide better protection against oxidative DNA damage than a single dose does cannot be answered unambiguously. selenium (100 mg). Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont.6. 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. lutein (4.5 mg/10 MJ).

assessment of the DNA damage occurring showed the levels of ENDOIII to be reduced [21]. 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.23–44]. 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. 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). 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. 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. The one investigation.Vitamins and selenium: Antioxidant vitamins and cancer risk 57 bread (76.or FPG-sensitive sites. was negative with respect to effects on the ENDOIII and FPG sites [20]. number of subjects. It can be speculated that the subjects suffered a slight. or to have any effect on the ENDOIII sites [17]. a cross-over study of male smokers.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. and their effects in the gastrointestinal tract are easier to comprehend. . Anthocyanines have low bioavailability and the dose provided here was rather high. no appreciable difference was present in terms of duration of the intervention period. or power to detect a 50% change between these studies reporting beneficial effects and those reporting no effects. 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 example). whereas 13 studies reported null effect. 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. 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. The other. An overall summary of the studies showed that six investigations reported beneficial effect of antioxidant supplementation. showed no effect on the ENDOIII sites of ingesting 500 g/d of such food for 3 wk [19]. Two studies concerned the effects of an intervention of providing vegetables and fruits. which showed the numbers of ENDOIII and FPG sensitive sites to be reduced [22]. 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. For 24 studies of the effects of antioxidant supplementation on the urinary excretion of 8-oxodG in which a controlled design was employed [20. unintentional intoxication of the gastrointestinal tract (some of those in the active groups complained of nausea.

44. whereas in one of the other two studies .0 mg). bixin (11. In the subsequent study. 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]. Rafa∏ Ró˝alski.39].7 mg). neither supplementation of vitamin C or vitamin E or a combination of them was found to have any effect in healthy subjects [24. Peter Møller.32.36]. 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]. cabbage. and vegetables. Mixed results concerning the effects of a dietary supplementation of Brussels sprouts (300 g/d) were obtained in two studies [40. kale.38. 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.31.35]. β-carotene (6. in which both sexes were included. 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 [23.4 mg). tea. A positive effect of vegetable juice consumption on 8-oxodG excretion was also observed in subjects enrolled in a soccer summer training camp [26]. In four additional studies.37] or in subjects undergoing cold-weather field training at a moderate altitude [33. 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]. the difference between results at the end of supplementation and at baseline) but no difference between the two groups at baseline [30].35. Investigations of the effects of natural food products are distributed about equally between studies reporting beneficial and those reporting null effects. No effect on the urinary excretion of 8-oxodG was found for the ingestion of capsules containing juices and powders of fruit (apple.2 mg)) revealed a statistically significant difference between the active and the placebo group in the delta values obtained (i.5 mg). fruits. and peach) and of vegetables (carrot. lycopene (4. the effect was less clear. In the first study.45]. spinach. Cook. cranberry. broccoli. parsley. Marcus S.4 mg). that involved only male subjects. beet. whereas this intervention was found to have a pronounced period effect [20]. pineapple. In the one study a beneficial effect was found for drinking 300 ml/d for a week [31].41]. 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. Further studies showed multi-vitamin tablet supplementation to provide no beneficial effect either in normal subjects [20. and paprika carotenoids (2. A number of studies have involved supplying antioxidants in the form of berries.Steffen Loft. there being a tendency for only the male subjects to benefit from ingestion of Brussels sprouts. 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]. Three additional studies in this area concerned the effect of drinking green tea [see 27. orange. the Brussels sprouts supplementation was found to lower the urinary excretion of 8-oxodG [41]. A comparable study involving supplementation of a mixture of carotenoids (daily intake: α-carotene (1.28. On the other hand. and tomato) [29]. papaya. lutein (4.34.e.

Interestingly. Under some circumstances. which might fail to be recognized. such as those of regulating changes in gene expression. revealed a statistically significant positive effect of drinking green for 4 months [27. including baseline 8-oxodG levels. The absorption of non-haem iron has also been found to be affected by ascorbic acid [49]. therefore. drinking soya hypocotyl tea was found to be associated with a lower urinary excretion of 8-oxodG. 3. 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. In still a further study. 2) the putative active constituents (isoflavones) were not measured in the plasma. It is possible that a preventive effect of the vitamins can be only be seen when their basal levels are very low. a positive correlation has been shown between LIP and the oxidatively modified nucleoside in human lymphocytes [48]. that are separate from their direct antioxidant effects [51]. 5. such that iron catalytic to free-radical reactions (a so-called labile iron pool — LIP) is present in the blood plasma [47]). 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. adjustment of the data for a number of variables. Paradoxically. and 3) the alterations in the urinary concentration could not be assessed due to insufficient information [42]. the prooxidative properties of certain of the antioxidant vitamins (vitamins C and A) may take effect. 4. which leads to iron overload. although it should be emphasized that the fate of the antioxidants in this investigations was inconclusive since 1) the plasma concentration of carotenoids decreased. 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].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]. antioxidant vitamins may have biological activities. It is possible. that the presence of oxidative stress. could increase the likelihood of detecting a protective effect. 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. Although the unadjusted data of the third study indicated no beneficial effect of drinking green tea. 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. such as in the case of severe oxidative stress. It is worthy of note in this context that presumably healthy men may have the hereditary disease idiopathic haemochromathosis. Experimental data suggest that supplementation of vitamin C to iron-overload subjects may increase the oxidative DNA damage that occurs [46].45]. . Indeed. 2.

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

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

. 0 min ELISA. 10 min Calibrators and controls in the serum matrix. requires luminometer Comments (nmol/l) CV (%) CV (%) ≤6 <8 <12 RIA. as well as being fast. 10 min Uses vitamin D binding protein * Represents the initial starting volume of the sample.4 9 11 5 h. 100% cross-reactivity with 24. CPB — competitive protein binding. 24. Sample type Manufacturer and volume* RIA. 25 (OH2) D. 24. 0 min ELISA. Biomedica Proprietary extraction reagent Unknown 6. and are thus more frequently used. RIA — radioimmunoassay.2 75 min Acetonitrile and C18 cartridge extraction Acetonitrile 15—150 4. Radioimmunoassays (RIA).9 3 h.25-dihydroxyvitamin D. Serum.2 8. DiaSorin Serum or plasma.7. ELISA — enzyme-linked immunosorbent assay. however.7. IDS Serum or plasma 50 µL Two-step reagent extraction 4—400 ≤5 5.6 11. no yield determination required Calibrators and controls in the serum matrix.4 20 min The laboratory must have an HPLC unit with a silica column CPB. Table 2. 0 min ChemilumiSerum nescence. counting or microplate reading times. Table 2. CV — coefficient of variation. IDK Serum 500 µL 17. Commercially available 25-hydroxyvitamin D assays [6] Range of Test principle. no yield determination required No radioactive waste. Immundiag. IDS Serum or plasma 50 µL Serum or plasma 50 µL None 6—360 ≤5 <6 <9 3 h.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 65 methods. HPLC — high-performance liquid chromatography. There are important differences in the performance of the various detection methods.0 5.5 7. enzyme immunoassays (EIA) and chemiluminescence assays (CLPBA) are easier to handle.5 9.3—250 2.9 14 1 h.5—300 18 6.plasma nostics or urine 500 µL 8—312 2. ** Does not include extraction. 50 µL Extraction detection (nmol/l) Acetonitrile Sensitivity Intraassay Interassay Assay time** 2 h.25 (OH2)D The primary antibody is the vitamin D binding protein from serum Fully automated. or plasma Nichols 20 µL Institute Diagnostics HPLC. summarizes some of the performance characteristics of most of the assays that are available commercially.

). and the proportion of subjects below an arbitrary threshold of insufficiency (32 ng/ml) varied between 17 and 90%.8. Mean (SEM) results for the serum 25-(OH)D concentrations for 10 subjects as estimated by six different laboratories (for abbreviations see Table 2. 2. RIA — radioimmunoassay.Jakob Linseisen.4. The mean serum 25-(OH)D concentrations differed 2-fold between laboratories (Figure 2. After spiking of the serum samples with a defined quantity of 25-(OH)D.4. Table 2. the serum of 10 subjects was sent to six different laboratories.8.).6 ng/ml (p < 0. Binkley et al. Fig.005) [11].).8. In their study. HPLC — high-performance liquid chromatography. . Binkley et al. lists the methods and the normal range for 25-(OH)D measurement in the laboratories that participated.1 to 35. [11] reported recently not only immense variation between different assays but also variability between different laboratories in using a given assay. On the basis of their results.5. Table 2. Sascha Abbas 66 Validity of different assays Only few studies have compared the different commercially available assays [9–13]. a broad range (17–95%) of the expected concentration was found by the different laboratories [11] (Figure 2. The means varied widely between laboratories from 17. 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.

2. 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]. 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]. Figure 2. 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. However.6 ng/ml (p < 0. Patients.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 67 Fig. However. 59% of the participating laboratories were found to have met the target [12. however. This suggests that the contribution of 25-(OH)D2 to the overall 25-(OH)D supply is less important than had been previously thought.7 to 18 ng/ml. the greatest increment being detected by HPLC [11]. .005) from 7. 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).13].8). as estimated by five different laboratories (for abbreviations see Table 2.05). In accordance with these findings. the authors of the report state that the validity of the 25-(OH)D results that are obtained will justifiably continue to be questioned. Another study compared the DiaSorin RIA assay with the Nichols chemiluminescence assay. There has been discussion regarding the importance of the assays detecting 25-(OH)D2 as well as 25-(OH)D3. especially those in the US. The means varied widely between laboratories from 27. 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]. Each of the laboratories was given five serum samples quarterly. Both assays also underestimated the 25-(OH)D-concentration as compared with the HPLC-method [10]. The increment was less than anticipated and varied between laboratories (p < 0. According to the latest DEQAS (Vitamin D External Quality Assessment Scheme) report. finding discordant serum 25-(OH)D-values.5. Mean (SEM) results for the 25-(OH)D concentrations in the serum samples of 10 subjects spiked with the 25-(OH)D standard. These give the impression that only method 6 exceeded the limit. shows the latest results of this external quality control project.6.4 to 44. several authors have called for international standardization of the vitamin D assays that are available and that are affordable for practicing clinicians.

season. It is important to note that “normal” is not defined as the median population level.15. In addition to the high degree of uncertainty that the divergence of the results for different techniques and different laboratories creates. but there may also be a significant proportion of the population that exhibits asymptomatic subclinical vitamin D insufficiency. that set the cutoff points at much lower levels. HPLC. 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. DiaSorin RIA. dietary intake. as shown by the seasonal variation in vitamin D status that occurs [17. around 25–40 nmol/L [1. method 4. . and the composition of the population being studied.18].Jakob Linseisen. method 6. age. the serum 25-(OH)D concentrations in different populations vary with latitude. IDS RIA. 2. Sascha Abbas 68 Fig. Apart from the analytical problems. Accuracy of 25-(OH)D methods used by the different DEQAS participants [13].16]. This is in contrast with most of the published studies. The method means of samples known to contain 25-(OH)D2 were excluded. Each column shows the mean deviation (% bias) and SE from the target value (ALTM) during the period of 2000–2004. Recent reports suggest European and North American populations to have a higher degree of insufficiency than had previously been thought [15. method 3. Nichols automated chemiluminescence assay. suggested cut-offs for insufficiency range from 10 to 30 ng/ml (25–80 nmol/l). In epidemiologic studies. A summary of studies on vitamin D status in the elderly is given elsewhere [1]. In a recent publication.6.18–23]. IDS EIA. epidemiological variability needs to also be taken into account. method 2. competitive protein-binding assay. Method 1. Populations at risk include nursing home residents and the elderly. Hollis defined 25-(OH)D levels below 80 nmol/L as being deficient. race. 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. due to the serious health risks encountered at levels less than this [7]. There is growing evidence that there is not only a specific seasonal decline in serum 25-(OH)D during the winter months. method 5. especially the home-bound elderly.

2. Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D. J Clin Endocrinol Metab 1971. Lake E.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 69 Conclusions In conclusion. Ann Clin Biochem 2004. 6. Potts JT. Immunodiagnostic Systems (IDS). References 1. J Nutr 2005.46:756–65. Competitive binding assay for vitamin D and 25-OH vitamin D. Taranto M. Which circulating level of 25-hydroxyvitamin D is appropriate? J Steroid Biochem Mol Biol 2004. Clin Chem 2000. 10. Endres D. Krueger D. Belsey R. vitamin D3).135:317–22. 11. Caldas AE. DeLuca HF. Binkley N. 3. This makes strong scientific research efforts needed. J Clin Endocrinol Metab 1986. . Hollis BW. and problems in defining the appropriate threshold for vitamin D sufficiency. 5. Noble JM.63:656–60. 4. Glendenning P. Plum L.46:1657–61. DeLuca HF. vitamin D metabolites have various important biological effects. The measurement of vitamin D: analytical aspects. Ann Clin Biochem 2003. Myles M.33:554–7. Issues of methodology.40:546–51. the determination of vitamin D status is still a challenging task. Editorial: The determination of circulating 25-hydroxyvitamin D: no easy task. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. McGuiness M.89:3152–7. May 2005. 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.89–90:611–4. Several problems are important to bear in mind including the different forms of vitamin D (vitamin D2. Smith GA.41:272–81. Epidemiologic studies in particular can contribute knowledge concerning the association between vitamin D insufficiency and the risk of disease. J Clin Endocrinol Metab 2004. et al. 8. the measurement variation introduced by different assays and different laboratories. 9. Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects. Comparison of commercially available (125)I-based RIA methods for the determination of circulating 25-hydroxyvitamin D. including the risk of cancers at various sites. Musk AA. Zhou XY. Nonetheless. Review SeriesVolume 2. Metabolism and excretion of 3H-1. Gray RW. 7. Hansen KE. Zerwekh JE. Goldswain PR. Lips P. epidemiologic variation in vitamin D status and its determinants. et al. Lindsay R. Hollis BW. Cowgill CS. Overview of Vitamin D Measurement and Methodologies.25-(OH)2-vitamin D3 in healthy adults. Clemens TL. J Clin Endocrinol Metab 2004. Hollis BW. Hart GR. Wilz DR. J Clin Endocrinol Metab 1978.89:3149–51. Lemann J.

Fenske JS. . Scheidt-Nave C. Prevalence and predictors of vitamin D deficiency in five immigrant groups living in Oslo.71:1577–81. Armas LA. Horowitz M. 20. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. Beard MK. Arnaud S. 15.59:57–63. Osteoporos Int 1997. Lund E. apparently healthy urban European population. Morris HA. 16. 25-dihydroxyvitamin D in postmenopausal women. 17. with particular reference to European countries. MacFarlane GD. Heaney RP. et al. Makin HL. Binkley N. Jones J. Hanley DA. Galan P. J Clin Endocrinol. J Steroid Biochem Mol Biol 2004. Woitge HW. Sascha Abbas 70 12.62:813–21. Vitamin D insufficiency in North America. Prevalence of vitamin D inadequacy among postmenopausal North American women receiving osteoporosis therapy. Sackrison JL. et al. 14. 19. Berry J. Body JJ. Norway: the Oslo Immigrant Health Study. 23. Need AG. Hercberg S. Vitamin D2 is much less effective than vitamin D3 in humans. Vitamin D status of middle-aged women at 65–71 degrees N in relation to dietary intake and exposure to ultraviolet radiation. Chapuy MC. Carter GD.Jakob Linseisen. Brunvand L. Khan A.135:332–7. Geographical differences in vitamin D status. Carter CR. Holvik K. Haug E. Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation. Andersen R. Katzer JT. J Clin Endocrinol Metab 1998.83:68–75. Jones G. Meyer HE. J Nutr 2005. Siris ES.89–90:467–71. Hollis BW.7:439–43. Gunter E. Alsaker E. Ovesen L. Proc Nutr Soc 2003. Nordin BC. 18. Miller AB. Vitamin D status: effects on parathyroid hormone and 1. Holick MF. Obstet Gynecol Surv 2005. Davison KS. Maamer M. Aksnes L. Prevalence of vitamin D insufficiency in an adult normal population.89:5387–91. Jakobsen J. Clin Chem 2004. Engelsen O. 22. Carter GD. et al. Leidig-Bruckner G. Ersfeld DL. Hypovitaminosis D in a normal.89–90:621–2. Am J Clin Nutr 2000. Seasonal variation of biochemical indexes of bone turnover: results of a population-based study. Meyer K. Brustad M. Preziosi P. Eur J Clin Nutr 2005. Carter R. Kissling C.50:2195–7. Grauer A. Metab 2004. J Steroid Biochem Mol Biol 2004. 13. et al.7:327–35. Jones J. 21.60:658–9. Public Health Nutr 2004.

