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: Website:


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

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

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

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

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

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

The strongest evidence for the anticancer effects these can have comes from animal studies. Even with the above reservations and the limited number of human studies available in mind. growth arrest or apoptosis is to occur are determined. Many healthful diet effects are attributed to probiotic bacteria. . The synergistic effects of dietary phytochemicals need to be investigated further. promising and as deserving of closer scrutiny. 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. In some systems. 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. Much hope is attached in this connection to the powerful techniques available today within the field of nutritional genomics. 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. It is important here to have insight into recent developments in analytical technology and thorough knowledge of biomarker kinetics. 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 appears that many combinations of complementary modes of action may be involved. and green tea flavonoids can also show increased activity when present together with other phytochemicals. Several possible mechanisms may explain how lactic acid bacteria can protect against tumour development in the colon.Executive summary 9 being activated. This can well contribute to cancer prevention. 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. The development of ideas regarding this has also been stimulated by findings that dietary supplements of only a single compound may have negative effects. As has been indicated here. 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. It is presently unclear how these different types of responses are coordinated by the cell and how matters of whether adaptation. Although our understanding of the molecular mechanisms behind the observed combinational effects is still limited. Different strains of the bacteria may possibly target different mechanisms. 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. one can regard the use of lactic cultures for human cancer suppression as interesting. 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. evidence from human studies still being limited. combinations of green tea flavonoids are more active than single compounds of this sort.


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

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

Biomarkers in studies of that type have been used either alone. There are particularly good reasons for using the 25-hydroxy vitamin D as a biomarker in extended studies in the future. The assessment of dietary intake in epidemiological studies has been a major use of biochemical markers of nutritional status. As summarised by Hunter [9]. faeces. Also. urine. The two contributions mentioned also illustrate the difficulty in defining valid cut-off points. For this reason. This topic is only taken up briefly in the present volume. . 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. Although the use of the biomarker concept for these purposes is very attractive. 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. buccal mucosa.12]. however. as exemplified in the present volume by Linseisen and his colleagues in connection with vitamin D and polyphenols. These matters are outside the scope of the present review. 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. Folate has served as a prototype here for demonstrating how genetic make-up can influence the nutrient status of an individual. it should be borne in mind that there are very few (if any) “ideal” biomarkers that have been validated for studies of different types. A number of statistical methods for the correction and evaluation of such data are available [8]. if the analytical methodology improves so that smaller amounts of sample suffice. or for the calibration of the dietary assessment methods [8]. Some of these samplings require much skill and effort and can only be used in small studies. 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]. Their use may increase. 2) help establish causality of food and nutrient associations in epidemiology studies. information on the interactions between dietary intake and gene polymorphisms “can serve to 1) define susceptible subpopulations. An overview of this matter is provided in [7]. 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. thus strengthening dietary associations. 3) aid in distinguishing causal components of complex dietary mixtures. methods for measuring nutrients or nutrient-dependent variables in cells in the blood (mainly leucocytes). although epidemiological findings on the association between the selenium level and cancer are reviewed by Gromadziƒska et al.Introductory overview and future prospects 13 Although several of the biomarkers mentioned here are widely used. 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. and 4) eventually provide the basis for gene-based screening tests”.

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

Introductory overview and future prospects

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

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

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.

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. Lund. and avoiding of similar articles by any given author. 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. Overview of possible anticarcinogenic food components Per Mercke Biomedical Nutrition. These protective compounds are found basically in all types of food. The criteria used for the selection were their relevance as adjudged from their title. Plant tissues can also vary enormously in the store of such compounds. This information was collected with use of a PubMed search conducted in 2005. a list of putative anticarcinogenic food components has been generated (Table 1.Per Mercke 18 Appendix 1. but plant-based foods appear to be their richest source.2. emphasis on recent reviews. 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. for example. depending on the plant variety and the growth conditions.2.3. Different plant families harbor different mixtures of compounds of this sort. Lund University. Table 1. . 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.). Sweden There is a vast range of dietary compounds proclaimed to have anti-carcinogenic properties as investigated by the scientific community.

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

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

dietary fat and antioxidants. 6. McGlynn AP. An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected — cont. Biomarkers in disease and health. Bartsch H. 11. Turmeric Branca et al. Manson [18]. Manson MM. Rowland IR. Class of compound Methylxanthines Example of bioactive substance Caffeine Theophylline Theobromine Limonene Perillyl alcohol Geraniol Menthol Carvone Hydroxytyrosol Curcumin Resveratrol Food source Tea. Hematol Oncol Clin North Am 2002. Br J Nutr 2002. Primary prevention: phytoprevention and chemoprevention of colorectal cancer. DuBois RN. Bartsch et al.Overview of possible anticarcinogenic food components 21 Table 1. Milner JA. 9. Nair J. Mutat Res 1999. Rafter JJ. Br J Nutr 2002. Cancer risk factors for selecting cohorts for large-scale chemoprevention trials. Owen RW Exocyclic DNA adducts as oxidative stress markers in colon carcinogene. et al.88 Suppl 1:S73–87. Wiseman [33] Grapes. Pannemans D. Prospects for cancer prevention. Hanley AB. Scientific basis of biomarkers and benefits of functional foods for reduction of disease risk: cancer. 5. 4. Steward WP. Br J Nutr 2004. black-berries. 10. J Cell Biochem 1996. Mathers JC. 2. walnuts.3. Sanderson P. pecans. Biol Chem 2002. et al. Emerging dietrelated surrogate end points for colorectal cancer: UK Food Standards Agency diet and colonic health workshop report. Rafter et al. Turini ME. cacao Manson [18] Reference Monoterpenes Citrus fruits (peel) Ferguson [3]. Pool-Zobel B. Ferguson LR. [2]. PASSCLAIM — dietrelated cancer. Diet and cancer: assessing the risk. Verhagen H. Rafter J. . Farmer PB. 8.428:329–38. Johnson IT. Greenwald P.166:257–75.88 Suppl 2:S219–24. Downes CS.86 Suppl 1:S55–S92. Br J Nutr 2001.43 Suppl 2:II47–84. [8] Other non-flavonoid phenolics Lignans Olive oil Adlercreutz [31]. Gill CI. raspberries. Innovative agents in cancer prevention. Branca F. Martel P. coffee. 3. olive oil References (selected from the PubMed search described above) 1. Powers HJ. [16]. J Nutr 2003. 7. Kelloff et al. cola.133 Suppl 1:3820S–6S.16:811–40. Govers M. Gescher A.383:915–21.25 Suppl:29–36. strawberries. Incorporating basic nutrition science into health interventions for cancer prevention.91:315–23. Pool-Zobel B. Cereals. Recent Results Cancer Res 2005. Eur J Nutr 2004. sis: potential role of lipid peroxidation. [1]. Rechkemmer G.

47:13–23. O'Brien NM. Srivastava S. Crozier A. Granado F. 30. Bowen P. Br J Nutr 2001. Clin Cancer Res 2004. 26. Maiani G.86 Suppl 1:S5–35. Crit Rev Oncol Hematol 2003. Milner JA. Poulsen HE. Getzenberg RH. 31. Malone WA. Duthie G. et al. Alink G. Vlastos AT. Kelloff GJ. Stacewicz-Sapuntzakis M. Cancer prevention — the potential for diet to modulate molecular signalling. Anderson DE.3:447–51. Sharma RA. Crews H. Urol Clin North Am 2002. 27.86 Suppl 1:S37–53. 20. Konety BR. Cancer Res 2003. 15. 16. Woods JA. Wilson L. Adlercreutz H. A critical assessment of some biomarker approaches linked with dietary intake. Tea beverage in chemoprevention of prostate cancer: a mini-review. McDonald SS. 32. Holst B. 13. J Nutr 2004. 19. 29.48:169–88. Phytoestrogens and breast cancer. Elmadfa I. et al. Lubet RA.133 Suppl 3:941S–7S.Per Mercke 22 12. Curr Opin Clin Nutr Metab Care 2000. Ghosh L. Sharifi R. Mutat Res 2004. Drug Metab Rev 2004. Vainio H. 2002. Duncan C.90:487–502. Cancer risk and oxidative DNA damage in man. J Nutr 2000. J Nutr 2003. Mayne ST. Farmer PB. J Mol Med 1996. Breast Cancer Res. et al.10:4901–12. Manson MM. Steele VE. Andersson C.63:4295–8. Greenwald P. Biological relevance of adduct detection to the chemoprevention of cancer. Trends Mol Med 2003. Follen M. 25.29:95–106. 24. 17. Isothiocyanates in cancer prevention.9:11–8. Nutritional and clinical relevance of lutein in human health. Plant-derived phenolic antioxidants. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. J Steroid Biochem Mol Biol 2002.130 Suppl:467S–71S. J Nutr 2003. Nutr Cancer 2003.555:173–90. Progress in cancer chemoprevention: development of diet-derived chemopreventive agents. Mukhtar H. 23. Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis. Wagner KH. Biomarkers and their use in cervical cancer chemoprevention. Saleem M. Boone CW. . Vitamin D and prostate cancer. Seifried HE. 28. Loft S.133 Suppl 3:933S–40S. Braesco V. The antioxidant conundrum in cancer. Mammographic breast density as a biomarker of effects of isoflavones on the female breast. Kamal-Eldin A.83:113–8. Biomarkers for cancer diagnosis: implications for nutritional research. A critical evaluation of the application of biomarkers in epidemiological studies on diet and health. Zhang Y. 22. 21. Exp Biol Med (Maywood) 2002. Schottenfeld D. Br J Nutr 2001. Atkinson C. Bingham SA. Chen L.36:655–67. Blanco I. Andersen R. Maruvada P.74:297–312.134 Suppl:1640S–5S. Bianchini F. Mason JB. Gamma-tocopherol — an underestimated vitamin? Ann Nutr Metab 2004. Olmedilla B. Br J Nutr 2003.46:261–73.4:1–4. Biomarkers of nutrient exposure and status in one-carbon (methyl) metabolism. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. Wild CP. 14.227:886–93. Crowell JA. Adhami VM. 18. Siddiqui IA.