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

Björn Åkesson. Metabolic pathways of selenium.9. 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. The major selenium metabolite excreted in urine is selenosugar. selenium is removed as dimethylselenide via exha- Fig.). selenocysteine and selenite can be converted into the key metabolite hydrogen selenide (H2Se). Katharina Bruzelius 72 Table 2. At toxic doses.7. dimethylselenide or trimethylselenonium ions for excretion. a much lesser amount being excreted as trimethylselenonium ions [7]. which is turn is the precursor of selenocysteine in selenoproteins and various excreted forms of selenium. Selenate and selenite are reduced by glutathione to hydrogen selenide.7. Several compounds can be converted into methylselenol (from [8]). . 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). Selenomethionine and selenocysteine can also be converted to hydrogen selenide. Selenomethionine. 2.

9].) [1. found in embryos and in the olfactory epithelium Unknown Unknown Unkown. a membrane protein Involved in protein folding in the ER Unknown. Several of these compounds can be converted to methylseleninic acid or methylselenol which have anticarcinogenic effects in vitro [8]. yet the functions of many of the proteins involved are still unknown (Table 2. involved in many biological pathways Thioredoxin/glutathione reductase found mainly in testes . Selenoproteins A total of 25 selenoprotein genes were discovered in the human genome several years ago by sequence analysis.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 73 lation. such as Se-methylselenocysteine and selenomethionine.10. are the selenium compounds most effective in cancer prevention [8]. only found in the testes Can reduce peroxides using glutathione as an electron donor Catalyses the formation of selenophosphate Cytoplasmic thioredoxin reductase. involved in the elimination of misfolded proteins from the ER Unknown Unknown. mainly in the gastrointestinal tract Reduction of phospholipid hydroperoxides Peroxide reduction. mutations in gene associated with muscular diseases Unknown An antioxidant and selenium transport protein Reduces methionine-R-sulfoxides A membrane protein. 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.10. involved in many biological pathways Mitocondrial thioredoxin reductase. The distribution and concentrations of selenoproteins Table 2. Several studies have suggested that methylated Se derivatives.

Several other glutathione peroxidases containing selenocysteine have been found since then. The cytosolic or cellular form of glutathione peroxidase (cGSHPx) was the first selenoprotein identified [10. serine is bound to tRNASec and then transformed to selenocysteine [13]. Except for the SECIS element there are no features in the DNA-sequence that selenoprotein genes are known to have in common. Katharina Bruzelius 74 in different tissues are also not well known. The SECIS consensus sequence consists of two helices.17]. 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. Biosynthesis of selenoproteins Translation of the codon UGA to selenocysteine (Sec) and its insertion into proteins is a complicated process (Figure 2. but the mechanisms involved have not yet been elucidated (Figure 2.12]. In addition to the SECIS element. which has two [15]. additional factors are required for the insertion of Sec in eukaryotes such as the SECIS binding protein 2 (SBP2). 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. . one apical loop. catalysing the reduction of oxidised thioredoxin and other substates [1. Several of the selenoproteins have an homologue protein containing Cys instead of Sec. This structure is located in the 3’-untranslated region of the mRNA in eukaryotes and immediately after the UGA codon in prokaryotes [14. All known eukaryotic selenoproteins have one SECIS-element. There are no selenoproteins known to be present in yeast or higher plants. which is a short sequence of non Watson-Crick paired nucleotides that appear to exist in all selenoprotein mRNA:s. and the SECIS core. The other two major groups of known selenoprotein enzymes are the iodothyronine deiodinases.). and the thioredoxin reductases. 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]. Many of the known selenoproteins catalyse redox reactions in which selenium is at the active site. that regulate thyroid hormones. these organisms tending to have homologue proteins containing Cys instead of Sec [13]. In the first step. but the catalytic ability of these latter proteins is much lower.15]. the Sec-specific elongation factor (EFsec). and the recently discovered L30 protein.8.Björn Åkesson.11]. except for SeP.). In eukaryotes the Sec codon and the SECIS element are located at some distance from each other in the mRNA. The current conception is that the mRNA and the SBP2-EFsec loops back towards the translation complex [13.8. one internal loop.

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

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

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

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

83.11. selenium and eGSHPx in different studies (from ref.85) 1.86) 0. 4. c Data from subgroups having (top to bottom) low.0.11.41. 346)a 6. 1.34.32) 3.46—1.6.38 (1.52 (0.47 (1. Study European countries Prior to LDL-apheresis After LDL-apheresis Finland.08.30—1.14) 0.55 (0.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].21.41 (0.74)b — — — 126—205 38 21 16 31 1.44.07 (0. Trial I baseline Finland. 1.20—4. a GSHPx activity. U/l.18 (0. mU/mg protein. 0. 1. 0.60.23 (1.24) 1.56) 4. 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. 1.63—1.66) 1.51 (6.65) 3.43. 1.51. glutathione peroxidase and protein-bound selenomethionine are the major selenium fractions contained in plasma [56].21) 1.u.16) 0.51) 0.64) 0.28. .11.16. respectively.27) 1.).22) 0.47) 1.2 mmol/l [55]. Selenoprotein P.91 (0.75—2.03 (0. 1. medium and high fish intake. 397)a 302 (259.69 (0.66—1.) 1. Supplementation by selenate resulted in glutathione peroxidase activity plateauing at a plasma selenium concentration of 1.98.00 (0. 1.73 (0.83) 0.69—5.56. [63]). Selenoprotein P accounts for at least 40% of the plasma selenium [37].77 (1.38 (0.95 (2.88) 1.07) 1.31—4. 2.12) 0. In the following various results from their use in the authors’ laboratory are summarized (Table 2.69 (0.83 (0.52) 0.39.86 (0.31) 2.44) 1.83) 1. Table 2.91 (1.14 (1. b GSHPx activity.25) Plasma selenium (µmol/l) 1.20 (1.69. 0. 1. Plasma concentrations of SeP. Median values (and range) are given. 1. 0.38 (2. 1.43) — — eGSHPx (mg/l) — 352 (306.13 (4.10 (1.41 (1.15) 1.54—1.78 (1. 0. 0.73) Values are means (95% CI).33. Immunoassays for measurement of this protein have been developed [57–59].92.

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]. with some indication of a plateau. 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. studies of lead-exposed children in Katowice.9. eGSHPx and plasma selenium as markers of selenium status. Regarding the relationships between SeP. 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. A summary of the responses shown by the different selenium indices in the first of these two studies is provided in Figure 2. the selenoprotein P values increased after selenium supplementation. and also between fish intake versus serum and urinary selenium were found. In a recent study of Chinese subjects of low selenium status given different doses of selenite and selenomethionine. in which the subjects were low in selenium status. total selenium and selenoprotein P than control subjects did [64].68]. however. Poland revealed an inverse relationship between the blood lead level and the level of selenoprotein P and of extracellular GSHPx [65]. For women. In the first study. . Concerning the association of selenium status and exposure to toxic metals. Katharina Bruzelius 80 Selenoprotein P as a biomarker Selenoprotein P levels were measured in healthy adults from 17 European Regions [60]. 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. 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. In the Malmö Food Study [61] the relationship between the intake of different foods and selenium status was investigated. Home parenteral nutrition patients with a variety of gastrointestinal diseases showed much lower levels of extracellular GSHPx. but that at normal selenium status selenoprotein P usually correlated more highly with plasma selenium than eGSHPx did. plateauing within two weeks [69] (Figure 2. In a study of Latvian fisherman [62]. significant relationships between milk intake versus selenoprotein P and urinary selenium. The levels of selenoprotein P and eGSHPx were found to vary markedly for subjects from different regions and with different diseases.9. performed after the introduction of selenium-enriched fertilizers in Finland.). the selenium status of the subjects thus being higher. Since the direction of causality was uncertain. The scientists there compared the responses to oral supplementation of 200 mg selenium per day in healthy subjects on two occasions. In the second study. There was a close correlation between selenoprotein P and plasma selenium values. 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].Björn Åkesson.

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. Interest in the preventive role of selenium supplementation in humans was stimulated markedly by results from two intervention studies. 2. whereas in other studies no significant differences in this respect were found between cases and controls [82–86].). In a number of case-control studies. 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. particularly among men [75–81]. The strongest associations between premorbid plasma selenium levels and risk of cancer were observed for cancer of the respiratory and digestive tracts. Fig.87]. .9. β-carotene and α-tocopherol reduced the total cancer mortality and stomach cancer rate in a Chinese population. 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. The calculated degree of protection was found to differ between cancer sites [81. Generally speaking.72]. Full expression of selenoprotein P was not achieved at the highest dose of either form (66 µg/d). Blot and coworkers [73] found that a mixture of selenium. in turn found selenium supplementation to reduce total incidence and mortality of cancer in an American study group (see below). This suggested that selenoprotein P is a better indicator of selenium nutritional status than glutathione peroxidase is [70]. Biomarkers of selenium status and cancer Experimental studies have shown that the addition of high (0. lower prediagnostic plasma selenium was found for cases of cancer than for controls. Clark and associates [74]. 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.69]).5–2 ppm) levels of selenium to the diet has a carcinostatic effect in animals treated with carcinogenic chemicals [71. These matters are reviewed in greater detail in the chapter that follows (Gromadziƒska et al.

The circles represent selenium and the squares SeP. and toenail selenium [90–92] were observed in smokers than in non-smokers. the ORs (adjusted for smoking) in tertiles of the SeP level were calculated for the respiratory. plasma selenium has been used as a marker of selenium status. respectively (p for trend = 0. and 0.90]. in the lowest tertile as compared with the cases in the highest tertile. 2. 2.05.89].0 and 1. 0.10.0. normal contour: p ≥ 0. Smoking and biomarkers of selenium status Several factors other than selenium intake may affect biomarkers of selenium status. 2.10. This is probably due to SeP constituting at least 40% of the total selenium in human plasma [37]. the case-control differences of plasma selenium and SeP are compared for major cancer sites in several different study populations. 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. The factors contributing to the lower selenium status in smokers are unclear. The individual references are cited in [63].0. The association between the relative risk of getting cancer and the SeP concentration found was also estimated from quintiles of the SeP level. the ORs (adjusted for smoking) were 5. Percentage differences in the prediagnostic selenium and SeP plasma levels between cancer cases and controls (set at 0) for cancer at different sites. 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. 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]. 3. .9. Fig. the SeP levels for cancer of the respiratory tract were found to be significantly lower than in matched healthy control subjects. These were 6. digestive and urinary tract and for cancer of other sites. 2. The previously reported association of plasma selenium levels with cancer risk can very likely be explained by the corresponding association of SeP levels.85.2. lower values of plasma selenium [77.01). Bold contour: p < 0.05. Smokers were found to have significantly lower levels of selenoprotein P than nonsmokers [88]. In other studies. whole blood selenium [89. When cases were divided into subgroups according to cancer site.Björn Åkesson. In addition. For increasing quintiles. In Figure 2.2. erythrocyte selenium [89].6 respectively.4.3.

91). whereas the concentration of cadmium in the blood is significantly higher in smokers [95]. in particular mortality due to stomach cancer [73]. and that these correlations are higher (more significant) in smokers. a result that could possibly be explained by the covariance of lead and selenium in the blood [65]. It has also been shown that selenoprotein P plays a role in the defence against peroxynitrite [39]. It has been reported that smokers eat less selenium than non-smokers do. a small but significant reduction in total cancer mortality was obtained (RR = 0. both being acute-phase reactants. In addition.74]. 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. 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. selenomethionine and glutathione peroxidase can scavenge peroxynitrite. Patients with a history of skin carcinomas who . In the Linxian trial involving supplementation of β-carotene. in a meta-analysis of 51 published nutritional surveys of the relationship between smoking status and nutrient intakes. In another study. which could probably in part explain their lower selenium level [90]. Multiple regression analysis also indicated the blood cadmium to increase significantly with a decrease in the selenoprotein P level. As reported by Sies and coworkers [94]. although this association disappeared when lead was included in the model. several intervention studies having indicated beneficial effects [73. Multiple linear regression analysis of the data also suggested a depressive effect of cadmium on the concentration of selenium in the blood. suggests that the selenoprotein P levels are reduced by inflammatory activity. α-tocopherol and selenium given for a 5. Smoking may also increase oxidative stress since cigarette smoke is a rich source of reactive nitrogen species. whereas smoking alone did not serve as a true predictor of this effect. 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].25-year period. which together with superoxide can produce peroxynitrite [39]. smoking was found to be significantly associated with an unhealthy pattern of nutrient intake. which suggests a repression of selenoprotein P expression during an acute phase reaction. 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. This agrees with findings of Dreher and co-workers [93] indicating that the human selenoprotein P promoter is rendered less active by cytokine treatment. The natural presence of cadmium in tobacco smoke may also contribute to the lower selenium status found in smokers.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. This may also explain the slightly lower selenoprotein P concentration in smokers. which could exacerbate the risk of cancer associated with smoking [96].

The type of selenium supplements best employed is also in need of further investigations.S. Hansen M.51:45–9. Vahl M. It is apparent from this review that selenium can play an important role in cancer prevention. Speciation of selenoamino acids. Bansal MP. 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]. Sunde RA. evaluation of health claims by the FDA in the U. Effect of dietary methionine on utilization of tissue selenium from dietary selenomethionine for glutathione peroxidase in the rat. Experimental findings also indicate that in some experimental systems selenium can both promote and inhibit cancer [98]. Katharina Bruzelius 84 were treated with 200 mg selenium per day showed significant reductions in total cancer mortality (RR = 0.54). 3. 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. Mukhopadhyay T. Food Chem 1994. 5. Kyriakopoulos A. 2. References 1. selenonium ions and inorganic selenium by ion exchange HPLC with mass spectrometric detection and its application to yeast and algae. Waschulewski IH. . Fan T. DNA sequencing of a mouse liver protein that binds selenium: implications for selenium's mechanism of action in cancer prevention. Srikumar TS.118:367–74. Åkesson B.37) cancer [74].102]. Recent results of the SUVIMAX study showed supplementation with vitamin C. Behne D. 4.50) and in incidence of lung (RR = 0.42) and prostate (RR = 0. 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]. Occurrence of low-molecular-weight and high-molecular-weight selenium compounds in fish. Cook RG. but additional studies are needed to determine whether there is also an increased risk of some forms of cancer after selenium supplementation.16:1403–8. In another Chinese investigation. selenium and zinc to reduce the rate of prostate cancer in men having normal levels of prostate-specific antigen in their plasma [99].Björn Åkesson.21:453–73. Larsen EH. Annu Rev Nutr 2001. Medina D. J Nutr 1988. 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.11:2071–3. 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]. In addition. vitamin E. Mukhopadhyay R. colorectal (RR = 0. Mammalian selenium-containing proteins. Scott J. β-carotene. Carcinogenesis 1990. J Anal Atomic Spectrom 2001.

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and in epidemiological studies of risk of cancer — and the outcome of long-term trials concerned with the disease-preventive potential of selenium supplementation. [5] published the alarming finding that feeding rats 0. In the previous chapter the forms and metabolism of selenium. equivalent to inclusion of 103–207 ppm Se in the diet for a 10-day period.1% bis-(4-acetamine)-phenylselenyl hydroxide. in addition to the occurrence of different selenoproteins. interest in selenium (Se) as a toxic trace element dates back to 1937 [1]. whereas such tumours were found in less than 1% of the animals in the control group. In 1963.3 ppm level.05–0. and concern for it as a carcinogenic agent dates to 1943 [2].5. It should be noted. induced thyroid adenoma. including the forms of selenium present in the diet and in the body. Researchers of the former Soviet Union [4] feeding Se (as selenite) to rats at a 4. In the present chapter the mechanisms behind the role that selenium can play in cancer prevention. . their functions and mechanisms of action. Wojciech Wàsowicz Department of Toxicology and Carcinogenesis. Poland Introduction There are many different aspects of the relationship between selenium and cancer. Edyta Reszka. however. [2] observed that liver tumours occurred in rats fed protein-deficient fodder and grains originating from Serich (5–10 ppm) regions. Nofer Institute of Occupational Medicine. Two of the chapters deal with these and related matters. Anticancerogenic activity of selenium — molecular mechanisms and epidemiological data Jolanta Gromadziƒska. suggested that the pathological changes that occurred reflected regeneration of the liver rather than a carcinogenic process. and intervention trials to investigate the effect of selenium supplementation on the incidence of cancer are summarized. it is quite possible that the organic selenium compound employed there had carcinogenic properties not linked with the element per se. observed the development of different forms of liver tumours. which suggests that the neoplastic changes that occurred could be attributed to Se. that since no studies have been reported of animals administered an analogous compound that did not contain selenium.3 ppm Se. ¸ódê. however. Tscherkers et al. [6] observed the development of tumours in male rats fed a diet containing 4. Nelson et al. their use as biomarkers of selenium status. Seifter et al. 11 were found to develop liver tumours. History Historically. Of 53 animals that survived for 18 months when thus fed. the methods used in assessing the nutritional status of selenium — in general. nevertheless.Vitamins and selenium: Anticancerogenic activity of selenium 91 2. as well as epidemiological findings regarding the relationship between selenium level and risk of cancer are reviewed. Shapiro [3]. in response to selenium supplementation.

Another early observation of such an effect was that of Clayton and Bauman [8]. 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]. respectively. Wojciech Wàsowicz 92 The anticancerogenic effect of selenium in animals was first reported in 1911. a supply of 0. In both groups the incidence of cancer was found to decrease with increasing level of Se in the diet.12-dimethylbenzantracene (DMBA). Providing selenium at a still higher level (2.0 ppm Se in their drinking water. [15]. Edyta Reszka.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). An interesting study of the anticarcinogenic role of selenium was carried out by Schrauzer et al. the first tumours developed when the animals were 4 months of age. in addition to this. a control group receiving a basal diet that contained 0. 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].1 to 0. The maize oil content in the one case was high (25%) and in the other case low (5%). At the same time. 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].15 ppm Se and the other groups receiving. when Wassermann et al. [10] showed that adding 0. In the control group. particularly when it was provided at both the initiation and the promotion stage of chemical carcinogenesis. using female mice of the C3H strain. in rats to which either of two different forms of Se were administered [9].Jolanta Gromadziƒska. Ip and Sinha [13] investigated the effect of various concentrations of Se on the development of breast cancer induced in rats by 7. These results indicated that supplying Se in larger amounts protects animals from developing cancer or delays .4-dimethylaminobenzene (3’-MeDAB). The animals were divided into several groups. [7]) managed to inhibit the development of placental tumours in mice by use of Se compounds. whereas in the group receiving the largest amount of Se in its drinking water (1. who reported that in rats a diet enriched with 5 ppm Se decreased the incidence of liver tumours brought on by 3’-methyl-1. In the groups given extra Se in their drinking water.1 to 1. 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%. Two groups of animals received a diet containing maize oil. Supplying Se was found to decrease the number of tumours induced (from 97 to 46%). to 3%. a strain characterized by the spontaneous development of breast adenoma in 80% or more of the cases. 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. tumours not only developed later.5 ppm) reduced the incidence of cancer even more. Harr et al. (cf. but they also decreased in number with an increase in the amounts of selenium provided. Se supplied during only one of these two phases had a much weaker effect [14].0 ppm) they first developed at 17 months of age.

It has also been shown. in the majority of cases the reduction being quite significant [19]. that selenium compounds can induce cell death through a mechanism distinct from oxidant toxicity [22. 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]. 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.12.25]. it has been postulated that the chemopreventive effect is related to the toxicity of selenium and the oxidative stress it induces.23]. 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. The effects can occur at a systemic. The activity of many cellular targets. Glutathione . affecting the cell cycle. including their providing protection against oxidative damage. Cells adequately supplied with Se have been shown to be less sensitive to effects of both endogenous and exogenous carcinogens [17].). and inhibiting angiogenesis [29. In several studies. can prevent mutations by serving as free radical scavengers [26–28]. The anticarcinogenic effect of Se has been found to depend on the chemical form in which the element is administered. It is also possible that selenium in the form of glutathione peroxidases. Thus a number of mechanisms for the anticarcinogenic effects of selenium and of many selenocompounds have been proposed. 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). Experiments in which no effect of Se was observed were rather rare. in which the metabolism.30] (Tables 2. cellular or nuclear level. and perhaps selenoprotein P and other selenoproteins as well. resulting in more efficient carcinogen detoxification [24. and 2. 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. enhancing immune responses. proliferation and differentiation of the cells can be affected. Regarding the mechanisms involved. Some reports suggest that the action of selenium toward cells that have been transformed earlier and toward normal cells differ [32]. 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. however. viruses or transplanted tumours [18]. depends on the cellular GSH/GSSG ratio. The anticarcinogenic effects of Se depend upon the chemical form of the Se-compound involved and the nature and dosage of the carcinogen. since reactive oxygen species can promote apoptosis in vitro [21]. the activity of xenobiotic-metabolizing enzymes in vivo has been reported to increase when selenium compounds are given. In addition. At the same time.13. one should bear in mind that the results of animal studies cannot be directly extrapolated to humans.Vitamins and selenium: Anticancerogenic activity of selenium 93 the carcinogenic processes. altering the metabolism of carcinogens. its dose and the agent that induces the development of cancer [16].

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

cell detachment Yes ++++ ++++ S/G2-M ++++ Necrosis Late Fibronectin is an extracellular component that plays an important role in intercellular communication. W — weak. Form of selenium Selenite p-XSC Selenite. Selenite activates the protein kinases involved in the pathways of cellular response to stimulation by inflammatory agents.13. Inorganic Se compounds such as selenite reduce the number of fibronectin receptors at the cell surface. BSC p-XSC. selenic acid Ebselen P-XSC.4-phenylenebis(methylene)selenocyanate. JNK — Jun-N-kinase. NE — no effect. AP-1 — activator protein 1. 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. BSC — benzyl selenocyanate. whereas these processes can be inhibited by selenate [34]. it cannot come about through activity of the selenoproteins. BSC. The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis (ref. selenium dioxide. Since this is an immediate action. PKA. p-XSC — 1. Table 2. TK — thymidine kinase.12. COX-2 — cyclooxygenase. E — enhancement.Vitamins and selenium: Anticancerogenic activity of selenium 95 Table 2. ACF — aberrant crypt foci. selenite Se-methylselenocysteine Se-methyselenocysteine. Oxidation of the thiol groups . NF-κB — nuclear factor κB. 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. PKC — protein kinase A and C. [16]) — cont.

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

the concentration of Se in the tissues also being found to be higher after the administration of selenomethionine than after selenite supplementation. The anticancerogenic activities of various Se compounds are summarized in Table 2. AP-1. Selenite. From this it can be concluded that a high concentration of Se is not crucial for the inhibition of carcinogenesis. It was also observed that methylated Se derivatives can induce apoptosis [48]. When selenides are generated in sizeable amounts. such as protein kinase A.54. 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]. Some low molecular weight Se compounds have been shown to influence the regulation of gene expression. modulation of tumour angiogenesis. Ca+2-dependent and -independent kinase C (PKC).12. not only the selenocysteine incorporated into the protein structure. ref-1. [50.49]. and the glucocorticoid receptors [34. .g. At the same time. Selenite has been shown to be more effective than selenomethionine in the inhibition of cancer cell growth during chemically induced carcinogenesis [31. affect cellular metabolism. [16]. a key enzyme in the deoxyribose formation needed for DNA synthesis. 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]. 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.55]. cellular growth. high concentrations of selenides tend to also be generated. In cells with a high Se level. reduction in the oxidative DNA damage that occurs. etc.. whereas Trx/TrxR activates ribonucleotide reductase. The various chemical forms of Se have been shown to differ widely in their anticancer properties. they react with oxygen to produce superoxide and hydrogen peroxide [56]. diacylglycerol kinase and thymidyl kinase [48]. p53. selenodiglutathione. but also its metabolites. Se is also known to arrest several kinase pathways important in signal transduction. Trx limits DNA synthesis. It is important to note that different selenocompounds are essential for the growth and differentiation of both normal and neoplastic cells [52]. ASK1) directly.54]. TrxR also regulates gene expression by affecting the cellular redox status and activating numerous DNA-binding transcriptional factors: NF-κB. selenomethionine and Se-methyloselenocysteine can be transformed into selenides.51]. but it is likely that one of Se metabolites is essential to this process.Vitamins and selenium: Anticancerogenic activity of selenium 97 and AP1 [47]. 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]. Effect of different selenium compounds in cancer prevention The chemoprevention of cancer by selenium can involve the action of different selenometabolites. Trx inhibits certain kinases of the MAPK family (e.

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

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.65]. an important epigenetic mechanism that exerts control over gene expression [30]. It has been found that Se derivatives can activate the p53 gene. and 2-nitrophenylselenocyanate) were found to cause a dose-dependent decrease in the activity of formamidopyrimidine-DNA glycosylase (Fpg). one of the enzymes involved in DNA repair processes.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]. which is a dominant mechanism of the cell death these compounds bring about [70]. In investigating the ability of inorganic Se compounds to induce apoptosis. Limitations in the DNA repair capacity appear to be a key determinant of predisposition to cancer [49. DNA methylation is one of the first steps in the carcinogenesis induced by certain chemicals. 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. Reducible selenium compounds (phenylseleninic acid. selenocystine. One possibility is that a small fraction of the SeMet is metabolized to a low-molecular-weight compound [32]. Selenium can also affect DNA methylation. This decreased GSH concentration in the cells induced apoptosis. 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. but that the induction of apoptosis is not a simple result of the regulation of p53 by selenium [16]. [67] described the incorporation of Se into the factors involved in DNA repair regulation. and to be involved in the pathway connected with the p53-dependent activation of DNA repair processes [64]. 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. The postsynthetic methylation of DNA is catalyzed by the family of S-adenosylmethionine-dependent DNA methyltransferases [71]. ebselen. which has an essential role in recognizing DNA lesions in the nucleotides and in the excision of damaged nucleotides in mammalian cells [69]. The methylation of Se compounds to Se(CH3)2 and Se(CH3) results in a decrease in methyl donation. Blessing et al. as well as damaging the integrity of the genes of the DNA repair enzymes [67. 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.66].68]. They also affect the integrity of the XPA protein (xeroderma pigmentosum group A protein). selenomethionine protects the cells from DNA damage through the induction of DNA repair. Seo et al. .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. this preventing DNA methylation from occurring. [49] showed that in normal human fibroblasts. In vitro studies of a mammary cell line exposed to DMBA showed 1. such as benzo(a)pyrene and arsenic [30].

NK cells and macrophages. due to the large amounts of unsaturated fatty acids present in their structure.74].Jolanta Gromadziƒska. a statistically significant increase in morbidity from oesophagus and stomach cancer was noted in a population with low levels of selenium. however. such as suppression of the host immune response to bacterial and viral infections. micronutrients and phytochemicals [81.g. An inverse association between selenium and risk of cancer has been reported both in case-control studies and in followup cohort studies. Se can stimulate the expression of IL-2 receptors found on activated T lymphocytes and on NK cells [79].76]. including nutrients. the inhibition of prostaglandin and immunoglobulin synthesis. 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. The anticarcinogenic effect of selenium is also partly based on its ability to produce antitumour metabolites (e. Low plasma selenium concentrations are thought to be associated with increased morbidity and mortality from cancer. Insufficient dietary intake of selenium results in many types of defects of the immune processes. but no such relationship was found for lung cancer [83]. Research over the last decade points to a significant protective role of Se in preventing the development of malignant neoplasms. such as in connection with antibody production and specific cell immunity [77]. Selenium compounds can activate cytotoxic cells. and impairment of the body’s ability to reject implants and to destroy neoplastic tumours [75.80]. 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. Wojciech Wàsowicz 100 Effects of selenium on immune functions Certain anticancer properties of the selenocompounds may be associated with their effects on cellular immunity. stimulate the expression of cytokine receptors and the proliferation of lymphocytes [73. Low Se concentrations in the tissues and in human body fluids also appear to be associated with numerous changes in the immune system. Edyta Reszka. 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.82]. Case-control studies tend to reflect the short-term Se concentrations in the organism. 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. To date. In a Chinese study. Selenium regulates the immune response by stimulating natural killer cells and activating antigens of various types to destroy the tumour cells [78]. reduction in the activity of T lymphocytes. methylselenol). such as the Se level associated with the dietary pattern at the time of sampling in cases and controls. the results of epidemiologic studies . Cellular membranes of the T lymphocytes are particularly sensitive to Se deficiency. 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.

97) (Table 2. the mean value obtained for groups showing a high basic concentration of Se was RR = 0. A review of epidemiologic studies of lung cancer and of the selenium concentration found in biological material.Vitamins and selenium: Anticancerogenic activity of selenium 101 have been rather inconsistent.87] in which Se in both serum and toenail samples was analyzed.).86 (95% CI: 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. representing a daily Se intake of 55 mg. The findings for three major forms of cancer are summarized below.61–1. 95% CI: 0.85].22).30–0.77–1. the risk of lung cancer at high Se concentrations was found to be RR= 0. those with high toenail Se levels were found to have a particularly high relative risk of lung cancer (RR: 4. whereas others have obtained null results [84.14. 95% CI: 0. Of the studies considered in the meta-analysis. These finding confirm the assumption that the Se level in the toenails can be used as a marker of long-term Se concentration [91]. to 0.09–0.80 (95% CI: 0.87) when toenail Se was used as a marker of the concentration of Se in the body.).17–0. The significant.60) after adjustments for smoking status were made [90]. . The protective effect of Se in connection with lung cancer could also be noted in studies examining Se concentration in the toenails. It is notable that of the women investigated within the Nurses Health Study. whereas no association between selenium level and risk of lung cancer was found in two studies of non-European populations [88.89]. 95% CI: 0.14. when the study population was divided up according to Se level. to 1. Interestingly.44) and for the Dutch population (RR = 0.24–0. The total RR values for lung cancer in patients showing high Se levels ranged from 0.30) in studies of Se intake based on use of a questionnaire.41.20.81) revealed a statistically significant protective effect of high Se concentrations. only the findings for the two Finnish populations (RR = 0.45–1. 11 of which had a prospective design. 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. In the control group of non-cancer patients the mean serum Se concentration was 100 ng/ml blood serum.16). undertaken by Zhou et al [91]. but only in populations with a low mean Se level.00 (95% CI: 0. 95% CI: 0. indicated selenium to have a certain protective effect.74 (95% CI: 0.57–0.94 and RR = 0. Some authors have reported there to be an association between cancer risk and Se status.46 (95% CI: 0. dosedependent protective activity of Se was documented in Finnish and Dutch studies [86. In a metaanalysis of 14 epidemiologic studies. whereas the value for groups with a low basic level of Se was RR = 0.72 (95% CI: 0.50. In most of the reports.58–1.54–34.33.10) for the serum Se level.