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4] or reversed-phase [5–7] liquid chromatography . 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. The possibility of using dried blood spots.1. a technique that was later superseded by various chromatographic and fluorescent techniques. 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. which are dealt with elsewhere in this volume. menadione and phylloquinones.2. the development of fast and simple methods for the determination of vitamin A status has long been given a high priority. 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. although reversed-phase methods able to separate certain of the retinol isomers have been published [8]. xanthophylls. biological markers of the dietary exposure to these vitamins is of importance. This chapter is concerned with such markers. With the advent of high performance liquid chromatography (HPLC). 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. 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]. except for markers of DNA damage. The structure of vitamin A and pro-vitamin A carotenoids is shown in Figure 2. Denmark Since antioxidant vitamins can affect an organism’s capacity for defence against reactive oxygen species. The direct-phase methods can also typically measure a large number of other lipid-soluble vitamin isomers in the same run.2]. Biomarkers of exposure to and effects of vitamins A. which is advantageous from a collection standpoint. tocopherols and tocotrienols.[3.1. Copenhagen. the retinol binding protein (RBP) [1. such as pro-vitamin A carotenoids. Vitamins and selenium 2. has also been demonstrated [1]. C and E Lars O. Due to worldwide concern for vitamin A deficiency (VAD). these techniques took over and today retinol can be determined in serum routinely by direct. Dragsted University of Copenhagen. .

Lars O. Dragsted

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)

CV% 4 4 <10 9

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

Speed (samples/day) 25 20 50–100 150

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

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

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.

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].

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

The performance of different methods for the measurement of vitamin C in plasma is summarized in Table 2. In another study an interlaboratory difference of about 15% was observed. irrespective of the concentration. The assays employed to this end here are the following: plasma thiobarbituric acid reactive compounds (TBARS). . following HPLC separation.3. In a European study of laboratories carrying out population surveillance. antioxidant capacity markers. to detect malondialdehyde.5 mg/l 3 mg/l Speed (samples/day] 40–50 40–50 500 Reference [36] [37] [38.1 mg/l 0. Dragsted 30 use of the HPLC and CZE methods and good linearity is obtained even at low ascorbate concentrations. This marker is highly controversial since it is variable both within and between laboratories. CV% AA interday < 4% 1. and plasma or urinary isoprostanes. plasma lipid hydroperoxides. either directly or online. with or without calibration.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. which led to relatively larger relative errors being registered at low concentrations [21]. In interlaboratory comparisons. since it may partially measure aldehydes deriving from endogenous metabolism. Table 2. whereas the intralaboratory variation was about 2 µmol/l. and since there is no generally accepted assay procedure [31]. ex vivo LDL oxidation. 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]. Only randomised study designs are included in this review. depending on the method employed for hydrolysis.3. The product can be determined spectrophotometrically. but as already mentioned this relatively simple method has an odd tendency of sometimes giving spurious results and of varying from one analytical series to another. also within the same laboratory. 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. quite disparate results have been obtained with use of these techniques. The interday coefficient of variation (CV) for TBARS with use of HPLC is in the order of 10–20 %. These flaws have caused some journals to generally regard the method as being invalid as a lipid oxidation biomarker [32]. Typical performance of different methods for determination of plasma vitamin C Test method Reversed phase HPLC Capillary electrophoresis Automated colorimetry ND — not determined. The most commonly used marker is determination of TBARS.Lars O.3% < 5% CV% DHAA interday < 6% ND ND Limit of detection 0.

In a smaller parallel study of vitamin C supplementation (1 g/d) versus placebo. Another controversial marker used by many laboratories is the ex vivo LDL oxidation assay. No differences between groups in plasma malondialdehyde concentrations were observed either before or after supplementation [34]. 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. 25 males were exposed to vitamin C (500 or 1000 mg/d) and/or to exercise. 182 mg/d dl-α-tocopherol alone. In a third study of this sort. The lag-time to oxidation and/or the slope of the oxidation curve are used as end points. or a placebo in a parallel design. the oxidative formation of conjugated dienes being followed spectrophotometrically at 234 nm [42]. No effect of either treatment on plasma MDA was observed [35]. The interday CV for this latter assay is less than 10%. as compared with placebo treatment. 30 mg beta-carotene and 100 µg organic selenium. no change in whole plasma ex vivo oxidation was observed in this group [44]. in which isolated LDL is exposed to copper chloride or to a semistable radical such as AAPH. 250 mg/day) was determined in 20 subjects each showing normal (67 µmol/l) or below average (32 µmol/l) plasma vitamin C concentration at baseline. In a larger study involving 56 smokers. the intake of 500 mg/d of vitamin C was found not to affect MDA as determined by HPLC [37]. 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]. by a decrease in plasma MDA concentration [41]. 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]. in which 19 smokers participated for 4 weeks following a 2-week . randomised double-blind crossover study the effect of vitamin C supplementation (six weeks. vitamin C plus 182 mg/d dl-α-tocopherol. In a group of 48 middle-aged male and female participants in a 36-month intervention study receiving 500mg/d of either vitamin C.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. but MDA to be unaffected by a slow-release formulation as compared with placebo treatment [33]. given in a normal formulation. given for 3 months. Oxidative stress in 10 volunteers as determined by increased plasma MDA induced by infusion of free fatty acids was also found to be unaffected by high-dosage vitamin C infusion [39]. The method is disputed because the outcome depends on the antioxidants present in the LDL and these may be lost during LDL isolation. In another. 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. Oxidative stress induced by Zn deficiency was found to respond to 250 mg/d vitamin C. 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]. 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. In addition. acute doses. 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]. In another counterbalanced design.

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

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

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

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

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.

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

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

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

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

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. 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]. In a trial involving 80 men who had an increase in plasma lipid oxidation following exposure to olive oil or to menhaden oil. or LDL-hydroperoxides induced ex vivo [87]. no effect on plasma antioxidant capacity was observed at 12 or at 36 months [44]. In 49 diabetic patients given 504 mg/d d-α-tocopherol or a placebo for 6 months. In a double-blind controlled intervention study of 56 patients with congestive heart failure. In a double-blind controlled intervention study of 56 patients with congestive heart failure. . no difference was observed in LDL-MDA. who were given 800 IU/d vitamin E or a placebo for 2 months prior to the race. and the strength of this effect was correlated with the level of plasma α-tocopherol [114]. Non-induced lipoprotein oxidation ex vivo was found to be delayed in the group given the vitamin supplement. 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]. In a dose-response study of 40 healthy men assigned doses of 60–1200 IU of vitamin E for an 8-week period.Lars O. vitamin E given at doses higher than 200 IU/d was found to affect the kinetics of ex vivo oxidation of LDL [113]. 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 change in these groups in whole plasma ex vivo oxidation at 36 weeks was likewise observed [44]. or placebo. LDL-dienes. Dragsted 40 182 mg/d dl-α-tocopherol alone. 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]. In a study without a parallel control group. In a group of 45 randomised healthy males and females. 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. In a blinded intervention study of 33 triathletes participating in a world championship. the treatment with vitamin E was found to significantly increase the level of plasma F2-isoprostanes as well as of several markers of inflammation. the effects of giving mixed supplements of 200 mg/d vitamin E. a 1000 IU/d vitamin E supplement for a 10 d period was found to reduce the exhalation of breath pentane [91]. 182 mg/d dl-α-tocopherol alone. this together with 182 mg/d dl-α-tocopherol. In a study of 49 HIV patients randomised for supplementation with a placebo or with 800 IU/d of vitamin E and 1000 mg/d of vitamin C over a 3-month period. In a 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. 900 mg/d vitamin C and 18 mg/d beta-carotene for 6 months were compared with those of a placebo. including IL-6 [115]. 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 vitamin supplement was found to prevent this [117]. no effect of either vitamin treatment was observed. 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]. the adding of vitamin E was found to have no further effect on the urinary excretion of F2-isoprostanes [122]. Running as such was found to increase the plasma isoprostane level.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. In 43 hypercholesterolemic patients randomised to taking simvastatin. 200. In an intervention study of 46 healthy smokers given 0. In another double-blind placebo-controlled trial with a crossover design. In a small non-blind study of cirrhotic patients. This was not observed for the young men. No other significant differences were observed between the two groups [116]. 800. 21 ultramarathon runners were given either a combination of 300 mg/d d-α-tocopherol and 1000 mg/d vitamin C. groups of 5 healthy individuals received either 0. The inflammatory markers were also affected by running. no effect on the excretion of F2-isoprostane could be observed for any of these interventions [119]. 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]. In a double-blind controlled intervention study of 56 congestive heart failure patients. In a group of 33 sclerosis patients. which may have caused the difference. 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]. The study design could not control for period effects. or a placebo during a 28 d period on the plasma isoprostane concentrations induced by exercise. 1200 or 2000 IU/d of vitamin E for a period of 8 weeks. 600 or 1200 IU/d of vitamin E for 3 weeks. C and E 41 In another study. 400. irrespective of the treatment [124]. 10 of them were given 500 mg/d and 10 of them 1000 mg/d vitamin E for a period of 3 weeks. or a placebo during the 6 weeks prior to the race. No effect on urinary excretion of F2-isoprostanes was observed [121]. 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. Following vitamin E supplementation a significant lowering of the plasma isoprostane level both before and 24 h after the exercise was found for the older men. although the treatment by g-tocopherol was found to affect the exercise-induced increase in plasma and muscle heat shock protein (HSP72) and also the expression of HSP72 in muscle [118]. In a third study of the effects of vitamin E on oxidative damage induced by exercise. particularly in the males. the rest being given a placebo. simvastatin together with 600 mg/d vitamin E or a placebo for 2 months. vitamin C (500 mg/d) together with d-α-tocopherol (290 mg/d) and d-γ-tocopherol (130 mg/d). In a study without a control group. No significant change in the plasma F2-isoprostane level could be detected by GC-MS at any time point. but this was not changed by the vitamin supplements. followed by 8 weeks of washout. In a study of the combined effects of giving either vitamin C (500 mg/d) together with d-α-tocopherol (400 mg/d). 300. In a dose-response study. 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]. .