41 (0.20 (0.40) 4.109) µg/g female) 227 (33.575 (0.6 (15. [88] NCC Serum Garland et al. [87] cohort Toenail All The Netherlands 3. c data from 91 individuals.126) µg/g male.20) 63 (NA) 290 (36.001 0.76 (0.7) µg/l)b 153 (61.52 0.080) µg/g female)g 0.134) mg/kg) China 4—5 108 (46.166) µg/g) 250 (0. e male subjects randomized in the earliest in the trial.110 ppm) Study Se index Gender Population Years of follow up No.70–2. .3) µg/l)c Finland 8—12 153 (57.1 (19.7 (18.5) µg/l) USA 15—18 258 (0.1 (2.41) f 0. [98] CC Diet Jolanta Gromadziƒska.308) µg/g) 250 (0.60–2.20 (0.41–2.65 (0.30 (0.2 (2.897 (0. 2459 (0.17–0.94) 1.529 (0.5 µg/l) Finland 16—19 77 (53.537 (0.41–1. controls (mean Se level) RR (95% CI) (high vs.2) µg/day) 290 (39.8 (16.37–1.44)e 0.33 (0. [86] NCC Serum Ratnasinghe et al.811 (0. [90] NCC Toenail Hartman et al.550 (0.19) 0.17 NA Knekt et al.0) µg/l USA 5—14 356 (119.0) µg/day) 0.108 ppm) 515 (0.20–1. 112.50 (0. d data from 177 individuals.129) mg/kg) Japan 11—13 77 (113.81) 0. [91]) Author All All All M.43) P for dose-response trend 0.56 (0.48 >0.3 317 (0.49 NA Comstock et al. 0. [97] cohort Diet Zhou et al.537 (0.6) µg/l) 356 (117. [96] Male Female China The Netherlands CC Diet All China — 25 — 1.30–0.547 (0. [93] NCC Serum Knekt et al. f male subjects randomized in the 5th year.36) 0.206) µg/g male. [95] NCC Toenail Van den Brandt et al. Epidemiological studies of the selenium level in the body and risk of lung cancer (according to Zhuo et al. low Se) 0.0) µg/day) 870 (64.3) µg/l F) Male All Male Female USA Male Finland 5—8 3.2 (24. b data from 189 sets.60) 0.66 (0.15) 0.88) 0.006 Hu et al.9) µg/l)d 216 (45 µg/l) 47 (0.14. cases (mean Se level) No. g data from 370 individuals.46 0.08 0.20 (0.02) 1.0 (13. [94] NCC Serum 0.0 µg/l) 120 (119.80 (33.77–1.09–0.12) µg/day 227 (33.102 Table 2.54–34.47–1.07 µg/day) 0. [89] NCC Serum Kabuto et al. Wojciech Wàsowicz a Nested case-control.5 47 (0.61 (0.10 (16.01 0.11 Kromhout et al.5) µg/l)b 145 (57. Edyta Reszka.0 (16.27–1. [92] NCCa Serum Goodman et al.98 (0.

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 2.38. In the Netherlands Cohort Study. colorectal cancer patients with low Se concentrations were found to have a significantly lower survival time .15.61-0. In addition. Several studies have shown that selenium can be of specific help in combatting prostate cancer [99].72 (95% CI: 0.49 (95% CI: 0. 95% CI: 0. 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].17–0. 95%CI: 0. indicating that the intake of selenium was able to reduce the risk of prostate cancer [107].).16.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 2.39) in case-control studies. The findings of this review showed that the pooled RR of prostate cancer for a particular Se intake. especially men who had a PSA concentration equal to or higher than 4 ng/ml [106]. in which the Se concentration in the toenails served as a measure of long-term Se intake.103]. the odds ratio (OR) for the development of cancer that was associated with a high level of Se intake was 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]. 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. 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. A high prediagnostic Se level was found.3–0. which involved a 6. In two large case-control studies of male populations in Great Britain and Canada. Also. to be associated (at a 13-year follow up) with a significantly reduced risk of prostate cancer. 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 = 0.25–0. was 0.9) [105].96) [100]. 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.85).84) in cohort studies and 0. high Se concentration in the toenails was associated with an appreciably lower risk of prostate cancer [101]. no protective effect of higher Se levels was found in either of the groups investigated [108]. however. in the Physicians’ Health Study. defined as the average of the 1st and 4th quintiles or the 1st and 3rd quartiles. although a protective effect of a high Se level (fifth quintile) was found (OR = 0.74 (95% CI: 0.3 years’ follow-up period. In the Health Professionals Follow-Up Study. In a US case-control study of the relation between the risk of colonic malignant or benign tumour and the serum Se level.5.61–1. 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].

611 µg/g) 0. [106] NCC Plasma 577 (0.16 0. b data from 51 sets.15.8 (19.02 Nomura et al.3—0.090) µg/g) NCC Van den Brandt et al.6) µg/l) 456 (114.3 (20.126) µg/g) 249 (131.42—2. cases (mean Se level) No.69 0.4) µg/l) Finland 8—12 46 (59.82 µg/g) 181 (0.05 0.02 (0.60) 0. [103] Case-control Nails Jolanta Gromadziƒska.77 µg/g) 233 (0.104 Table 2.106 (0.96 µg/g) USA 5—14 235 (114. low Se) 1.24 (0. [104] (2000) Toenail USA (JapaneseAmericans) USA UK — 300 (0.13) 1.48—0. [100] NCC Helzlsouer et al.85) P for dose-response trend 0.65—1.99) 0.17—0. Epidemiological studies of the selenium level in the body and risk of prostate cancer Author Serum Serum Toenail Toenail USA 1—6 117 (0.9) 0. Wojciech Wàsowicz a Nested case-control. Edyta Reszka.00 (0.73—2.8) µg/l)b Study Se indicator Population Years of follow up No.622 µg/g) 13 586 (0.018) µg/g) — 249 (128.12 Knekt et al.008 0. [105] Case-control Serum Li et al. [89] NCC Yoshizawa et al.6 µg/l) 0.707 0.54—1. .10) 0.4) µg/l)b 46 (58.40) 1. controls (mean Se level) RR (95% CI) (high vs.5 (0.38 (0.108 (0.018) µg/g) 300 (0.78 (0.581 Allen et al.39 (0.18—0.0 µg/l) The Netherlands 6.3 540 (0.84) 0.530 (0. [94] NCCa Goodman et al.547 (0.3 (14.79 µg/g) USA 5 181 (0.6 (19.69 (0. [101] Cohort 1211 (0.

cases (mean Se level) No.43—1.146) µg/g) 47 (0.88—4.2) µg/l)b 0.7) µg/l) 276 (130. low Se) P for dose-response trend Knekt et al.0 (18.3 (17.091)) The Netherlands 3.3 Colon 121 1209 USA 3. Epidemiological studies of the selenium level in the body and risk of and colorectal cancer Author Male Female 1.7 (0. c data from 59 sets. [112] NCC Serum Nelson et al.41—2.575 (0.75) 0.7) µg/l)c 48 (65.724 0.3 (15. 105 .9) µg/l)b 1.58) 0. [94] NCC Serum Wallace et al.3 Colon 112 1246 (0.186) µg/g) 2.535 (0.733 (0.30) 1.3) µg/l)c Finland 8—12 29 (63.12 0. [108] Case Serum -control Female Male (0.91 (0.863 (0.44—1.593 (0.131 0.65) 29 (64.411) µg/g) Female (0. [94] NCCa Serum Knekt et al.18—5.0 (18.Table 2.00) 0.3 (18.123) µg/g) The Netherlands 3.273 0.04 (0.50 NA Study Se indicator Gender Population Years of follow up No.42—2.01 (0.9) All All USA — 25 (138 µg/l) 138 (134 µg/l) USA 1—4 276 (131. [90] NCC Toenail Van den Brandt Cohort Toenail Vitamins and selenium: Anticancerogenic activity of selenium et al.22) 0.326 Garland et al.16. b data from 32 sets.8) µg/l) Finland 8—12 48 (64.10 (0.77 (0.5 (19.58 (0.5 89 (0.547 (0.82 (0.643 0.578 (0.560 (0. [111] a Nested case-control.5—5. [111] Van den Brandt Cohort Toenail et al.108)) 1.092) µg/g) Rectal 76 (0.76 (0.45) 0.59—4.106)) Rectal 36 (0. controls (mean Se level) RR (95% CI) (high vs.41—1.843 (0.92) 0.

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. However.57. however. Edyta Reszka.118]. There is a need of clarifying the role of Se . However. and the like need to be taken into account. To sum up.3-year follow-up study of a Dutch cohort in which toenail Se level was used as an indicator [111]. 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. No inverse association of Se level and cancer risk was found for lung cancer in females. The majority of studies on relations between selenium level and occurrence of colorectal cancer have yielded null results for both males and females.Jolanta Gromadziƒska. as found in the Polyp Prevention Study [113]. prostate or colorectal cancer. a number of epidemiologic studies show a low Se level. gender.19–0. to be associated with an elevated risk of lung and prostate cancer. smoking status and study site. especially in males. 95%CI: 0. However. A similar result was obtained in a 3. breast cancer was not found to be influenced by the selenium level [114–116].34–0. A low level of selenium concentration in the body was shown to be associated with a higher risk of lung. 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. a significant inverse association was obtained between toenail Se level and risk of colon cancer (OR = 0. 95% CI: 0. possibly due in part to the rather low proportion of women in the study population [94].112]. Summary Results of epidemiologic and laboratory studies have indicated selenium to have a protective role in counteracting or preventing the development of cancer. genetic susceptibility. and the Polyp Prevention Study. The selenium level was measured in blood specimens of 1763 trial participants. In contrast to prostate cancer. no significant association being found between risk of cancer and toenail Se status. The potential role of dietary Se in the early prevention of colorectal neoplasms would need to be confirmed. selenoprotein P (SeP) concentration or glutathione peroxidase activity in the plasma [110]. To gain further insight into the anti-carcinogenic potential of selenium. the Polyp Prevention Trial.42. Colorectal cancer risk was also analyzed in a 41-month follow-up of the Nurses’ Health Study [90]. age.95) [113]. environmental exposure. After adjustment for age. and the preventive role of Se regarding cancer of this type. gender. a variety of confounding factors such as geographical location.93) [102]. a pooled analysis of data from three clinical trials of colorectal adenoma was conducted: the Wheat Bran Fiber Trial. although this result was only statistically significant in the case of the Polyp Prevention Study (OR: 0. In a Canadian case-control study. would need further verification as well. there are some reports of a significant inverse relationship between blood Se levels and the prevalence of adenomatous polyps [117. Wojciech Wàsowicz 106 and a lower cumulative cancer-related survival rate than such patients with higher Se concentrations did [109].

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

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

↑Bax.↓HIF-1α. PC-3. ↑p21. ↓TNF a-induced NF-κB activation Apoptosis. ↑p53. NOS-2. ↓CDK2/4/6. ↓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. ↓ colony formation [19] PC-3 prostate cancer cells ↓p50. p16. ↓p65. p27. LAN-5. ↓Bcl2. ↑p21. ↑HER2 degradation Apoptosis [22] A549 lung cancer cells in vitro ↓Akt. ↑cleavage of caspases 3. MMP9. ↓p70S6K1. ↑cleavage of caspase 3. ↓p70S6K1. ↓HER2/neu phosphorylation. ↓IκBα degradation and phosphorylation. COX-2. VEGF Apoptosis [20] DU145 prostate cancer cells ↓cyclin D1/2 & E. ↑IκBα . ↑IκBα Apoptosis Effect Reference [15] PWR-1E. altered Bax:Bcl2 ratio. HT-29 colon cancer cells ↓CK2. LNCaP. [26] . apoptosis HER2-overexpressing breast tumor cells ↓PI3K & Akt activity. ↓TNFα activation of NF-κB ↓Bcl2. cyclin D1. ↑p27 Apoptosis. ↓IKKα actvity. ↑DFF-45 cleavage.Bioactive components in foods: Anticarcinogenic effects of flavonoids 117 Table 3.↓HIF-1α. ↑Bax. ↓VEGF and in nude mice OVCAR-3 and A2780/CP70 ovarian cancer cells ↓Akt. SK-N-BE neuroblastoma cells Breast cancer cells ↑ROS.7.9 and cIAP-2 ↓fatty acid synthase activity Apoptosis [16] [17] Growth inhibition and apoptosis ↑p53. DU145 prostate tumour cells Breast and prostate cancer cells NUB-7. ↓PI3K-HER2 docking.1. ↓VEGF. ↑cleavage of caspase 3 [18] Apoptosis (p53-dependent) ↓Her2.8. ↑APAF-1. p18. ↓cyclin D1/D3 & cdk4. G1 arrest. ↑cyt c release. Chemopreventive suppressing effects of apigenin Tissue/cell type Jurkat T cells Mechanism ↓chymotrypsin-like activity of 20S and 26S proteosomes. ↓Akt. ↓NF-κB p50 & p65 [21] Growth inhibition. ↓NF-κB-DNA-binding.

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

Cancer Prevention — the potential for diet to modulate molecular signalling. Phosphorylation of JNK1/2 and p38 was increased by the combination. [39]. Mut Res 2004. Manson MM. including cyclins.9:11–8. nor quercetin stability were affected by ellagic acid. and induction of apoptosis. Li YW.41:1842–53. mitochondrial membrane depolarisation. Suppressing activities include inhibition of signalling pathways responsible for cell proliferation and survival. Dodson VL Quercetin and myricetin protect against hydrogen peroxide-induced DNA damage (strand breaks and oxidised pyrimidines) in human lymphocytes. Duthie SJ. Mut Res Gen Toxicol Environ Mutagenesis 1997.Bioactive components in foods: Anticarcinogenic effects of flavonoids 119 acid receptor (RARβ). which. They can also induce cell cycle arrest by modulating key components of cell cycle regulation.555:53–64. in several different cancer cell types. involved direct interaction with the enzyme. 3. while quercetin alone only induced p38 phosphorylation. Collins AR. exhibit a wide range of potentially useful activities for cancer prevention. Cell signaling pathways altered by natural chemopreventive agents. cytochrome c release and activation of caspases. For example Mertens-Talcott et al. Surh Y-J. mainly through intrinsic pathways involving Bcl family members. Their blocking activities include antioxidant effects. Ellagic acid potentiated the inducing effect of quercetin on levels of p21 and phosphorylation of p53 at serine 15. Conclusions Flavonoids. Neither the generation of ROS. Duthie GG. Inhibition of survival signalling by dietary polyphenols and indole-3-carbinol.3:768–80. synergistic or antagonistic effects of combinations of more than one chemopreventive agent. Trends Mol Med 2003. cyclin dependent kinases and inhibitors. modulation of drug metabolising enzymes and multidrug resistant genes.393:223–31. using MOLT-4 human leukaemia cells. showed that quercetin and ellagic acid acted synergistically in inducing apoptosis. Cancer chemoprevention with dietary phytochemicals. Combinations of flavonoids were found to have an inhibitory effect on the breast cancer resistance protein (ABCG2). Nature Rev Cancer 2003. . like other types of dietary chemopreventive agents. Eur J Cancer 2005. References 1. in the case of EGCG. Sarkar FH. Manson MM. 4. suggesting the potential use of ‘flavonoid cocktails’ to reverse multi-drug resistance in treatment of this cancer [40]. The mechanism proposed was through inhibition of DNA methyltransferase. 5. 2. Combined effects There is now accumulating evidence for the additive.

Greaves P.551:245–54. Heiss E. Nutr Cancer 2004.Margaret M. Chen S. Hladky SB. Inhibition of P-glycoprotein function and expression by kaempferol and quercetin. Individual and interactive effects of apigenin analogs on G2/M cell cycle arrest in human colon carcinoma cell lines. Knauft J. 10. Alt A. et al. 7. Verschoyle RD. Chen D.272:4725–40. 16. Modulatory effects of plant phenols on human multi-drug resistance proteins 1. Watson RWG. a natural product derived from hop. 9. Shukla S. 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. Gerhauser C. Biochem Pharmacol 2005. Mut Res 2005. Steward WP. Morrissey C. Daniel KG. Prostate 2005. Gamal-Eldeen A. Cancer preventive activity of xanthohumol. Kaplan DR. Mut Res 2004.91:1364–71. Yeger H.4:1287–92. Wu CP. 13. Dou QP. Brusselmans K. 20.582:155–62. Vrolix R. Kuhn DJ. Cai H. Effect of flavonoids and vitamin E on cyclooxygenase-2 (COX-2) transcription. Platton S. Verhoeven. Febs J 2005. Kleinjans JCS. Manson 120 6. Dietary flavonoids as proteosome inhibitors in human leukemia cells. Williamson G. 11. p53-mediated apoptosis by apigenin in human neuroblastoma. Boots AW. Steward WP et al. Mol Cancer Ther 2005. Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells. Calcagno SB. Khantamat O. Christoffel V. Spengler B. Stewart JW. Qin CH. 8. Mol Cancer Ther 2002. Irons KA. Manson MM. Tunstall RG. Zhang S. 4 and 5 (ABCC1. 4 and 5). J Biol Chem 2005. VanAlstyne PC. Gupta S. Chen MS. 17. Safe SH Flavonoids as aryl hydrocarbon receptor agonists/antagonists: effects of structure and cell-context.69:1421–32. Brit J Cancer 2004. Bao YP. 18. Torkin R. Klimo K.10:3169–78. 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. Fitzpatrick JM. O’Brien NM. de Pascual-Tereasa S. Lavoi JF. Al-Fayez M. Kao MC. O’Neill A. Hollman PCH. Wilms LC.579:145–52. Hudson EA. et al. Wang WQ.17:86–95. Lin JK. Barrand MA. 12. Clin Cancer Res 2004. Limtrakul P. .1:959–69. Birt DR.63:131–42. Env Health Persp 2003. 14. Pintha K. Mol Cancer Ther 2005.111:1877–82. J Chemotherapy 2005. O’Leary KA. The rice bran constituent tricin potently inhibits cyclooxygenase enzymes and interferes with intestinal carcinogenesis in Apcmin mice.280:5636–45.4:1–11. Protection by quercitin and quercitin-rich fruit juice against induction of oxidative DNA damage and formation of BPDE-DNA adducts in human lymphocytes. Cai H. Swinnen JV. Greaves P. Febs Lett 2005. Landis-PiWowar KR. 15. Induction of caspase dependent. Ambudkar SV. Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity. 19. Way TD. Degradation of Her2/neu by apigenin induces apoptosis through cytochrome c release and caspase 3 activation in HER2/Neu overexpressing breast cancer cells.48:106–14. Neumann I.

31. Phytoestrogen exposure elevates PTEN levels. Kim HK. Targeting of focal adhesion kinase by flavonoids and small interfering RNAs reduces tumor cell migration ability. Sulikova M. Gong AY. Muga SJ. Flavonoids promote cell migration in nontumorigenic colon epithelial cells differing in APC genotype: implications of matrix metalloproteinase activity.14:1457–63. Pelling JC. 28. Human Mol Genetics 2005. Liu HF. Jiang BH. Flavonoid quercetin. Lee PPH. Wargovich MJ.279:4479–89.19:342–53. Mol Pharmacol 2005. Quercetin. Salh B. Farah M. 39:114–24. silymarin. Gupta 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.52:273–9. Calderwood SK. Bodo J. Shukla S. The 70 kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer. . Eivemark S.6-dichloro-ribifuranosylbenzimidazoleand apigenin-induced sensitization of colon cancer cells to TNF-alpha-mediated apoptosis. Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells. curcumin. 36. Huang YT. a potent inhibitor against beta-catenin/Tcf signaling in SW480 colon cancer cells. Fang J. Knudson A. but not apigenin or luteolin. thelial growth factor and angiogenesis in human lung cancer cells. Moussavi M. 23. induced apoptosis in human myeloid leukemia cells and their resistant variants. Hu XW Shi XL. Jung KC. Int J Hyperthermia 2004. Parhar K. Young CYF. Mol Carcinogenesis 2004. Zhou Q. Fang J. 35.25:2017–25. J Nutr Biochem 2005.26:793–801. Davenport DM. Lee MT. Kim WK. Park CH. Apigenin inhibits expression of vascular endo.Bioactive components in foods: Anticarcinogenic effects of flavonoids 121 21.279:55875–85. Zheng JZ. J Biol Chem 2004. Cao ZX. Reed E. Sedlak J. 29. Yang CH. 30. Modulation of aberrant crypt foci and apoptosis by dietary herbal supplements (quercetin. Kim ES. Van Dross R. J Nutr 2003. Quercetin decreases the expression of ErbB2 and ERbB3 proteins in HT-29 human colon cancer cells. 5. Lin JK. Duraj J. Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells. Carcinogenesis 2005. Kang NE.48:182–8. Am J Physiol 2003. Fenton JI. Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells. Neoplasma 2005. Zazrivcova K. The chemopreventive bioflavonoid apigenin modulates signal transduction pathways in keratinocyte and colon carcinoma cell lines. 26. 27. Park S. Cho HJ et al. Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K and HDM2/p53. 34. Xue Y. Kuo PC. Waite KA.328:227–34. Biochem Biophys Res Comm 2005. Jones EL. 24. Hahm ER.20:835–49.68:635–43. Yuan HQ. Chang JY. J Biol Chem 2004.16:155–62.285:G919–28. Volate SR.26:1450–6. 25. Way TD. Hord NG. 22. Zhao MJ. Lee LT. Anticancer Res 2005. Bang MH. Eng C. Lui LZ. FASEB J 2005. Stevenson MA. Lin YS. 33. Carcinogenesis 2005. Nutr Cancer 2004. Sinden MR. 32. ginseng and rutin).133:3800S–4S. Jiang BH. Chao JI. Kao MC.

Yang CS. Reversal of hypermethylation and reactivation of p16 ink4a. Mertens-Talcott SU. Fang MZ. Wang Y.11:7033–41. 39. . Cancer Res 2003. Bomser JA. Christman JK. Clin Cancer Res 2005. Talcott ST. Ai N. Chen D. Romero C. Combined effects of multiple flavonoids on breast cancer resistance protein (ABCG2)-mediated transport. Hou Z. Sun Y.135:609–14. Fang MZ. Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Percival SS. 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. Lu H. Yang XN.21:1263–73. et al. J Nutr 2005. Morris ME. Zhang SZ. Manson 122 37. Pharmaceutical Res 2004.63:7563–70. 40. RARβ and MGMT genes by genistein and other isoflavones from soy. Sun Y. 38.Margaret M.

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

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

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

8–7.3 0.0 5.27) 2.46 (0.8 (0.3 (0. Phenolic acids Flavonoids.5 42.4–9.Jakob Linseisen.7–9. Most of the studies concerned only one or some few compounds within a given subclass of polyphenols.2 1.8) Range 3. even after the administration of polyphenol preparations or polyphenol-rich food corresponding to 50 mg aglycone equivalents. Phenolic acids Flavonoids.8–29.3 5.04 1.7) 7.4.87 8. Sabine Rohrmann 126 Table 3.3 (3.88 (0.50 Urinary excretion2 (% of intake) Mean(SEM) Range Elimination half-life (h) Mean (SEM) 5.4–62. Table 3.6 (4. GC — gas chromatography.21–0.8) 8.0) 6. (according to [7]).0–9. [7]) Tmax (h) Mean (SEM) Range Daidzin Daidzein Genistin Genistein Glycitin Hesperidin 6.26 42.25) 1.5 (0.6 6.8 4.2 Cmax (µmol/l) Mean(SEM) 1.9 (1.4.52) 1.0–6.21) Range 0.76–3.9 2.26–4. Phenolic acids Flavonoids.8) 8.92 (0.6 4.1 (0.9 (12.6) 4. 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.4–5.5 15.1) 5.4 .1 (0. Relatively low plasma concentrations were obtained in each case.6) 4. Differences between the various compounds and of classes of polyphenol are striking nevertheless. Kinetic data from the experiments in question are summarized in Table 3.3) 8.6 (1.4–8.0) 21.50–2.00 0.46–4.14 0.5 (0.0–55.5 (0.0 27.0) 19.57 (0. lignans GC LC Immunoassays Mass spectrometry Mass spectrometry Time-resolved fluorescence HPLC — high-performance liquid chromatography.6) 5.0 7.0 3.3 (0.84 (0.7 7.0) 3–24.1 6.56 (1.36–3.7–10.3. 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]. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al. Phenolic acids Phenolic acids Flavonoids. Phenolic acids Isoflavones. LC — liquid chromatography.00) 1.3 3.38) 0.0–5.

02) 0.06) 0. The magnitude of the variation in the plasma polyphenol concentrations found between free-living subjects following their usual diet habits was .00 (0.07–1.1–30. 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].10 0.5–2. [7]) — cont.51–3.02 (0.7–2.5–5.03 (0.5) Range 1.5 (1.5 0.20 0.7–4.4.2 1.5–2.50 0.06 (0.1 1.2) 10.70 Urinary excretion2 (% of intake) Mean(SEM) 8.01) 0.5 (0.7 0.46 (0. EGC — epigallocatechin.6 0.03) 2.0 2.7) 11.4 (0.8 2.9 (2.09–1.7 5.1–61.1–1.4 0.8 (0.20 (0. Tmax (h) Mean (SEM) Range Naringin Quercetingluc.1 1. 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.80 0. Tmax — time to reach Cmax. 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) 3. although one study in particular failed to support this conclusion [12].5 (0.7 27.1) 2.4 (0.12 (0.0 (0.3 (0.03) 4.1 Elimination half-life (h) Mean (SEM) 2.1) 1.7 (0.50 (0.5 (0.1) 11.96 (0.0 1.31–6.6 (0.4 (0.001–0. Usually represent 24-h urine samples.5–2.0 4.3) 18.5 (5.9 (8.6) 2. flavanones or isoflavones) as biomarkers of polyphenol intake [9–16].1–55.8–28.38 0.2 0.6 (17.45–1.1 (0.3–9.004–5.3 (0.09) 1.10 (0.09–0.45) 0. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al. The authors examined evidence relating to the effects of these polyphenols had on biomarkers used to test various pathophysiologically relevant hypotheses in vivo.7) 1.4) 2.0–0.57) 0.3) 0.13–1.0) 36.0 4.7 (1.0 0.3) 6.3 0.26) 0.35 10.7–2.03–0.4–39.0 0.33) 1.9–28.3–1. AUC — area under the plasma concentration-time curve.0 1.03 0.6 0.6–5.1 0.3) 1.9 4.2) 1.2–15.3–2.1 (0.03 All the data were converted so as to correspond to a supply of 50 mg aglycone equivalent.4) Range 1. Rutin (Epi)catechin EGC EGCG Gallic acid Chlorogenic acid Caffeic acid Ferulic acid Anthocyanins Proanthocyanidins 1 2 Cmax (µmol/l) Mean(SEM) 0.52 0.1–4.6–3.2) 0.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 127 Table 3.5 (0.008–0.0 1.4) 2.70 37.40) 0.1) 1.1 (3.1) 1.26 Range 0.8 (3.1 2.2) 1.3 0. EGCG — epigallocatechin gallate.6) 3.17) 2.57–4.3 (0.30–2.5 0.5 0.5 17.40 (0.0 19.

9 7103.0a 58.6 339. 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.2) 1031.9 372.0 742.0 6103.1) 520.1 (94.9) 267.3 644.5 114.6 460.3 388.4 59.3 (153.8 158.9) 0. 91.7 (78. a precondition most likely to be fulfilled by compounds such as kaempferol that are widely distributed in plant foods.0a 0.6 413.7 2290. Due to the short half-life time of most polyphenols.4 2904.9 2641.9) 140.8 0.0a 0.8) 108.5.9 563.0a 0. Table 3.5 Mean (SD) Min.3) 652.2 (17. (nmol/l) Max.8 (77.Jakob Linseisen. close to of steady-state plasma concentrations of them can only be achieved if the substance in question is consumed regularly.0a 0.5) 1304. 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 68.7 1348. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet.8 82.4) 107.3 1253.7) 277.4) 10.6) 2160. and if the bioavailability of the compound is not particularly low.0 0.2) 260.0a 0.1 (203.7 (48.0 (3077.8 .1 1357. Sabine Rohrmann 128 found to be rather high (Table 3.6 (275.8 1121.3 (67.1 (2.0a 0.7 561.0a 0.0a 0. participating in a cross-sectional study (according to Bolarinwa and Linseisen [16]. the intra-individual variation also being high [14].3 (42.).4 104468.6) 6990.1 (41.0 478.6 (254.7 (15.6 2542.0a 78.0a 19.4 159.0a 48. and that the correlations are much lower when intake calculations are based on data obtained either three or seven days prior to blood sampling.2 0.75.8 26.1 6849.5) 908.8 (15.5.6) 477.0a 0.9 4528.1 53. 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.