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

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

proliferating stem cells have to be affected. In addition. Although studies that indicate this support the hypothesis that oxidative DNA damage can be an important risk factor for carcinogenesis. Marcus S. Again. although their suitability for this has yet to be verified in prospective studies of cancer risk. For DNA mutations to arise through oxidative damage. . 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. An elevated level may be due to some well-established characteristic of the tumour. On the basis of an extensive investigation by the European Standards Committee on Oxidative DNA Damage (ESCODD) it has been concluded that the true background level of 8-oxodG in mammalian cells is approximately 0. 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. the analytical procedures in current use are unable to quantify the lesion levels found in the most important ‘target’ cells. Ultimately. but also the damage must be within a coding region of the DNA. connected with the spurious oxidation of DNA during sample preparation. This raises a number of different issues: 1. Rafa∏ Ró˝alski.2 lesions per 106 unaltered guanines and that reports of values outside this range should be viewed skeptically [6]. 3. 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. Issues of this sort have to be addressed before a causal link between oxidative DNA damage and cancer can be established. Oxidative DNA damage may be an epiphenomenon in relation to the ongoing pathophysiological process. At the same time. In order for a mutation to come about. This. such as a high level either of metabolism or of cell turnover. not only must the DNA of the target cells be affected. or (ii) there being reduced DNA repair [5]. Elevated ROS levels may activate transcription factors and the corresponding genes being permanently activated. the levels of damage having no causal role in carcinogenesis. Cook.3–4. creates a selection pressure for the malignant phenotype seen in the case of cancer. there are serious problems in the chromatographic measurement of 8-oxodG. 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. the nuclei of the undifferentiated. 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]. regarding the question of ‘cause or consequence’. Peter Møller. 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.Steffen Loft. 2. Ryszard Oliƒski 52 of a potential disease endpoint such as that of cancer. it has been argued that the mere presence of 8oxodG in DNA is unlikely to be a necessary and sufficient condition for tumour formation.

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

and 25 mg β-carotene) for 20 wk Multi-vitamin (250 vitamin C. 280 mg vitamin E. provides an overview of intervention studies involving optimal design in which the effects of antioxidants or antioxidant-rich food supplements on oxidative damage to DNA in WBC or in urine have been investigated.6 g) for 1 mo 116 M (S) 30—65 No effect on the 24-h urinary excretion of 8-oxodG (HPLC) 13 M (NR) 30±3 Lower 24-h urinary excretion of 8-oxodG (HPLC) in the active group of HIV-infected patients receiving zidovudine therapy 184 MF 58±14 No effect on the 24-h urinary excretion of 8-oxodG (ELISA) (NS) 48 M (22S) 30 NR (NR) 45—69 No effect on the 24-h urinary excretion of 8-oxodG (HPLC) 22±1 No effect on the 24-h urinary excretion of 8-oxodG (ELISA) in subjects undergoing cold-weather training at a moderate altitude 7±2 No effect on the excretion of 8-oxodG (ELISA) in spot urine samples 35 25 28 2x2 parallel study of vitamin C (500 mg) and vitamin E (400 IU) for 2 mo 2x2 parallel study of vitamin C (500 mg) and vitamin E 182 mg) for 12 mo Multivitamin tablete for 2 wk 34 33 Multivitamin tabletf for 21 d 39 MF (NR) 37 Multivitamin tabletg for 24 d 40 M (NR) 18—40 No effect on the overnight excretion of 8-oxodG (ELISA) in subjects undergoing cold-weather training at a moderate altitude 59 MF (11S) 50±6 No effect on the excretion of 8-oxodG (ELISA) in spot urine samples 36 Fruit and vegetable capsulesh for 7 wk 29 . 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. 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. Marcus S. however. Table 2.Steffen Loft.6. Rafa∏ Ró˝alski. 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. Peter Møller. Cook. 200 IU α-tocopherol.6. Table 2. 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. from use of a non-optimum design. but a decline in the course of the trial in both groups 32 MF (NS) 32±11 No effect compared with baseline. Ryszard Oliƒski 54 Many studies suffer.

but a decline in all the groups 21—45 No effect on FPG sites (Comet) in WBC 19 20 18 MF (NR) 14 MF (NS) 10 MF (NS) 10 M (NS) 15 26—54 A decrease at the ENDOIII and FPG (Comet) sites 22 Brussels sprouts (300 g) for 1 wk NR No effect on the 24-h urinary excretion of 8-oxodG (HPLC) A decrease in the 24-h urinary excretion of 8-oxodG (HPLC) in the active group 40 Brussels sprouts (300 g) for 12 d NR 41 Fruit juice (480 ml)j for 4 d 11 M (NR) 21±1 A decrease in the 12-h urinary excretion of 8-oxodG (ELISA) in the active group 57 MF (6S) 19—52 No effect on ENDOIII and FPG sites (Comet) in WBC 26 Parallel study of blackcurrant juice or anthocyanin drink (475—1000 ml) for 3 wk Green tea or black tea (4 cups) for 1—4 mo 18 120 MF (S) 18—79 A decrease in the excretion of 8-oxodG (ELISA) in spot urine samples in the green tea group after 4 mo. but a post-supplementation decrease in the 24-h urinary excretion of 8-oxodG (ELISA) in the active group NR No effect on ENDOIII sites (Comet) in WBC 32 Rye crisp bread (76.5 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. 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 . 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. No effect on the 24-h urinary excretion of 8-oxodG (HPLC). but not earlier 68 MF (13S) 18—45 A decrease in the 12-h urinary excretion of 8oxodG (HPLC) 27.6.Vitamins and selenium: Antioxidant vitamins and cancer risk 55 Table 2.