In the following.7 151.0a 0. and total flavonoids as being 25(23). are taken up. kaempferol.0a 0. some of which appear very promising.7 1356. Plasma and urinary polyphenol concentrations are not expected to reflect long-term or habitual dietary intake. .24 and 0.1 (40.8 (80.6 36. Hertog and colleagues were the first to analyze commonly consumed foods in terms of their flavonol and flavone content by means of HPLC.3 (1.2 (4. One study reported correlation coefficients of between 0. several studies on associations with the risk of cardiovascular diseases and with cancer at different sites.8 5. 50(32).74 for plasma isoflavone concentrations.7) 545. 701(659). 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. 76(110). participating in a cross-sectional study (according to Bolarinwa and Linseisen [16] — cont.1) 37.0 108.5 204. 1638(1316) µg/24 h. Compound Naringenin Luteolin Genistein Hesperetin Kaempferol Apigenin Isorhamnetin a Median 79.2 Mean (SD) Min. The urinary polyphenol excretion rates show a high degree of variability.0a 0.4) 9. In large-scale epidemiologic (etiological) studies of disease-related effects of dietary polyphenol levels. naringenin.9) 31. little use has been made of biomarker measurements.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 129 Table 3.8 Below the detection limit.38 [13]. (nmol/l) Max.8 52.0a 0.8) 157. 122.0a 0. although this has not been investigated tested extensively.1 27.28 and 0.9) 126.1 2555.3 14.0a 0. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet. Only few studies concerning the use of biomarkers of polyphenol intake were found to be available (Table 3. the dietary intake estimates being based on FFQ data [11].0 (7. respectively.1 (24.7 388. just as was indicated already for the plasma concentrations.6). To give an example of the polyphenol concentrations found in 24-h urine samples of subjects on a habitual diet.5 (21.3) 0. the correlation coefficients for selected flavonols and flavanones being between 0.0a 533. Nielsen and coworkers [13] reported average (SD) concentrations of quercetin. Their work provided the basis for the estimation of dietary flavonol and flavone intake.2 639. phloretin. except as regards the intake of phytoestrogens (isoflavones and lignans).5.

total phenols Breast NS Sun et al. isoflavones Urine. [23] Pietinen et al. enterolactone Result Significant inverse association NS Significant inverse association Significant inverse association Significant inverse association Significant inverse association NS 248/492 Plasma. lignans Higher risk when isoflavone estimates are higher CCS — case-control study. 1999 [18] CCS 60/60 Urine. enterolactone Serum. [22] Dai et al. [26] Type CCS CCS CCS CCS CCS CCS Cohort: nested CCS Cohort: nested CCS N. Table 3. enterolactone Urine. NS — not significant. [18] Murkies et al. Urine. [24] den Tonkelaar et al. Phytoestrogen Urine. cases/ controls 250/250 Specimen. [17] Piller et al.) may be the ready availability of appropriate immunoassays suitable for measurements on large numbers of samples. Author Ingram et al. tea polyphenols Gastric and esophageal Significant inverse association CCS — case-control study. isoflavones Urine. enterolactone Urine. One of the major reasons for the frequent use of biomarkers of phytoestrogen intake in epidemiological studies (see Table 3. Sabine Rohrmann 130 Table 3.6.7. isoflavones.Jakob Linseisen. genistein. enabling the attainment of the requirement of sufficient statistical power. 2002 [19] Cohort 232/772 Urine. Epidemiological studies of the risk of breast cancer risk in which biomarkers of dietary isoflavone and mammalian lignans intake were employed. enterolactone for highest and lowest categories Significant positive association Grace et al. . NS — not significant. equol.7. Flavonoid Urine. [21] Zheng et al. cases/ controls 144/144 60/60 18/20 250/250 220/237 194/208 88/268 Specimen. citrus flavonoids Cancer site Breast Result NS Zheng et al. isoflavones. [25] Hulten et al. 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. [27] Cohort: nested CCS 97/187 114/219 Serum. 2002 [17] Type CCS N. lignans Plasma.

except for some few small studies [14]. the concentrations were found to be high enough to enable the characteristic mass fragments to be identified. To the best of our knowledge. in which the sample volumes available are usually very small. However. The higher values here are those for the MS-based techniques (coefficient of variation < 5–7%. LC-MS. confirmation of their results by use of MS-based techniques is necessary. MS-based systems also have their problems in connection with concentrations close to the detection limit. For all these methods. Reproducibility. Specificity and sensitivity In applying mass-selective detection methods at least to standard mixtures or biological specimens spiked with the compound in question. such as HPLC-UV/VIS. . GC-MS and HPLC with use of fluorometric detection is slightly lower. The sensitivity achievable with HPLC-MS. the immuno-assays being located at the other end of the scale. and reports on the quality of the method in question are usually obtained clearly above the detection limit. HPLC-MS-MS. MS-based systems also have their problems when the concentrations involved are very low. having coefficients of variation close to or above 10% and recovery rates frequently at < 90%. Antibody-based assays are also subject to failures in specificity due to cross-reactions with other matrix compounds. the analysis of polyphenol content is restricted in most cases to only a few compounds or classes of compounds. Detection limits very close to 1 nmol/l of polyphenols have been found for techniques involving HPLC-ECD. validity and reliability For each well-developed method. however. This differs from other methods. Sensitivity is a major issue with regard to analytical methods for determining polyphenols in biological specimens. ECD (electron capture detection). In epidemiological studies in particular. working at the detection limit of a method is always a challenge.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). the degree of sensitivity a method provides can be decisive for whether it can be used or not (along with other factors. recovery 90–105%). no systematic findings on repeated within-subject samples or on losses during sample storage are available. where the characteristic fragments may be absent. such as analytical time and costs). Accounts of analytical procedures that permit a wide variety of polyphenols to be determined in a single run were published recently [16.20]. As already mentioned. or use of fluorescence detectors. satisfactory figures concerning the analytical precision and accuracy are available. in which peak identity cannot be confirmed with sufficient certainty.

Merken HM. Manach C. Plasma concentrations of the flavonoids hesperetin. Am J Clin Nutr 2005. Application of Biomarkers in Cancer Epidemiology.41:203–9. Blake A. Flavonoids in human urine as biomarkers for intake of fruits and vegetables. Nielsen SE. Bioavailability and bioefficacy of polyphenols in humans. et al. Kesaniemi YA. 7. Wang GJ. 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. Lapcik O. Bioavailability and bioefficacy of polyphenols in humans.56:891–8. J Agric Food Chem 2000. Beecher GR.. Pharmacokinetics and metabolism of dietary flavonoids in humans. Williamson G.823:143–51.Jakob Linseisen. Kaaks R. Prediction of dietary flavonol consumption from fasting plasma concentration or urinary excretion. Noroozi M. IARC Scientific Publications No. Linseisen J. Wolfram G. Rantala M. Mutanen M. Steroids 2000. Manach C. 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). Jimenez L.38:771–85. Key TJ. 2. Freese R. 11. Free Radic Res 2004. Am J Clin Nutr 2004. . 13. Allen NE. Scalbert A.86:415–21. Am J Clin Nutr 2005. and diets high or low in fruit and vegetables. van Staveren WA. Morand C. Plasma and urinary phyto-oestrogens as biomarkers of intake: validation by duplicate diet analysis. Hampl R. 5.81 Suppl 1:243S–55S. Bolarinwa A. 12. Verkasalo PK. Uehara M. 4. Eur J Clin Nutr 2002. Morand C. Eur J Nutr 2002. Cancer Epidemiol Biomarkers Prev 2002. Sabine Rohrmann 132 References 1. Al-Maharik N. Scalbert A. Phenolic acids in foods: an overview of analytical methodology. Remesy C.91:447–57. Aro A. Kleemola P. Alfthan G. Hollman PC.65:339–48. Cummings JH. Lean ME. Measurement of food flavonoids by high-performance liquid chromatography: A review. Ritchie MR. editors. In: Toniolo P et al. Crozier A. Remesy C. Deighton N. Adlercreutz H. I. Eur J Clin Nutr 2000. J Chromatogr B Analyt Technol Biomed Life Sci 2005.11:459–66. J Agric Food Chem 2003. Zock PL. Riboli E. Br J Nutr 2004. 16. Buysman MN. Sinha R. Review of 97 bioavailability studies. Meyboom S. Lyon: IARC. Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake. et al. Am J Clin Nutr 1998.68:60–5. II. 6. Linseisen J. Morton MS. Davey G. Stumpf K. 1997. 142. Biochemical markers of dietary intake. Robbins RJ. 8. Silaste ML.51:2866–87. 14. Fasting plasma concentrations of selected flavonoids as markers of their ordinary dietary intake. Radtke J.48:577–99. Manach C. Erlund I. Donovan JL. Williamson G. de Vries JH. Kelly IE. 10. Polyphenols: food sources and bioavailability. 15. Br J Nutr 2001.81 Suppl 1:230S–42S. 9. 3. naringenin and quercetin in human subjects following their habitual diets. Appleby PN.54:143–9. Time-resolved fluoroimmunoassay of plasma daidzein and genistein. Burns J. Review of 93 intervention studies. Manach C.79:727–47.

23:1497–503. Pietinen P. Yuan JM. Custer LJ. Jin F. Dai Q. et al.7:289–96. Hallmans G. Manach C. Wahlqvist ML.10:223–8. Urinary excretion of phytoestrogens and risk of breast cancer among Chinese women in Shanghai. Gonthiera MP. Menopause 2000. Shu XO. Biofactors 2004. Stumpf K. Mulligan AA. Arts CJ.15:225–32. Mennen L. High-throughput profiling of dietary polyphenols and their metabolites by HPLC-ESI-MS-MS in human urine. Gao YT. Sun CL. Briganti EM.10:339–44.22:241–3. Phytoestrogens and breast cancer in postmenopausal women: a case control study. Jin F. Franke AA. Adlercreutz H. Ito H. Johansson R.13:698–708. Kolybaba M. Urinary phytoestrogens and postmenopausal breast cancer risk. et al.350:990–4. Cancer Epidemiol Biomarkers Prev 2001. Serum enterolactone and risk of breast cancer: a case-control study in eastern Finland. An incident casereferent study on plasma enterolactone and breast cancer risk. China. Linseisen J.8:35–40. Adlercreutz H. Taylor JI. Ross RK. Eur J Cancer Prev 2006. 23.41:168–76. Wen WQ. Cancer Epidemiol Biomarkers Prev 2004. et al. Dai Q. Yang CS. Urinary excretion of isoflavonoids and the risk of breast cancer. Low YL. Veer PV. Cancer Epidemiol Biomarkers Prev 2002. Hebert JR. et al. Kataja V. et al. 18. et al. Grace PB. Botting NP. 19. Lee MJ. Carcinogenesis 2002. Healy DL. Luben RN. Sanders K. Adlercreutz H. Chang-Claude J. Ingram D. 27. Piller R. Remesy C.11:815–21. Case-control study of phyto-oestrogens and breast cancer.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 133 17. Morand C. 20. Den Tonkelaar I. Shu XO. Urinary tea polyphenols in relation to gastric and esophageal cancers: a prospective study of men in Shanghai. Burger HG. 24. . Zheng W. 21. Winkvist A. Uusitupa M. Thijssen JH. 22. 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. et al. 25. Hulten K. Murkies A. 26. Lenner P. Lancet 1997. Dalais FS. Mannisto S. Cancer Epidemiol Biomarkers Prev 1999. Lopez D. Cancer Epidemiol Biomarkers Prev 2001. Plasma enterolactone and genistein and the risk of premenopausal breast cancer. Eur J Nutr 2002. Custer LJ. Keinan-Boker L.

HValc in urine reflects the in vivo .Theodore G. Soterios A. 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. Sotiroudis. Phenolic compounds HPLC chromatography of the methanol extract of virgin olive oil reveals seven major polyphenol peaks corresponding to hydroxytyrosol. Anticarcinogenic compounds of olive oil and related biomarkers Theodore G. A brief overview is presented of recent findings concerning the bioavailability of certain minor but important olive oil minor components (polyphenols. The National Hellenic Research Foundation. a statistically significant negative correlation has been found between homovanillyl alcohol (HValc. Sotiroudis and Soterios A. These include tocopherol and carotenoid antioxidants. A plethora of minor constituents in olive oil have been identified as being effective agents mitigating against the initiation. a major metabolite of hydroxytyrosol. promotion and progression of multistage carcinogenesis.). together with homovanillic acid — HVA) and isoprostane excretion. a number of simple and bound phenolics (tyrosol. which represent major antioxidants in olive oil. Greece Olive oil is an important ingredient of the Mediterranean diet.3. the aglycone of ligstroside. squalene and β-sitosterol). lignans. In addition.2]. are dose-dependently absorbed in humans after the ingestion of realistic doses of virgin olive oil. This indicates olive oil phenolics to maintain their antioxidant activities in vivo. Kyrtopoulos 134 3. the excretion of both HValc and HVA also being significantly correlated with the dose of administered hydroxytyrosol. and a peak containing the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol [1–3] (Table 3. When olive oil samples containing increasing amounts of a phenolic extract of olive oil were administered to human volunteers. Athens. Kyrtopoulos Institute of Biological Research and Biotechnology. tyrosol.2.8. considered as putative nutritional biomarkers in relation to cancer the incidence of cancer. secoiridoids and lignans). 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]. Epidemiological studies demonstrate rather conclusively that populations within Europe consuming this diet have a particularly low incidence of a number of common cancers [1. was observed. the triterpene hydrocarbon squalene and the phytosterol β-sitosterol [1–3]. two secoiridoids (dialdehydes related to oleuropein and ligstroside but lacking the carboxymethyl group at C4). a biomarker of in vivo lipid peroxidation processes. hydroxytyrosol. Oleuropein and its metabolites tyrosol and hydroxytyrosol. Thus. oleuropein.. Figure 3. a dose-dependent decrease in the urinary excretion of the F2-isoprostane 8-iso-PGF2α. that have been thoroughly studied.

8. mg/kg 27. Concentrations of the major phenolic compounds found in virgin olive oil Compounds Tyrosol Hydroxytyrosol Oleuropein aglycone Total secoiridoids Lignans References [2] and [3].Bioactive components in foods: Anticarcinogenic compounds of olive oil and related biomarkers 135 concentration of hydroxytyrosol [5–11]. but this was not observed after the intake of olive oils of moderate to high phenolic content [12]. After the ingestion of olive oil of low phenolic content plasma glutathione peroxidase activity was found to decrease postprandially. (+)-pinoresinol (C). oleuropein aglycone (B). 2. An HPLC method for the simultaneous determination of oleuropein and of its metabolites hydroxytyrosol and tyrosol in human plasma has been developed [13. a biomarker of the intake of tomato-rich food and hypothesized to be responsible for reducing the risk of various .72 [2] 41. Table 3.71 [3] 103–205 [3] 27. 3. Recent findings suggest that olive oil may also affect the bioavailability of other food bioactive components with a chemopreventive potential.45 [2].75 [3] 14. It was observed in this respect. that the concentration in human plasma of lycopene. Structures of certain phenolic compounds detected in olive oil.83–4.14].42 [2]. 1.2.65–4. 38–65 [3] Fig.53 [2]. Hydroxytyrosol (A).

Nevertheless. was found to improve the antioxidant activity of plasma [16]. the squalene intake would be more than 200 mg/d [22].Theodore G. The consumption of tomato products prepared together with olive oil. leading to a reduced farnesyl pyrophosphate availability for prenylation of the ras oncogene [26]. restricted almost entirely to estrogen-receptor alpha negative breast cancer has been found. but not with sunflower oil. which expands the plasma pool of this metabolically active hydrocarbon [25]. and that the epithelial cells in the colon may be responsible for this metabolism [19]. 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]). Phytoestrogens have an anticarcinogenic potential through the anti-estrogenic. Recent findings suggest that enterolactone is more rapidly metabolized in human colon epithelial cells and/or excreted by them than enterodiol is. proapoptotic and anti-oxidant mechanisms established for them [17. its content ranging from 800 to 12. 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. Lignans are plant compounds metabolized in the gut to produce the phytoestrogens enterolactone and enterodiol. Nevertheless. that the phase II metabolism of enterolactone and enterodiol already may take place during their uptake in the colon. as compared to the consumption of tomatoes cooked without olive oil [15]. Experiments in vitro and animal models suggest squalene to play a tumour-inhibiting role. It has been observed that postprandial squalene metabolism is age dependent [23]. Kyrtopoulos 136 cancers.000 mg/kg. Sotiroudis. suggesting that dietary lignans may be important in the etiology of breast cancer. 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. Although animal studies have enhanced our understanding of the possible . 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. which is most probably based on its strong inhibitory action on the catalytic activity of beta-hydroxy-beta-methylglutaryl-CoA reductase. particularly in premenopausal women [21]. while virgin olive oil is a major source of phytosqualene. Soterios A. the prostate and the colon. anti-angiogenic. very little is known concerning the postprandial metabolism of squalene. increased dramatically after the consumption of tomatoes cooked in olive oil. If virgin olive oil were the sole source of dietary fat. This triterpene hydrocarbon is found mainly in nonedible shark liver 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]. whereas the findings of epidemiological studies are controversial [18]. their reaching a steady state. a tendency for a lower risk of breast cancer to be associated with higher plasma concentrations of enterolactone.18].

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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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Ninewells Hospital and Medical School. Cruciferous vegetables uniquely contain glucosinolates at approximately 20 µmol/g dry mass of vegetable [8. lung cancer [5].17]. Claverton Down. though certain broccoli . Glucosinolates are substituted β-thioglucoside N-hydroxysulfates. such as broccoli. methionine. this variable elongation of amino acid side chains entails repetitive additions of methyl groups through a series of transamination. Michael O.John D.4.12–15]. Kelleher1 and Ian M.11].20]. Glucoraphanin has been reported to be abundant in broccoli [9]. The glucosinolate content is primarily under genetic control. valine.. used in their biosynthesis.9. Eggleston 140 3. Bath BA2 7AY. Hayes1. phenylalanine. University of Bath. Furthermore. Eggleston2 1 2 Biomedical Research Centre. University of Dundee. the olefinic glucosinolates sinigrin. namely. though it can be influenced by environmental factors [16. swede and turnip. phenylalanine and methionine. condensation. Subsequently. As shown in Figure 3. and the aromatic glucosinolate gluconasturtiin (Table 3. Kelleher. Anticarcinogenic effects of glucosinolate breakdown products John D. The most common of these are the methylsulfinylalkyl glucosinolates glucoiberin and glucoraphanin. Brussels sprouts. This endeavour is simplified to some extent by the fact that relatively few glucosinolates are present in the human diet. United Kingdom Glucosinolates and their association with cancer chemoprevention Epidemiological studies have revealed that regular consumption of cruciferous vegetables. which is in turn conjugated with glucuronic acid to form a desulfoglucosinolate before finally being sulfated to yield the glucosinolate [2]. tyrosine and tryptophan [2].9]. Feeding experiments in animals have also suggested broccoli can protect against liver cancer [7]. the oxime is metabolised to a thiohydroximate. and possibly prostate cancer [6]. kale.) [9. greater health benefit may be obtained from raw as opposed to cooked vegetables [3].2]. The types of neoplastic disease in man that these vegetables appear to protect against include colorectal cancer [4]. isomerisation and decarboxylation reactions [18]. Much of the diversity amongst glucosinolates arises from the addition of different sized alkyl groups to the side chain of those amino acids. leucine. the synthesis of glucosinolates proceeds through the conversion of elongated amino acids to their oxime derivatives. Hayes. Each cruciferous vegetable contains a mixture of glucosinolates that varies according to the strain of the plant [8.3. Michael O. principally valine. The task of establishing a link between the ingestion of particular glucosinolates and their possible health benefits is not straightforward. alanine. cauliflower. formed by the plant from any one of eight amino acids. catalysed by members of the cytochrome P450 (CYP) 79 family [19]. glucobrassicanapin and progoitrin. Dundee DD1 9SY Department of Pharmacy and Pharmacology. Ian M. isoleucine. Over 115 naturally occurring glucosinolates have been identified. gluconapin. is associated with a reduced incidence of cancer [1. 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. cabbage.

9. cabbage. Synthesis of glucosinolates. gluconapin is also found in high levels in Brussels sprouts [9].Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 141 strains also contain substantial amounts of glucoiberin [21]. Sinigrin has been reported to be the predominant glucosinolate in Brussels sprouts. The indolyl glucosinolate glucobrassicin is present in Savoy cabbage. cauliflower and kale [9]. 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. Table 3. The aromatic glucosinolate gluconasturtiin is present in watercress. 3. Substantial amounts of progoitrin are present in many cruciferous vegetables [9].22]. Brussels sprouts and cauliflower [9.3. The R group is derived from the original amino acid and is highly variable.

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. thiocyanates. The outcome of the reaction with myrosinase depends on the nature of the aglycone. Fig.4A. .3. At high or neutral pH the formation of isothiocyanates is favoured while at low pH the formation of nitriles is favoured.John D. Ian M. or during chewing whilst eating. In addition. EC 3. 3. Eggleston 142 Production of isothiocyanates. Hayes. 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.4A. during food preparation. Michael O. as well as the reaction temperature. the pH and the presence of ferrous ions (Figure 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. during freeze-thawing. for example during harvesting. Hydrolysis of these phytochemicals is catalysed by myrosinase (β-thioglucoside glucohydrolase. Hydrolysis of glucosinolates. but rather it appears to be due to certain of their breakdown products. myrosinase activity may be present in human colonic microflora.). Kelleher. myrosinase is released from the “myrosin” cells and is able to hydrolyse glucosinolates within the damaged plant.2.25]. nitriles.147). resulting in the formation of an epithioalkane. and 3.4B. 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]. Upon wounding of the vegetable. suggesting that glucosinolates can be hydrolysed in the gastrointestinal tract during digestion of food [24.

to form their respective isothiocyanates (ITCs). glucoraphanin. Fig. give rise to a cyano-epithioalkane [27]. The glucosinolates glucoiberin. In this case. 4-methylsulfinylbutyl-ITC (sulforaphane). with the elimination of sulfate. Hydrolysis of sinigrin. . respectively. hydrolysis of glucosinolates with aliphatic or aromatic side chains gives rise primarily to isothiocyanates (ITCs). glucobrassicanapin and sinigrin yield 3-methylsulfinylpropyl-ITC. can convert spontaneously to form allyl isothiocyanate [26]. are unstable and can undergo a relatively slow spontaneous conversion to their respective isothiocyanate [26].Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 143 ESP — epithiospecifier protein. gluconapin and glucobrassicanapin) may. At low pH.g. in the presence of an epithiospecifier protein (ESP) and ferrous ions. thiocyanates or nitriles [10. The thiohydroximate-O-sulfates formed from methylsulfinylalkyl. olefinic and aromatic glucosinolates undergo a Lossen rearrangement. Following damage to the plant tissue the glucosinolate sinigrin is hydrolysed by myrosinase resulting in the formation of four distinct compounds. 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]. 3. On the right-hand side of the figure.23]. Thus. at pH 4 and in the presence of Fe2+ ions. the thiohydroximate-O-sulfates formed by myrosinase from glucosinolates with a side chain containing a double bond (e. formed from sinigrin. gluconapin. such as allyl-ITC. an arrow shows that allyl thiocyanate. Certain thiocyanates that are formed during a Lossen rearrangement. 4-pentenyl-ITC and 2-propenyl-ITC (allyl-ITC).4B. Elemental sulfur is also formed in certain circumstances. 3-butenyl-ITC. sinigrin. At neutral pH.

as a consequence of spontaneous cyclization. Likewise.36]. The best studied of these is glucobrassicin.John D. 3.5. Michael O. hydrolysis of glucobrassicin by myrosinase does not generate an ITC. ESP and Fe2+ ions to an epithionitrile [32] and in the case of epi-progoitrin ((2S)-hydroxy-3-butenyl glucosinolate). is also converted in the presence of myrosinase. and is diminished by heating [40].31]. a (2R)-hydroxy-3-butenyl glucosinolate. 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.5. glucoconringiin and gluconapoleiferin [2]. Production of indoles from glucosinolates. If the aglycone generated by myrosinase is from a glucosinolate with a side chain lacking a double bond.3-epithiopropane [29]. Gluconapin can similarly be converted by the combined actions of myrosinase and ESP to 1-cyano-3. Kelleher. but rather gives rise to indole-3-carbinol and a thiocyanate ion (Figure 3. this reaction probably proceeds Fig. Two cDNAs for ESP have recently been cloned from Arabidopsis and broccoli and the purified proteins characterized following their heterologous expression in E. Production of indoles from glucosinolates The indolyl glucosinolates glucobrassicin and neoglucobrassicin are synthesised by the plant from tryptophan. Eggleston 144 myrosinase and ESP convert sinigrin to 1-cyano-2. Examples of these include progoitrin. Upon hydrolysis by myrosinase. Hayes.34]. coli [35. A few glucosinolates produce thiocyanates though the mechanism involved is unclear [23]. This reaction may involve ESP.). Ian M. glucobrassicanapin can be hydrolysed to 1-cyano-4.4-epithiobutane [30. the sulfur atom may be lost and a nitrile formed [37–39]. those aglycones from glucosinolates that contain β-hydroxylated sidechains form oxazolidine-2-thiones. At neutral pH. it can be hydrolysed by myrosinase to crambene (1-cyano-2-hydroxy-3-butene) [33. .5-epithiopentane [31]. Progoitrin.

There is a dose-dependency in these responses: generally. cyanoepithioalkanes. hydrolysis of glucobrassicin yields indole-3-acetonitrile. At acidic pH. 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. Thus. both of which have potent pharmacological effects [41]. It can also combine with ascorbic acid to form ascorbigen [42] (Figure 3. Fig. hydrogen sulfide and elemental sulfur [22].2-b]carbazole and 3.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 145 through a Lossen rearrangement generating an unstable ITC intermediate [22]. A major problem exists in interpreting experiments utilizing vegetable extracts because of uncertainty about attributing biological effects to specific phytochemicals. induction of cytoprotective genes and inhibition of CYP activity occurs at relatively low concentrations of phytochemical.). 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. Structures of indoles produced from glucosinolates — 3.3’-Diindolylmethane (DIM). indole-3-carbinol condenses to form various compounds including indolo[3. nitriles. when compared with the data about thiocyanates. such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1). inhibition of CYP enzyme activity. 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.46]. indolo [3. and stimulation of apoptosis. cell cycle arrest.44]. and oxazolidine-2-thiones.6. These include induction of antioxidant and detoxifying genes. Agents such as ITC that increase GST and NQO1 . In the acidic environment of the stomach. whereas activation of cell cycle arrest and apoptosis occurs at higher levels of phytochemical [43.2-b] carbazole (ICZ) and ascorbigen (ASG). there is a relative abundance of information about isothiocyanates and indoles. 3. both in vivo and in vitro [45.6.3∂-diindolylmethane.

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

Whether indolo[3.2-b]carbazole and 3. western and northern blotting that CYP1A1 is inducible by indolo[3. Interestingly.69].2-b]carbazole and 3. 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. 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. . this may not necessarily lead to an increase in Nrf2 protein levels.3∂-diindolylmethane [2]. this seems unlikely [72]. and thereby generate reactive oxygen species. The indole-containing glucobrassicin breakdown products can activate gene expression by several mechanisms. the mouse nrf2 gene promoter also contains an XRE [71]. However. and it is therefore anticipated that activation of AhR by glucobrassicin-derived indoles will influence significantly the metabolism of xenobiotics (for a review. rat ALDH-3. Examination of the dose of indole required to double XRE-driven reporter gene expression shows that indolo[3. it is a good inducer of GST enzyme activity in mouse liver and small intestine [68]. mouse. cranbene was found to be an approximately equally potent inducer in the rat [66].Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 147 required for the ubiquitination of Nrf2 under homeostatic conditions [60–62]. and there is abundant evidence from enzyme assays. Consistent with this view. and this may also contribute to gene induction [46]. and rat UGT1A6 contain an XRE. rat UGT1A1. Amongst genes for drugmetabolising enzymes. Surprisingly. By comparison with sulforaphane.3∂-diindolylmethane. Although indole-3-acetonitrile has not been studied in the Nrf2 knockout mouse. This cytochrome has O-deethylase activity towards ethoxyresorufin.3∂-diindolylmethane can increase the stability of Nrf2 protein is not known.67]. indole-3-carbinol or ascorbigen [43. rat GSTA2. rat and human CYP1A1 are prototypic XRE-regulated genes.2-b]carbazole and 3. It has been found that administration of cranbene to Fischer 344 rats causes an elevation in hepatic GST and NQO1 enzyme activities. as does the mouse BAX gene. see [70]).2-b]carbazole is a much more potent inducing agent than 3. The identity of this metabolite is not known.3∂-diindolylmethane. allyl-ITC does not increase Nrf2 stability [64] and there may therefore be other factors involved in enzyme induction by these phytochemicals. but not CYP1A1. a fact that implies it works through Nrf2. Indole-3-carbinol is a modest inducer of Nrf2-dependent ARE-driven gene expression in the liver and small intestine of mice [52. indicating that AhR ligands could lead to an increased production of Nrf2 mRNA. 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. suggesting it is a mono-functional inducer [65].64]. but unless they are metabolised by CYPs. 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. The promoters of other genes including rat and human NQO1.

prior treatment of the LS-174 cells with both indolo[3. Most importantly.2-b]carbazole and sulforaphane before exposure to benzo[a]pyrene was found to confer substantial protection against genotoxicity. Each of the histone proteins contains an evolutionary conserved amino tail protruding from . Furthermore. Inhibition of CYP can also be achieved by sulforaphane [80. Kelleher.John D. Ian M. Within the cell. Inhibition of carcinogen activation by glucosinolate breakdown products Certain isothiocyanates can block the activation of several carcinogens to their ultimate carcinogenic forms. sulfotransferase and UGT1 [74]. In the case of the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. recognition of this possibility has lead to considerable recent interest in the ability of HDAC inhibitors to act as both chemopreventive and chemotherapeutic agents. such as CYP1B1 and CYP19 [73]. such as growth arrest and DNA damage (GADD) GADD34. Also. GADD45 and GADD153 [75. other cytochromes are inducible. 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. as are the drug metabolising enzymes AKR. Hayes. 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 [78.79]. p21 is induced by indole-3-carbinole [77]. Inhibition of histone deacetylase by isothiocyanates The isothiocyanate sulforaphane has been shown to inhibit histone deacetylase (HDAC) [83. Eggleston 148 In addition to induction of CYP1A1 by indoles. This viewpoint is probably an oversimplification and does not take into account the multiple changes in gene expression that indoles affect. Michael O. the basic structural unit of chromatin. 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. Indeed. Bonnesen et al [43] have reported that treatment of human colon LS-174 cells with indolo[3. DNA is tightly coiled around an octamer of histone proteins in a structure known as a nucleosome.81]. and this protection was greater than was achieved by either phytochemical alone [43].2-b]carbazole before exposure to benzo[a]pyrene provides a small measure of protection against DNA damage as measured by the comet assay.76]. the ITCs with highest lipophilicity and low reactivity of their NCS group had the greatest ability to inhibit lung tumorigenesis [82].84]. c-Jun.3∂-diindolylmethane can induce expression of the transcription factors ATF3. GST T1-1. 3. It is not however clear whether the genes for ATF3. NF-IL6 and the GADD proteins are regulated through XREs and whether the process is mediated by AhR. c-Jun and NF-IL6 as well as genes involved in cell growth.

allowing transcription factors access to the regulatory regions of genes.92].85]. p53 [86] and Bax [84. respectively. Equally. Inhibition of HDAC may prevent the removal of acetyl moieties from histones thus allowing transcription of the tumour suppressor genes. However. and was accompanied by its translocation from the nucleus to the cytoplasm [92]. Conversely.dependent fashion [87].Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 149 the nucleosome. removal of acetyl groups causes the tail to wrap tightly around the DNA thereby preventing interaction with the transcription machinery. In pre-cancerous and cancerous cells. mammalian HDAC is capable of downregulating p53 function. Relocation of Cdc25C was found to be controlled by its phosphorylation at Ser-216. mediated through activation of checkpoint kinase 2 (Chk2) [84]. 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. 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. along with other dietary HDAC inhibitors such as diallyl disulfide [88]. tumour suppressor genes are associated with deacetylated histones resulting in the inactivation of these genes. which can determine the accessibility of the DNA to transcription factors. resulting in a reduction in its transcriptional activity. Cell proliferation by ITCs may also be achieved by distrupting cytoskeletal structure and tubulin polymerisation [93.85]. apoptosis and differentiation [89]. The loss of Cdc25C was reported to be due to proteasomal activity. In human PC-3 prostate cancer cells. Thus the ability of sulforaphane. . human embryonic kidney cells [83] and human colorectal cancer cells [83]. 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. this was associated with a higher level of apoptosis and lower proliferation in animals on the ITC containing diet [90].94]. For example. the majority of the studies into mechanisms by which this class of chemical inhibit cell growth have focussed on sulforaphane and phenethyl-ITC. The addition and removal of the acetyl groups is carried out by HAT and HDAC. The tail is also subject to many post-translational modifications including acetylation. to affect chemoprevention via its ability to alter chromatin structure is of acute importance to the welfare of the cell. The link between inhibition of HDAC activity and the resultant increase in transcription of tumour suppressor genes has been reported for p21 [84. 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 shown to diminish HDAC activity with a concomitant rise in histone acetylation in prostate cancer cells [84]. sulforaphane has been found to cause a G2/M phase delay with an increase in apoptotic cell fraction in a timeand dose. by deacetylation of histones associated with the p53 gene. In addition.