Also.44 mg) per day. a protective effect could be shown after only 20 weeks of supplementation but no effect after 10 weeks (or 6 months of supplementation) [13. 3) 500 mg slow-release vitamin C. 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. Smokers (S) and nonsmokers (NS) are indicated in brackets. possibly because of differences in the respective rates of bioavailability and elimination. lutein (500 mg). Supplement given per day Subjectsa Age (yr)b Effect Ref Soy milk. or a carotenoid-rich diet.6. vitamin C (219 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). beet. and powder or extract of fruits and berries). bixin (11. capsules (containing 90 mg vitamin C. parsley. Ryszard Oliƒski 56 Table 2. 4) 90 mg coenzyme Q10 in oil.14]. The daily dose consisted of 200 mg vitamin C.2 mg). 4 mg β-carotene).4 mg β-carotene. kale. rice milk. Supplement consisting of β-carotene (6 mg). tocopherols (650 IU). Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont. 2) 500 mg plain-release vitamin C. papaya. The effect of a single dose of vitamin C seems to disappear after several hours. Consisted of 1000 mg extract/kg body weight in meat patties (total phenolics 23. Tablets containing β-carotene (12 mg). The vegetable capsules were made from carrots. selenium (167 mg). 2. Peter Møller. lycopene (100 mg). 6) placebo.16]. however. 150 mg vitamin E. Containing vitamin A (240 mg).Steffen Loft. being given rye crisp . and 15 mg β-carotene. Rafa∏ Ró˝alski. vitamin C (330 mg). selenium (100 mg). and zinc (30 mg). cranberry and peach.12]. spinach. vitamin E (1.12]. cabbage. respectively [15.4 mg). The six groups received daily supplements of 1) 200 mg vitamin E. Consisted of tablets containing vitamin antioxidants (400 mg vitamin C. and pomegranate extract (5 mg). The fruit capsules were made from juiced apple.5 mg). 5) 30 mg coenzyme Q10 granulate. vitamin E (400 IU). α-carotene (1. Accordingly. Tablets containing micronutrients similar to those of 3 fruits and 3 vegetables (containing 107 mg vitamin C and 83 mg vitamin E). catechin (13. Marcus S. and tomato. 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 [9.5 mg/10 MJ). broccoli. the question of whether multiple vitamin supplementation provide better protection against oxidative DNA damage than a single dose does cannot be answered unambiguously. orange. Age is shown as range or as mean ± standard deviation. and papika carotenoids (2. pineapple.4 mg). In two well-designed studies of the effects of administering a combination of antioxidant vitamins. Consisted of β-carotene (20050 IU). whereas tocopherols and carotenoids exert their effects somewhat longer.2 mg). β-carotene (399 mg). Among studies in which multiple doses of single antioxidants are given.7 mg). 18 IU vitamin E. Cook. 60 mg vitamin E. This suggests that the protective effect of a single antioxidant is relatively short. lycopene (4. lutein (4. there are fewer that reveal protective effects than those that report only negative results [9. vitamin C (500 mg). N-acetyl-1-cysteine (181 mg).

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

0 mg). whereas this intervention was found to have a pronounced period effect [20].34. beet.39].Steffen Loft. In the first study.4 mg). lutein (4. 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. parsley. cranberry. In the subsequent study. in which both sexes were included. bixin (11. Cook. 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].31.41]. In four additional studies. 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]. pineapple.35. and paprika carotenoids (2. Peter Møller. cabbage. In the one study a beneficial effect was found for drinking 300 ml/d for a week [31].45]. 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].38.35]. Marcus S.5 mg).37] or in subjects undergoing cold-weather field training at a moderate altitude [33. Investigations of the effects of natural food products are distributed about equally between studies reporting beneficial and those reporting null effects. A comparable study involving supplementation of a mixture of carotenoids (daily intake: α-carotene (1.36]. kale. broccoli. and tomato) [29]. papaya.32. lycopene (4.4 mg). there being a tendency for only the male subjects to benefit from ingestion of Brussels sprouts. the difference between results at the end of supplementation and at baseline) but no difference between the two groups at baseline [30]. Three additional studies in this area concerned the effect of drinking green tea [see 27. 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].7 mg).44.28. β-carotene (6. the effect was less clear. whereas in one of the other two studies . 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. On the other hand. that involved only male subjects. spinach. the Brussels sprouts supplementation was found to lower the urinary excretion of 8-oxodG [41]. fruits. 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. A positive effect of vegetable juice consumption on 8-oxodG excretion was also observed in subjects enrolled in a soccer summer training camp [26]. A number of studies have involved supplying antioxidants in the form of berries. No effect on the urinary excretion of 8-oxodG was found for the ingestion of capsules containing juices and powders of fruit (apple. and peach) and of vegetables (carrot. and vegetables. Rafa∏ Ró˝alski.e. orange.2 mg)) revealed a statistically significant difference between the active and the placebo group in the delta values obtained (i. tea. neither supplementation of vitamin C or vitamin E or a combination of them was found to have any effect in healthy subjects [24. Further studies showed multi-vitamin tablet supplementation to provide no beneficial effect either in normal subjects [20. Mixed results concerning the effects of a dietary supplementation of Brussels sprouts (300 g/d) were obtained in two studies [40.

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.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]. which leads to iron overload. could increase the likelihood of detecting a protective effect. The absorption of non-haem iron has also been found to be affected by ascorbic acid [49]. revealed a statistically significant positive effect of drinking green for 4 months [27. and 3) the alterations in the urinary concentration could not be assessed due to insufficient information [42]. Interestingly. Although the unadjusted data of the third study indicated no beneficial effect of drinking green tea. such as those of regulating changes in gene expression. It is worthy of note in this context that presumably healthy men may have the hereditary disease idiopathic haemochromathosis. such that iron catalytic to free-radical reactions (a so-called labile iron pool — LIP) is present in the blood plasma [47]). Indeed. 5. such as in the case of severe oxidative stress. the prooxidative properties of certain of the antioxidant vitamins (vitamins C and A) may take effect. It is possible. including baseline 8-oxodG levels. antioxidant vitamins may have biological activities. 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. 2. that the presence of oxidative stress.45]. therefore. Paradoxically. 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]. The question can be raised as to whether antioxidants in the blood and 8-oxodG in the DNA of lymphocytes and leukocytes are representative of the situation in the target tissue. vitamin supplementation of HIV-infected patients showing very low levels of antioxidant vitamins and significantly increased lymphocyte amounts of 8-oxoGua (as well as other base modifications) was found in one study to result in vitamin restoration to levels characteristic for the control subjects. Experimental data suggest that supplementation of vitamin C to iron-overload subjects may increase the oxidative DNA damage that occurs [46]. 2) the putative active constituents (isoflavones) were not measured in the plasma. 4. drinking soya hypocotyl tea was found to be associated with a lower urinary excretion of 8-oxodG. Under some circumstances. which might fail to be recognized. adjustment of the data for a number of variables. 3. 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]. It is possible that a preventive effect of the vitamins can be only be seen when their basal levels are very low.

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

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One of the major problems in measuring 25-(OH)D lies in the molecule itself. The biologically most active vitamin D metabolite is 1. the circulating 1. Measurement of serum 25-hydroxycholecalciferol as marker of vitamin D status Jakob Linseisen and Sascha Abbas DKFZ. Although such measurement is very accurate.). Vitamin D is metabolised by hepatic 25-hydroxylase into 25-hydroxycholecalciferol (25-(OH)D). 7-dehydrocholesterol. 25-(OH)D measurements are quite vulnerable to matrix effects. Germany Vitamin D3. the half-life time of its precursor. which is the major circulating metabolite. this metabolite is a good indicator of the vitamin D-stores obtained both from exposure to sunlight and ingestion of vitamin D [2]. to lipids.3. is synthesized in the skin. is converted by UV light from the sun (UVB 290–315 nm) into previtamin D3 which slowly isomerizes to vitamin D3. As compared with 25-(OH)D. There is overall agreement that 25-(OH)D is the appropriate biomarker to measure vitamin D-status in humans. Extraction from the matrix (serum or plasma) is thus required for most analytical procedures (see Table 2. vitamin D (cholecalciferol). Since the time of first assays in 1971. 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.25-(OH)2D.25-(OH)2D whereas 25-(OH)D has only limited biological activity [1.8]. Measurement by means of HPLC is considered by most to be the gold standard here [6. arising from the irradiation of ergosterol). Its precursor. or cholecalciferol. Considerable interest has developed in assays for measuring 25-(OH)D. First of all it is a very hydrophobic compound and secondly it exists in two forms.2]. strong efforts have been made to simplify them for routine use [6].Jakob Linseisen. Plasma 1. In the blood vitamin D and its metabolites are bound to the vitamin D binding protein. Sascha Abbas 64 2.7. Cancer Epidemiology. the determination of circulating 25-(OH)D is not an easy task [7].g. Further hydroxylation to 1. Such matrix effects can markedly diminish the validity of assays carried out [8]. is rather short (24 h in the blood circulation). Because of its hydrophobic nature. it has to be performed by experienced personnel. is also not regarded as a suitable biomarker of vitamin D status [5]. with an even shorter serum half-life time of 4–6 h. In addition.25-(OH)2D or calcitriol) occurs primarily in the kidney (1α-hydroxylase).25-(OH)2D level does not provide valid information on the individual’s vitamin D status [6]. Heidelberg. Because of its serum half-life time of about three weeks [3]. its thus being more strongly affected by very recent sun exposure or dietary intake of vitamin D [4]. e. Determination of 25-(OH)D As reflected in the recent scientific literature.25-dihydroxycholecalciferol (1. 25-(OH)D2 and 25-(OH)D3. requires expensive equipment (HPLC) and is more time-consuming than other . since its conversion from 25-(OH)D is also tightly regulated.

5 9. Table 2. enzyme immunoassays (EIA) and chemiluminescence assays (CLPBA) are easier to handle. requires luminometer Comments (nmol/l) CV (%) CV (%) ≤6 <8 <12 RIA. ELISA — enzyme-linked immunosorbent assay. Radioimmunoassays (RIA). 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. 100% cross-reactivity with 24. 0 min ELISA. CPB — competitive protein binding. Serum. as well as being fast. Commercially available 25-hydroxyvitamin D assays [6] Range of Test principle. 0 min ChemilumiSerum nescence.3—250 2. IDS Serum or plasma 50 µL Two-step reagent extraction 4—400 ≤5 5. 10 min Calibrators and controls in the serum matrix. IDS Serum or plasma 50 µL Serum or plasma 50 µL None 6—360 ≤5 <6 <9 3 h. There are important differences in the performance of the various detection methods.9 3 h.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 65 methods.6 11. however.7. 10 min Uses vitamin D binding protein * Represents the initial starting volume of the sample. Table 2. summarizes some of the performance characteristics of most of the assays that are available commercially. Immundiag. counting or microplate reading times.5 7. no yield determination required No radioactive waste. HPLC — high-performance liquid chromatography. or plasma Nichols 20 µL Institute Diagnostics HPLC.0 5. 50 µL Extraction detection (nmol/l) Acetonitrile Sensitivity Intraassay Interassay Assay time** 2 h.5—300 18 6.9 14 1 h. Biomedica Proprietary extraction reagent Unknown 6.25 (OH2)D The primary antibody is the vitamin D binding protein from serum Fully automated. 24.4 9 11 5 h. 25 (OH2) D.7.2 8. RIA — radioimmunoassay. DiaSorin Serum or plasma.2 75 min Acetonitrile and C18 cartridge extraction Acetonitrile 15—150 4.plasma nostics or urine 500 µL 8—312 2. and are thus more frequently used. 24.25-dihydroxyvitamin D. ** Does not include extraction. IDK Serum 500 µL 17. Sample type Manufacturer and volume* RIA. 0 min ELISA. CV — coefficient of variation. .

The mean serum 25-(OH)D concentrations differed 2-fold between laboratories (Figure 2. Binkley et al.Jakob Linseisen. lists the methods and the normal range for 25-(OH)D measurement in the laboratories that participated. On the basis of their results.8.). RIA — radioimmunoassay. 2.). .5.4. 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. a broad range (17–95%) of the expected concentration was found by the different laboratories [11] (Figure 2.8. Fig.1 to 35. Binkley et al. Mean (SEM) results for the serum 25-(OH)D concentrations for 10 subjects as estimated by six different laboratories (for abbreviations see Table 2. Sascha Abbas 66 Validity of different assays Only few studies have compared the different commercially available assays [9–13]. [11] recommended the use of RIA techniques for 25-(OH)D measurement until other methodologies have been developed further. The means varied widely between laboratories from 17.). [11] reported recently not only immense variation between different assays but also variability between different laboratories in using a given assay. Table 2.4. and the proportion of subjects below an arbitrary threshold of insufficiency (32 ng/ml) varied between 17 and 90%.005) [11].6 ng/ml (p < 0. In their study. the serum of 10 subjects was sent to six different laboratories.8. After spiking of the serum samples with a defined quantity of 25-(OH)D. HPLC — high-performance liquid chromatography. Table 2.

Patients. Figure 2.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 67 Fig. 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]. Both assays also underestimated the 25-(OH)D-concentration as compared with the HPLC-method [10].7 to 18 ng/ml. 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. The means varied widely between laboratories from 27. as estimated by five different laboratories (for abbreviations see Table 2. There has been discussion regarding the importance of the assays detecting 25-(OH)D2 as well as 25-(OH)D3. 59% of the participating laboratories were found to have met the target [12.5. This suggests that the contribution of 25-(OH)D2 to the overall 25-(OH)D supply is less important than had been previously thought. Each of the laboratories was given five serum samples quarterly.6 ng/ml (p < 0. However. however.05).005) from 7. Mean (SEM) results for the 25-(OH)D concentrations in the serum samples of 10 subjects spiked with the 25-(OH)D standard. 2. the greatest increment being detected by HPLC [11]. finding discordant serum 25-(OH)D-values. Another study compared the DiaSorin RIA assay with the Nichols chemiluminescence assay. .6. several authors have called for international standardization of the vitamin D assays that are available and that are affordable for practicing clinicians. shows the latest results of this external quality control project. The increment was less than anticipated and varied between laboratories (p < 0. 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]. 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). However. 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]. These give the impression that only method 6 exceeded the limit. In accordance with these findings.13].4 to 44. especially those in the US.8). According to the latest DEQAS (Vitamin D External Quality Assessment Scheme) report. the authors of the report state that the validity of the 25-(OH)D results that are obtained will justifiably continue to be questioned.

method 6. A summary of studies on vitamin D status in the elderly is given elsewhere [1]. 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. method 5. In epidemiologic studies. Apart from the analytical problems. The method means of samples known to contain 25-(OH)D2 were excluded. method 2. IDS EIA. that set the cutoff points at much lower levels.Jakob Linseisen. race. Sascha Abbas 68 Fig.6. the serum 25-(OH)D concentrations in different populations vary with latitude. age. and the composition of the population being studied. competitive protein-binding assay. 2. Recent reports suggest European and North American populations to have a higher degree of insufficiency than had previously been thought [15. 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. In a recent publication. method 4. season. DiaSorin RIA.16]. epidemiological variability needs to also be taken into account. There is growing evidence that there is not only a specific seasonal decline in serum 25-(OH)D during the winter months. due to the serious health risks encountered at levels less than this [7]. Nichols automated chemiluminescence assay.18–23]. This is in contrast with most of the published studies. method 3.18]. suggested cut-offs for insufficiency range from 10 to 30 ng/ml (25–80 nmol/l). Each column shows the mean deviation (% bias) and SE from the target value (ALTM) during the period of 2000–2004. IDS RIA. but there may also be a significant proportion of the population that exhibits asymptomatic subclinical vitamin D insufficiency. Method 1. around 25–40 nmol/L [1. Accuracy of 25-(OH)D methods used by the different DEQAS participants [13].15. HPLC. . It is important to note that “normal” is not defined as the median population level. dietary intake. Populations at risk include nursing home residents and the elderly. In addition to the high degree of uncertainty that the divergence of the results for different techniques and different laboratories creates. Hollis defined 25-(OH)D levels below 80 nmol/L as being deficient. as shown by the seasonal variation in vitamin D status that occurs [17. especially the home-bound elderly.

J Clin Endocrinol Metab 1986. Clin Chem 2000. Lips P. 4. Review SeriesVolume 2.46:756–65. 10. Hollis BW.135:317–22. Cowgill CS.63:656–60. DeLuca HF. Epidemiologic studies in particular can contribute knowledge concerning the association between vitamin D insufficiency and the risk of disease. Belsey R. 11. Plum L.89–90:611–4. May 2005. McGuiness M. Lake E. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J Nutr 2005. Goldswain PR. Overview of Vitamin D Measurement and Methodologies. vitamin D3). Musk AA. Competitive binding assay for vitamin D and 25-OH vitamin D. . Editorial: The determination of circulating 25-hydroxyvitamin D: no easy task. Ann Clin Biochem 2003. Myles M. 5. DeLuca HF. Ann Clin Biochem 2004. Wilz DR. References 1. J Clin Endocrinol Metab 1971.40:546–51. Hollis BW. Endres D. Several problems are important to bear in mind including the different forms of vitamin D (vitamin D2. J Clin Endocrinol Metab 2004. The measurement of vitamin D: analytical aspects. Issues of methodology. Taranto M. including the risk of cancers at various sites. Clemens TL. 8.89:3152–7.41:272–81. the determination of vitamin D status is still a challenging task. 9. Smith GA. Immunodiagnostic Systems (IDS). 3. 2.89:3149–51. Comparison of commercially available (125)I-based RIA methods for the determination of circulating 25-hydroxyvitamin D. Nonetheless. Binkley N. epidemiologic variation in vitamin D status and its determinants. Which circulating level of 25-hydroxyvitamin D is appropriate? J Steroid Biochem Mol Biol 2004. Metabolism and excretion of 3H-1. Potts JT. Zhou XY. and problems in defining the appropriate threshold for vitamin D sufficiency. Hollis BW. Hart GR. 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 1978.46:1657–61. 7. 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.33:554–7. Lemann J. Glendenning P. J Clin Endocrinol Metab 2004.25-(OH)2-vitamin D3 in healthy adults. the measurement variation introduced by different assays and different laboratories. et al. This makes strong scientific research efforts needed. Caldas AE. Krueger D. Zerwekh JE. Hansen KE. Lindsay R. Noble JM. vitamin D metabolites have various important biological effects. 6. Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 69 Conclusions In conclusion. Gray RW. et al.