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

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

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One of the key problems of research in this field. In this chapter. 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. The combinations of phytochemicals that natural foods contain can reduce the risk of cancer by affecting different overlapping and complementary mechanisms. 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. Combined action of different dietary compounds preventive of cancer Theo M. van Breda 160 3. Evaluating the effects of phytochemicals that act synergistically Carcinogenesis is an extremely complex multistep process in which numerous molecular mechanisms play an important role. In the research conducted. van Breda Department of Health Risk Analysis and Toxicology. 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.Theo M. Table 3. both in vitro and in vivo.6]. evaluation of the effects of synergistically acting phytochemicals is needed. and the like. evidence that bioactive compounds act synergistically is reviewed. de Kok. in contrast.5. whole grains. In addition to characterizing the chemopreventive effects of individual compounds. vegetables. 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. Maastricht. de Kok and Simone G.2]. 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. 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. Isolated and purified compounds. are far from certain. summarizes various mechanisms by which phytochemicals can modulate the risk of cancer [1. The Netherlands Introduction The relatively consistent epidemiological finding that the consumption of whole foods of different types such as fruits. Some studies show no reduced incidence of cancer as a result of taking supplements of vitamin C [3] or β-carotene [4]. . which are believed to have cancer-preventive effects. 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. Cancer-preventive dietary compounds may interfere at a variety of different levels with these processes. The intended positive effects of an increased intake of β-carotene or vitamin C as dietary supplements. Simone G. University of Maastricht. therefore. numerous bioactive compounds have been isolated and identified. Although relatively high doses of single bioactive agents may show potent anticarcinogenic effects. however.

[2]. 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. Table 3. 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. [7] reported that the effects of tritium-labelled epigallocatechin gallate (EGCG) being incorporated into human lung cancer cells were enhanced by epicatechin (EC).11. Suganuma et al.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 161 Table 3. another green tea polyphenol.10. one without a galloyl moiety.

[21] Liu et al. trap-ping of lipid peroxyl radicals and regene-ration of vitamin E Zhou et al. EC. reduced levels of estrogen receptor-α protein and of serum IGF-I. [12] Zhou et al. [18] hydroperoxides and malondialdehydes.Theo M. These effects. [19] Zhou et al. [22] EGCG. EGC and ECG Modulation of CYP1A1 expression in human hepatocytes Williams et al. -8 and -9. Simone G. A and ß-carotene Reduced oxidative stress Morré and Morré [16] Reduced formation of lipid Gorelik et al. vitamin E. [7] EGCG and EC Inhibition of cell growth and induction of apoptosis in gastric carcinoma cells Horie et al. enhanced apoptosis. de Kok. 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. [10. 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. EGC and ECG or gallic acid and α-tocopherol Reduced oxidative stress in micelles and human LDL . 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. [13] Green tea infusions and grape Reduced tumour cell growth or grape skin extracts Polyphenols. Extracellular production of oxygen species Antagonism of TCDD-induced transcription of human CYP1A1 via interaction with the Ah-receptor Suganuma et al. Table 3. sulindac or tamoxifen Synergistic effect Mechanisms involved Reference Inhibition of growth of human lung cancer cells Enhanced cellular uptake of EGCG. induction of apoptosis. reduced co-oxidation of vitamins E.11. Inhibition of tNOX. induced by a tea polyphenol having a galloyl moiety.11] Polyphenols and other phytochemicals Green/black tea and soy (SPC)* Green/black tea and soy (SPC) Inhibition of prostate tumours. [8] EGCG. Zhou et al. tumour weight and metastasis Inhibition of breast tumour cell growth Reduction in serum levels of testosteron and DHT Inhibition of tumour angiogenesis. were also enhanced in a dose-dependent way by EC. reduced release of TNF-α Increased production of caspases-3.

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

). In line with this. Simone G. Zhou et al. 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. such as by phytochemicals. which is found predominantly in the seeds. SPC in combination with green tea showed a synergistic inhibition of tumour cell growth. Recently. though to a lesser extent) [18]. They cease to divide and eventually undergo apoptosis. reduced expression of estrogen-receptor (ER) alpha and reduced serum levels of the insulin-like growth factor (IGF)-I. The modulation of these two different mechanisms may be an explanation of the synergistic effects of the combined phytochemicals [13]. Cells in which NOX activity is blocked. are unable to enlarge. whereas no activity was detected for extracts of grape seeds. 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. In the gastric fluid. van Breda 164 Synergistic effects of polyphenols and other phytochemicals In two different studies.13]. the latter a biologically more active metabolite of testosterone and a prerequisite for the development of benign prostatic hyperplasia and prostate cancer [12]. increasing at the same time the amounts of vitamin antioxidants reaching the blood system. the authors suggested that the antioxidant network in the stomach can decrease the level of hydroperoxides and other cytotoxic compounds. indicating that the effects were not caused by resveratrol. de Kok. In an immunedeficient mouse model in which MCF-7 human breast cancer cells were implanted. These results suggest that more effective cancer prevention and cancer therapy could be achieved by use of combinations of different phytochemicals. 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.Theo M. Polyphenols and dietary antioxidant vitamins can also have synergistic inhibitory effects on lipid peroxidation and on the co-oxidation of dietary antioxidants. 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]. NOX proteins are located at the cell surface and are responsible for the increase in cell size that occurs following cell division [17]. 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]. The authors indicated that the resulting synergistic increase . vitamin C can enhance the activity of polyphenols through a synergistic antioxidant effect. The strongest synergistic activity was found for ethanol extracts of grape skins.11. all of these being factors in breast cancer development. 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. A phytochemical soy concentrate (SPC) and green and black tea infusions were employed (Table 3. This was accompanied by inhibition of tumour angiogenesis.

In one study. 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. EGC. it was found that most of the genes that were differentially expressed before and after vegetables were consumed. When the contributions of the separate phenolic compounds were calculated. 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. In two recent animal studies on the effects of vegetable consumption on the modulation of gene expression. . 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]. 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•). By studying the kinetics of the reaction of α-tocopherolyl radicals with green tea polyphenols by means of stopped-flow electron paramagnetic resonance.22]. the synergistic effects of taking whole foods or complex mixtures of compounds have been reported. On the other hand. 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. 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. indicating that combinations of different foods containing different complex mixtures of phytochemicals can also have an antagonistic effect on gene expression. Taking account of the different mixtures found of the 12 phenolic compounds that were present. the individual vegetables were able to modulate genes which were not significantly modulated by the mixture. the Trolox equivalent antioxidant capacity (TEAC) value and phenolic composition were determined for a large number of pinotage wines [25]. Zhou et al. ECG and gallic acid) can effectively reduce α-tocopheroxy radicals so as to allow α-tocopherol to be regenerated. 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. 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.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. [19] demonstrated clearly that several green tea polyphenols (EC. This.24]. combined with an α-tocopherol regenerating reaction involving coexisting antioxidants. the changes in gene expression could be interpreted as a cancer preventive effect [23. plays a major role in enhancing the antioxidant efficiency of vitamin E.

Jørgensen et al. the effects reported by Suganuma et al. Use of retinoids in combination with such SERMs (selective estrogen receptor modulators) as tamoxifen or raloxifene [29. 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. its also reducing side effects.28]. 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]. Simone G. A review of the effects of various combinations of chemotherapeutic and chemopreventive approaches in dealing with cancer has appeared recently [28]. This suggests that the regeneration of phenolic compounds from phenoxy radicals is a mechanism that can contribute to the synergistic effects observed. de Kok. particularly those of sulindac. Various combinations of drugs have been proposed for further clinical development based on their synergistic activity as shown in vitro or in animal studies.Theo M. 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. [27] demonstrated. Other synergistic effects In addition to investigating the synergistic effects of various phytochemicals. it appears . Although our understanding of the molecular mechanism behind the observed combinatorial effects of these chemopreventive agents is still limited. without a loss in treatment potency. 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. Also. Administering multiple agents can increase the efficacy and potency of the chemopreventive measures taken and reduce toxic side-effects. EGCG and sulindac were also found to synergistically enhance apoptosis. the regeneration of quercetin from its phenoxyl radical by the presence of (+)-catechin. van Breda 166 indicated 16 to 23% of the antioxidant activity to be synergistic. 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]. In that study.30] are examples of this. in addition to the synergistic effects between the different phenolic compounds. [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. 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. however. synergistic effects between these and the other wine constituents may have contributed to the TEAC values. This implies that.

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Bifidobacterium spp. anticancer effects. 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. Administration of L. Probiotics and prebiotics — potential anticarcinogenic food components Joseph Rafter Department of Medical Nutrition. and Streptococcus spp. Karolinska Institute. 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. 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..and prebiotics). Diet makes an important contribution to CRC risk [6] implying that risks of CRC are potentially reducible. The vast majority of studies on the anticancer effects deal with colorectal cancer (CRC). epidemiology and human intervention studies.and prebiotics on cancer is derived from in vitro studies. serum cholesterol reduction. Evidence for protective effects of pro. e.g.2]. 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. Evidence from a wide range of sources supports the view that the colonic microflora is involved in the etiology of CRC. Overall. that can modulate gut microflora and its metabolism. animal models.or prebiotics alone. The list of healthful effects attributed to probiotic bacteria is extensive [3] and includes: alleviation of lactose intolerance symptoms. Lactobacillus spp. resulted in a lower faecal mutagenic activity after 3 days . The evidence from animal studies provides strongest support for anticancer effects and data from human studies (epidemiology and experimental) are limited. Huddinge. relieving vaginitis to name but a few. Sweden Introduction An example of a functional food. with defined gut survival properties and associated biological activities and which can be ingested in fermented milk products or as a supplement. 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. acidophilus to eleven volunteers on a fried meat diet known to increase faecal mutagenicity. Probiotics usually refer to highly selected lactic acid bacteria. such as probiotics. alleviating constipation.6. that have the potential to improve host health’) [3] and synbiotics (combinations of pro.Joseph Rafter 170 3. although there are some on breast [4] and bladder cancer [5]. This has led to intense interest in factors.

Once such markers are available. in the near future. two companion American prospective studies. the 1980–1988 follow-up of the Nurses’ Health Study and the 1986–1990 Health Professionals follow-up study. an inverse association was observed for yoghurt [12] and cultured milk consumption [13]. In conclusion. An epidemiological study performed in Finland demonstrated that. 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]. the most profitable approach would be to use combinations of pro. High levels of mutagenicity also appeared in urine on days 2 and 3 of the fried meat and ordinary fermented milk dietary regimen. prebiotics or fermented milk consumption are causally related to reduction in cancer risk. In two population-based case-control studies of colon cancer. casei on the urinary mutagenicity arising from ingestion of fried ground beef in the human. particularly in urine.11]. acidophilus administration. the opportunity to exploit genomics and proteomics in investigations of effects of pro/prebiotics on gene expression and post-transcription . There will also be. did not provide evidence that intake of dairy products is associated with a decreased risk of colon cancer [16]. this is an area of high priority for future studies. As yet.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]. In most cases. Thus. In summary. Hayatsu and Hayatsu [8] also demonstrated a marked suppressing effect of orally administered L.and prebiotics. On the other hand. 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. 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. 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. at-risk groups and patients.15]. the urinary mutagenic activity on days 2 and 3 was significantly lower compared to the ordinary fermented milk period. yoghurt. it will become possible to perform studies in healthy volunteers. there are few epidemiological studies addressing the association between fermented dairy products and colorectal cancer. adjusted for potential confounding variables. an increase in the number of faecal lactobacilli corresponded to a lower mutagen excretion. data from human intervention studies are of paramount importance in providing evidence that probiotics. despite the high fat intake. It will be important to define dose and time relationships and it would appear at present. it would appear that the case control studies indicate protective effects while the prospective studies do not. In another case control study. In a cohort study in the Netherlands. and other dairy products [10. colon cancer incidence was lower than in other countries because of the high consumption of milk. 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]. from animal studies. During L.

such as aberrant crypt foci (ACF). It will also be particularly important to use data on mechanisms of action to develop hypothesis based intervention studies in humans. acidophilus.2-dimethylhydrazine (DMH)-induced genotoxicity. while heat-treated L. L. Certain strains of lactic acid bacteria have also been found to prevent putative preneoplastic lesions or tumours induced by carcinogens. that Lactobacillus acidophilus. using these models. all of the ”state of the art” colon cancer risk biomarkers. using the comet assay. which clearly demonstrate a protective effect of dietary supplements of lactic acid bacteria against colon tumour development. longum were antigenotoxic toward N’-nitro-N-nitrosoguanidine (MNNG). induced by azoxymethane (AOM) [20]. there have been many studies. Among different cell fractions from L.syncan. It involves a twelve-week randomised. L. the peptidoglycan fraction and whole freeze-dried cells were antigenotoxic. [19] showed that a specific strain of L. Streptococcus thermophilus.22] or DMH [23. Although B. are being measured. acidophilus was not antigenotoxic.24]. and involving 8 research centres in Europe. feeding of bifidobacteria suppressed the ACF formation induced by AOM [21. In recent years. casei subsp. http://www. prevented MNNG-induced DNA damage.Joseph Rafter 172 events in colonic biopsies and to identify human groups responsive to pro/prebiotic intervention. Bifidobacterium Bb-12 and Raftilose Synergyl in adenoma patients. 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. End points used are the tumours themselves or early lesions.e. as well as an acetone extract of the culture. These bacteria were also protective toward 1. induced by chemical carcinogens. placebo controlled trial of a food supplement containing Lactobacillus GG. Consumption of B. gasseri. Metabolically active L. Oral administration of lactic acid bacteria has been shown to effectively reduce DNA damage. double blind. acidophilus also inhibited the formation of ACF. 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. faecal water markers and immunological markers. Bifidobacterium breve and B. to examine the effect of a synbiotic preparation on colon cancer risk biomarkers in humans (SYNCAN project. including colonic mucosal markers. ACF are putative preneoplastic lesions from which adenomas and carcinomas may develop. In this study. Pool-Zobel et al. Overnight cultures of L. adolescentis culture and its supernatant did not show an inhibitory effect in this study [20]. [18] reported. 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. funded by EU. longum or inulin was associated with a decrease in AOM-induced colonic small ACF in rats and combined administration . acidophilus cells.be). in gastric and colonic mucosa in rats. Goldin et al. Of relevance here is a clinical trial which is presently ongoing i. confusus.

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

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

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

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