J Clin Endocrinol Metab 1998. Khan A. Vitamin D status: effects on parathyroid hormone and 1. Clin Chem 2004. Aksnes L. Geographical differences in vitamin D status. Osteoporos Int 1997. J Steroid Biochem Mol Biol 2004.Jakob Linseisen. Am J Clin Nutr 2000. Woitge HW. Seasonal variation of biochemical indexes of bone turnover: results of a population-based study.135:332–7. Chapuy MC. Holick MF. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. Hollis BW. J Nutr 2005. Alsaker E. Proc Nutr Soc 2003. 20. Sackrison JL. 25-dihydroxyvitamin D in postmenopausal women. et al. Leidig-Bruckner G. Andersen R.89:5387–91. J Steroid Biochem Mol Biol 2004. Vitamin D2 is much less effective than vitamin D3 in humans. Binkley N. Fenske JS. Haug E. et al. Maamer M. et al. Heaney RP. Obstet Gynecol Surv 2005. Jones G. Vitamin D status of middle-aged women at 65–71 degrees N in relation to dietary intake and exposure to ultraviolet radiation. Jones J. 14. Davison KS. Ovesen L. Prevalence of vitamin D inadequacy among postmenopausal North American women receiving osteoporosis therapy. Hanley DA. with particular reference to European countries. Berry J. Makin HL. Brustad M.89–90:467–71. Engelsen O. Katzer JT. Siris ES. 19. 18. Prevalence and predictors of vitamin D deficiency in five immigrant groups living in Oslo. et al. Kissling C. Sascha Abbas 70 12. Hercberg S. Morris HA.89–90:621–2.50:2195–7. Ersfeld DL.59:57–63. Vitamin D insufficiency in North America. Eur J Clin Nutr 2005. Metab 2004. Public Health Nutr 2004. Carter R. J Clin Endocrinol. Miller AB. 17. . 15. 23. Lund E.60:658–9. Scheidt-Nave C. Carter GD. Need AG. Hypovitaminosis D in a normal. Holvik K. Meyer HE. Jakobsen J. MacFarlane GD. 21.71:1577–81. Carter GD. Armas LA. Body JJ.7:327–35. Norway: the Oslo Immigrant Health Study. Nordin BC. Carter CR. Brunvand L. Gunter E. Jones J. Arnaud S. 13. Horowitz M. Grauer A. Meyer K. Beard MK. 22.62:813–21. apparently healthy urban European population. 16. Galan P. Preziosi P. Prevalence of vitamin D insufficiency in an adult normal population.7:439–43. Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation.83:68–75.

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

Selenate and selenite are reduced by glutathione to hydrogen selenide. . which is turn is the precursor of selenocysteine in selenoproteins and various excreted forms 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. At toxic doses. selenocysteine and selenite can be converted into the key metabolite hydrogen selenide (H2Se).).Björn Åkesson. 2. Metabolic pathways of selenium. Selenomethionine. Several compounds can be converted into methylselenol (from [8]). dimethylselenide or trimethylselenonium ions for excretion. selenium is removed as dimethylselenide via exha- Fig. 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).7.7. Selenomethionine and selenocysteine can also be converted to hydrogen selenide. a much lesser amount being excreted as trimethylselenonium ions [7]. Katharina Bruzelius 72 Table 2. The major selenium metabolite excreted in urine is selenosugar.

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

This structure is located in the 3’-untranslated region of the mRNA in eukaryotes and immediately after the UGA codon in prokaryotes [14. Many of the known selenoproteins catalyse redox reactions in which selenium is at the active site. one internal loop. In addition to the SECIS element. This takes place when a special stem-loop structure called the selenocysteine insertion sequence (SECIS) is present in the mRNA. these organisms tending to have homologue proteins containing Cys instead of Sec [13]. Except for the SECIS element there are no features in the DNA-sequence that selenoprotein genes are known to have in common.Björn Åkesson. The cytosolic or cellular form of glutathione peroxidase (cGSHPx) was the first selenoprotein identified [10. and the SECIS core. All known eukaryotic selenoproteins have one SECIS-element. selenoproteins also differing markedly in the nucleotide sequence of their SECIS elements. The next major step is the interpretation of UGA as a signal to insert Sec instead of stopping the translation. that regulate thyroid hormones. . Katharina Bruzelius 74 in different tissues are also not well known. except for SeP. which has two [15]. but the mechanisms involved have not yet been elucidated (Figure 2. Biosynthesis of selenoproteins Translation of the codon UGA to selenocysteine (Sec) and its insertion into proteins is a complicated process (Figure 2. Several of the selenoproteins have an homologue protein containing Cys instead of Sec. The current conception is that the mRNA and the SBP2-EFsec loops back towards the translation complex [13. and the recently discovered L30 protein. The other two major groups of known selenoprotein enzymes are the iodothyronine deiodinases.).15]. The SECIS consensus sequence consists of two helices. catalysing the reduction of oxidised thioredoxin and other substates [1. but the catalytic ability of these latter proteins is much lower. serine is bound to tRNASec and then transformed to selenocysteine [13]. It is found in almost all tissues and is believed to play a part in the body’s antioxidant defence.17]. Several other glutathione peroxidases containing selenocysteine have been found since then. 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]. one apical loop.8.11]. which is a short sequence of non Watson-Crick paired nucleotides that appear to exist in all selenoprotein mRNA:s. the Sec-specific elongation factor (EFsec). and the thioredoxin reductases. In the first step. There are no selenoproteins known to be present in yeast or higher plants. In eukaryotes the Sec codon and the SECIS element are located at some distance from each other in the mRNA. additional factors are required for the insertion of Sec in eukaryotes such as the SECIS binding protein 2 (SBP2).12].).8.

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

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

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

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

41.41 (0.07) 1. a GSHPx activity. 1. 0. Selenoprotein P.33.60.75—2. 1.20—4.73 (0.20 (1. 1.16) 0. 1. 0. mU/mg protein.22) 0.38 (0.13 (4.54—1.11. 1. respectively.25) Plasma selenium (µmol/l) 1.11.66) 1.38 (2.86) 0.6.) 1. 0. Trial I baseline Finland.69 (0.83. glutathione peroxidase and protein-bound selenomethionine are the major selenium fractions contained in plasma [56]. Immunoassays for measurement of this protein have been developed [57–59].32) 3.18 (0.41 (1.08. 346)a 6.63—1.12) 0.23 (1.91 (1. 1. medium and high fish intake. 1.83) 0.86 (0.85) 1.00 (0.78 (1.95 (2. c Data from subgroups having (top to bottom) low.66—1. 2.31) 2.91 (0.15) 1. U/l.u. Selenoprotein P accounts for at least 40% of the plasma selenium [37]. .07 (0.51.51 (6. 1.46—1.69—5.77 (1. 0.56.55 (0. 1.10 (1.64) 0.83) 1.28.21) 1. 4.03 (0.98.44) 1.34.56) 4.31—4. 0.14) 0.69 (0.44.88) 1.27) 1.30—1. 397)a 302 (259.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]. [63]). Study European countries Prior to LDL-apheresis After LDL-apheresis Finland.43) — — eGSHPx (mg/l) — 352 (306.24) 1. 0. Median values (and range) are given. b GSHPx activity.43.73) Values are means (95% CI).16. Plasma concentrations of SeP.69. 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.51) (1. In the following various results from their use in the authors’ laboratory are summarized (Table 2.).2 mmol/l [55].83 (0.92.11. Supplementation by selenate resulted in glutathione peroxidase activity plateauing at a plasma selenium concentration of 1.47) 1. selenium and eGSHPx in different studies (from ref.52 (0.74)b — — — 126—205 38 21 16 31 1. Table 2. 1.14 (1.65) 3.21.38 (1.52) 0.

Concerning the association of selenium status and exposure to toxic metals. The levels of selenoprotein P and eGSHPx were found to vary markedly for subjects from different regions and with different diseases.68]. Home parenteral nutrition patients with a variety of gastrointestinal diseases showed much lower levels of extracellular GSHPx. the selenium status of the subjects thus being higher. significant relationships between milk intake versus selenoprotein P and urinary selenium.). Considerable attention has been directed at two studies conducted in Finland regarding the use of different forms of selenium [67.9. Poland revealed an inverse relationship between the blood lead level and the level of selenoprotein P and of extracellular GSHPx [65]. 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]. studies of lead-exposed children in Katowice. eGSHPx and plasma selenium as markers of selenium status. In a recent study of Chinese subjects of low selenium status given different doses of selenite and selenomethionine. 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. Regarding the relationships between SeP. For women. in which the subjects were low in selenium status. 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. and also between fish intake versus serum and urinary selenium were found. performed after the introduction of selenium-enriched fertilizers in Finland. the selenoprotein P values increased after selenium supplementation. plateauing within two weeks [69] (Figure 2. In the first study.Björn Åkesson. . with some indication of a plateau. Katharina Bruzelius 80 Selenoprotein P as a biomarker Selenoprotein P levels were measured in healthy adults from 17 European Regions [60]. There was a close correlation between selenoprotein P and plasma selenium values. In the second study. total selenium and selenoprotein P than control subjects did [64]. but that at normal selenium status selenoprotein P usually correlated more highly with plasma selenium than eGSHPx did.9. A summary of the responses shown by the different selenium indices in the first of these two studies is provided in Figure 2. it was found generally speaking that the selenoprotein P level correlated more highly with the plasma selenium and eGSHPx level at low selenium status than at high selenium status. In a study of Latvian fisherman [62]. 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. 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. however. 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].

lower prediagnostic plasma selenium was found for cases of cancer than for controls. The strongest associations between premorbid plasma selenium levels and risk of cancer were observed for cancer of the respiratory and digestive tracts. This suggested that selenoprotein P is a better indicator of selenium nutritional status than glutathione peroxidase is [70]. These matters are reviewed in greater detail in the chapter that follows (Gromadziƒska et al.). 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. Generally speaking.5–2 ppm) levels of selenium to the diet has a carcinostatic effect in animals treated with carcinogenic chemicals [71. Blot and coworkers [73] found that a mixture of selenium. whereas in other studies no significant differences in this respect were found between cases and controls [82–86]. in turn found selenium supplementation to reduce total incidence and mortality of cancer in an American study group (see below). Clark and associates [74]. Full expression of selenoprotein P was not achieved at the highest dose of either form (66 µg/d). 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. Biomarkers of selenium status and cancer Experimental studies have shown that the addition of high (0. Fig.87]. Interest in the preventive role of selenium supplementation in humans was stimulated markedly by results from two intervention studies. The calculated degree of protection was found to differ between cancer sites [81. In a number of case-control studies. β-carotene and α-tocopherol reduced the total cancer mortality and stomach cancer rate in a Chinese population. 2. .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.69]). particularly among men [75–81]. in view of the many regulatory mechanisms that exist for the incorporation of selenium into selenoproteins and the varying effects of different forms of dietary selenium on indices of the selenium status.9.72].

the ORs (adjusted for smoking) were 5. the SeP levels for cancer of the respiratory tract were found to be significantly lower than in matched healthy control subjects. and 0.Björn Åkesson. The association between the relative risk of getting cancer and the SeP concentration found was also estimated from quintiles of the SeP level.4. Percentage differences in the prediagnostic selenium and SeP plasma levels between cancer cases and controls (set at 0) for cancer at different sites.01). The individual references are cited in [63]. Bold contour: p < 0.0. The previously reported association of plasma selenium levels with cancer risk can very likely be explained by the corresponding association of SeP levels. 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. 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. The circles represent selenium and the squares SeP. When cases were divided into subgroups according to cancer site. 2.9.0. the ORs (adjusted for smoking) in tertiles of the SeP level were calculated for the respiratory. 2. For increasing quintiles. digestive and urinary tract and for cancer of other sites. 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.10. and toenail selenium [90–92] were observed in smokers than in non-smokers.85.05.89]. . This is probably due to SeP constituting at least 40% of the total selenium in human plasma [37]. In other studies. The factors contributing to the lower selenium status in smokers are unclear. 2.05.2. In Figure 2. 0.6 respectively. These were 6. normal contour: p ≥ 0. Fig. the case-control differences of plasma selenium and SeP are compared for major cancer sites in several different study populations. 3. lower values of plasma selenium [77. Smokers were found to have significantly lower levels of selenoprotein P than nonsmokers [88]. plasma selenium has been used as a marker of selenium status. In addition. whole blood selenium [89. 2. erythrocyte selenium [89]. respectively (p for trend = 0. in the lowest tertile as compared with the cases in the highest tertile.10. Smoking and biomarkers of selenium status Several factors other than selenium intake may affect biomarkers of selenium status.2.0 and 1.90].

which together with superoxide can produce peroxynitrite [39]. This agrees with findings of Dreher and co-workers [93] indicating that the human selenoprotein P promoter is rendered less active by cytokine treatment. As reported by Sies and coworkers [94]. smoking was found to be significantly associated with an unhealthy pattern of nutrient intake. although this association disappeared when lead was included in the model. Multiple regression analysis also indicated the blood cadmium to increase significantly with a decrease in the selenoprotein P level. selenomethionine and glutathione peroxidase can scavenge peroxynitrite. It has been reported that smokers eat less selenium than non-smokers do. α-tocopherol and selenium given for a 5. a small but significant reduction in total cancer mortality was obtained (RR = 0. In the Linxian trial involving supplementation of β-carotene. This may also explain the slightly lower selenoprotein P concentration in smokers. which suggests a repression of selenoprotein P expression during an acute phase reaction. whereas smoking alone did not serve as a true predictor of this effect. suggests that the selenoprotein P levels are reduced by inflammatory activity. 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]. which could exacerbate the risk of cancer associated with smoking [96]. in a meta-analysis of 51 published nutritional surveys of the relationship between smoking status and nutrient intakes.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. whereas the concentration of cadmium in the blood is significantly higher in smokers [95]. Patients with a history of skin carcinomas who . 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. 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]. in particular mortality due to stomach cancer [73]. several intervention studies having indicated beneficial effects [73.91).74]. 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. Multiple linear regression analysis of the data also suggested a depressive effect of cadmium on the concentration of selenium in the blood. In addition. The natural presence of cadmium in tobacco smoke may also contribute to the lower selenium status found in smokers. It has also been shown that selenoprotein P plays a role in the defence against peroxynitrite [39].25-year period. In another study. which could probably in part explain their lower selenium level [90]. Smoking may also increase oxidative stress since cigarette smoke is a rich source of reactive nitrogen species. both being acute-phase reactants.

2. References 1. colorectal (RR = 0. 5. β-carotene. J Anal Atomic Spectrom 2001. In addition. Sunde RA. 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]. Recent results of the SUVIMAX study showed supplementation with vitamin C. Carcinogenesis 1990. selenonium ions and inorganic selenium by ion exchange HPLC with mass spectrometric detection and its application to yeast and algae. Mammalian selenium-containing proteins.Björn Åkesson. Waschulewski IH.42) and prostate (RR = 0. J Nutr 1988.11:2071–3. 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. Bansal MP. concerning the purportedly positive effects of selenium in connection with the prevention of cancer provided certain evidence for permitting a qualified health claim regarding selenium and cancer [103]. Occurrence of low-molecular-weight and high-molecular-weight selenium compounds in fish. evaluation of health claims by the FDA in the U. Medina D. Srikumar TS. Mukhopadhyay R. The type of selenium supplements best employed is also in need of further investigations. 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]. .21:453–73. Annu Rev Nutr 2001. Larsen EH.37) cancer [74]. Effect of dietary methionine on utilization of tissue selenium from dietary selenomethionine for glutathione peroxidase in the rat. 4. but additional studies are needed to determine whether there is also an increased risk of some forms of cancer after selenium supplementation.51:45–9.S. Experimental findings also indicate that in some experimental systems selenium can both promote and inhibit cancer [98]. DNA sequencing of a mouse liver protein that binds selenium: implications for selenium's mechanism of action in cancer prevention. Fan T. Scott J. vitamin E.16:1403–8.50) and in incidence of lung (RR = 0. Katharina Bruzelius 84 were treated with 200 mg selenium per day showed significant reductions in total cancer mortality (RR = 0. 3. Cook RG. It is apparent from this review that selenium can play an important role in cancer prevention.118:367–74. In another Chinese investigation. Mukhopadhyay T.54). Vahl M. Åkesson B. Hansen M. Behne D. Food Chem 1994. Speciation of selenoamino acids. 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.102]. selenium and zinc to reduce the rate of prostate cancer in men having normal levels of prostate-specific antigen in their plasma [99]. Kyriakopoulos A.

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however. and in epidemiological studies of risk of cancer — and the outcome of long-term trials concerned with the disease-preventive potential of selenium supplementation. [2] observed that liver tumours occurred in rats fed protein-deficient fodder and grains originating from Serich (5–10 ppm) regions. It should be noted. however. and concern for it as a carcinogenic agent dates to 1943 [2]. [6] observed the development of tumours in male rats fed a diet containing 4. including the forms of selenium present in the diet and in the body.Vitamins and selenium: Anticancerogenic activity of selenium 91 2. Wojciech Wàsowicz Department of Toxicology and Carcinogenesis. Tscherkers et al. Nofer Institute of Occupational Medicine. which suggests that the neoplastic changes that occurred could be attributed to Se. equivalent to inclusion of 103–207 ppm Se in the diet for a 10-day period. Two of the chapters deal with these and related matters. Edyta Reszka.5. it is quite possible that the organic selenium compound employed there had carcinogenic properties not linked with the element per se. History Historically. [5] published the alarming finding that feeding rats 0.3 ppm level.05–0. suggested that the pathological changes that occurred reflected regeneration of the liver rather than a carcinogenic process. ¸ódê. and intervention trials to investigate the effect of selenium supplementation on the incidence of cancer are summarized. interest in selenium (Se) as a toxic trace element dates back to 1937 [1]. their functions and mechanisms of action. nevertheless. Shapiro [3]. their use as biomarkers of selenium status. the methods used in assessing the nutritional status of selenium — in general. as well as epidemiological findings regarding the relationship between selenium level and risk of cancer are reviewed. Anticancerogenic activity of selenium — molecular mechanisms and epidemiological data Jolanta Gromadziƒska. In 1963. in addition to the occurrence of different selenoproteins. Seifter et al. whereas such tumours were found in less than 1% of the animals in the control group. . observed the development of different forms of liver tumours. in response to selenium supplementation. Poland Introduction There are many different aspects of the relationship between selenium and cancer. Nelson et al. In the present chapter the mechanisms behind the role that selenium can play in cancer prevention. 11 were found to develop liver tumours.3 ppm Se. induced thyroid adenoma. that since no studies have been reported of animals administered an analogous compound that did not contain selenium. In the previous chapter the forms and metabolism of selenium. Of 53 animals that survived for 18 months when thus fed.1% bis-(4-acetamine)-phenylselenyl hydroxide. Researchers of the former Soviet Union [4] feeding Se (as selenite) to rats at a 4.

but they also decreased in number with an increase in the amounts of selenium provided.1 to 1. Providing selenium at a still higher level (2. whereas in the group receiving the largest amount of Se in its drinking water (1.1 to 0. who reported that in rats a diet enriched with 5 ppm Se decreased the incidence of liver tumours brought on by 3’-methyl-1.4-dimethylaminobenzene (3’-MeDAB). a supply of 0. [10] showed that adding 0. in rats to which either of two different forms of Se were administered [9]. respectively.15 ppm Se and the other groups receiving. using female mice of the C3H strain. particularly when it was provided at both the initiation and the promotion stage of chemical carcinogenesis. At the same time. Harr et al.12-dimethylbenzantracene (DMBA). Two groups of animals received a diet containing maize oil. Ip and Sinha [13] investigated the effect of various concentrations of Se on the development of breast cancer induced in rats by 7. a control group receiving a basal diet that contained 0.5 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).0 ppm) they first developed at 17 months of age. In the control group. These results indicated that supplying Se in larger amounts protects animals from developing cancer or delays . to 3%. In both groups the incidence of cancer was found to decrease with increasing level of Se in the diet. An interesting study of the anticarcinogenic role of selenium was carried out by Schrauzer et al. Supplying Se was found to decrease the number of tumours induced (from 97 to 46%). [15]. Wojciech Wàsowicz 92 The anticancerogenic effect of selenium in animals was first reported in 1911. 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]. in addition to this. Another early observation of such an effect was that of Clayton and Bauman [8]. In the groups given extra Se in their drinking water. 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]. The animals were divided into several groups. tumours not only developed later.Jolanta Gromadziƒska. when Wassermann et al. The maize oil content in the one case was high (25%) and in the other case low (5%). Ip [14] also studied the effect of selenium on different stages of cancer development in rats given 5 ppm Se at different time intervals after the administration of 10 mg DMBA.0 ppm Se in their drinking water. Edyta Reszka. [7]) managed to inhibit the development of placental tumours in mice by use of Se compounds. 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]. the first tumours developed when the animals were 4 months of age. Se supplied during only one of these two phases had a much weaker effect [14]. 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%.5 ppm) reduced the incidence of cancer even more. (cf. a strain characterized by the spontaneous development of breast adenoma in 80% or more of the cases.

Some reports suggest that the action of selenium toward cells that have been transformed earlier and toward normal cells differ [32]. depends on the cellular GSH/GSSG ratio. one should bear in mind that the results of animal studies cannot be directly extrapolated to humans. can prevent mutations by serving as free radical scavengers [26–28]. in the majority of cases the reduction being quite significant [19]. It is also possible that selenium in the form of glutathione peroxidases. The anticarcinogenic effects of Se depend upon the chemical form of the Se-compound involved and the nature and dosage of the carcinogen. Experiments in which no effect of Se was observed were rather rare. 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. Thus a number of mechanisms for the anticarcinogenic effects of selenium and of many selenocompounds have been proposed. 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]. cellular or nuclear level. and inhibiting angiogenesis [29. At the same time. Regarding the mechanisms involved. proliferation and differentiation of the cells can be affected. The anticarcinogenic effect of Se has been found to depend on the chemical form in which the element is administered. The effects can occur at a systemic. and perhaps selenoprotein P and other selenoproteins as well. affecting the cell cycle. its dose and the agent that induces the development of cancer [16].23].). 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. resulting in more efficient carcinogen detoxification [24. It has also been shown. and 2. that selenium compounds can induce cell death through a mechanism distinct from oxidant toxicity [22. In several studies. The activity of many cellular targets.30] (Tables 2. it has been postulated that the chemopreventive effect is related to the toxicity of selenium and the oxidative stress it induces. in which the metabolism. 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). altering the metabolism of carcinogens. In addition.Vitamins and selenium: Anticancerogenic activity of selenium 93 the carcinogenic processes. Glutathione .25]. including their providing protection against oxidative damage.13. 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. enhancing immune responses.12. Cells adequately supplied with Se have been shown to be less sensitive to effects of both endogenous and exogenous carcinogens [17]. 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. viruses or transplanted tumours [18]. since reactive oxygen species can promote apoptosis in vitro [21]. however. the activity of xenobiotic-metabolizing enzymes in vivo has been reported to increase when selenium compounds are given.

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

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

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

high concentrations of selenides tend to also be generated. but also its metabolites. [50. 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]. affect cellular metabolism. selenomethionine and Se-methyloselenocysteine can be transformed into selenides. At the same time. such as protein kinase A. ref-1. 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].49]. Trx inhibits certain kinases of the MAPK family (e. diacylglycerol kinase and thymidyl kinase [48]. ASK1) directly. reduction in the oxidative DNA damage that occurs.12. Some low molecular weight Se compounds have been shown to influence the regulation of gene expression. . Selenite. Se is also known to arrest several kinase pathways important in signal transduction. the concentration of Se in the tissues also being found to be higher after the administration of selenomethionine than after selenite supplementation. In cells with a high Se level. and the glucocorticoid receptors [34. It has been found that both sodium selenite and selenomethionine (SeMet) suppress tumour growth in many animal models involving a dose-dependent response [20.51]. AP-1. The anticancerogenic activities of various Se compounds are summarized in Table 2. 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]. but it is likely that one of Se metabolites is essential to this process.54. Trx limits DNA synthesis. When selenides are generated in sizeable amounts.Vitamins and selenium: Anticancerogenic activity of selenium 97 and AP1 [47]. cellular growth. a key enzyme in the deoxyribose formation needed for DNA synthesis.55]. not only the selenocysteine incorporated into the protein structure. the bioactivation of carcinogens. whereas Trx/TrxR activates ribonucleotide reductase. It is important to note that different selenocompounds are essential for the growth and differentiation of both normal and neoplastic cells [52]. they react with oxygen to produce superoxide and hydrogen peroxide [56]. modulation of tumour angiogenesis. etc. p53.g.. Selenite has been shown to be more effective than selenomethionine in the inhibition of cancer cell growth during chemically induced carcinogenesis [31. Ca+2-dependent and -independent kinase C (PKC). Effect of different selenium compounds in cancer prevention The chemoprevention of cancer by selenium can involve the action of different selenometabolites. [16]. TrxR also regulates gene expression by affecting the cellular redox status and activating numerous DNA-binding transcriptional factors: NF-κB.54]. 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]. The various chemical forms of Se have been shown to differ widely in their anticancer properties. selenodiglutathione.

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

66]. selenomethionine protects the cells from DNA damage through the induction of DNA repair. One possibility is that a small fraction of the SeMet is metabolized to a low-molecular-weight compound [32]. an important epigenetic mechanism that exerts control over gene expression [30]. selenocystine. The methylation of Se compounds to Se(CH3)2 and Se(CH3) results in a decrease in methyl donation. such as benzo(a)pyrene and arsenic [30]. DNA methylation is one of the first steps in the carcinogenesis induced by certain chemicals.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]. Seo et al.68]. [67] described the incorporation of Se into the factors involved in DNA repair regulation. which has an essential role in recognizing DNA lesions in the nucleotides and in the excision of damaged nucleotides in mammalian cells [69]. . They also affect the integrity of the XPA protein (xeroderma pigmentosum group A protein). The postsynthetic methylation of DNA is catalyzed by the family of S-adenosylmethionine-dependent DNA methyltransferases [71]. 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. This decreased GSH concentration in the cells induced apoptosis. Blessing et al. These selenocompounds affect the DNA repair processes through oxidation of the zinc finger structures and the release of zinc from DNA. Limitations in the DNA repair capacity appear to be a key determinant of predisposition to cancer [49. but that the induction of apoptosis is not a simple result of the regulation of p53 by selenium [16]. this preventing DNA methylation from occurring. It has been found that Se derivatives can activate the p53 gene. 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. Selenium can also affect DNA methylation.65]. Reducible selenium compounds (phenylseleninic acid. [49] showed that in normal human fibroblasts. In vitro studies of a mammary cell line exposed to DMBA showed 1. and 2-nitrophenylselenocyanate) were found to cause a dose-dependent decrease in the activity of formamidopyrimidine-DNA glycosylase (Fpg). 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. ebselen. 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. as well as damaging the integrity of the genes of the DNA repair enzymes [67.Vitamins and selenium: Anticancerogenic activity of selenium 99 gene. one of the enzymes involved in DNA repair processes. and to be involved in the pathway connected with the p53-dependent activation of DNA repair processes [64]. which is a dominant mechanism of the cell death these compounds bring about [70]. 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. In investigating the ability of inorganic Se compounds to induce apoptosis.

Selenium and cancer risk — epidemiological results Several studies have shown the development of cancer in humans to be inversely related to the intake of specific dietary components. The 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.Jolanta Gromadziƒska. but no such relationship was found for lung cancer [83]. The anticarcinogenic effect of selenium is also partly based on its ability to produce antitumour metabolites (e.g. Cellular membranes of the T lymphocytes are particularly sensitive to Se deficiency. Selenium regulates the immune response by stimulating natural killer cells and activating antigens of various types to destroy the tumour cells [78]. 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. the inhibition of prostaglandin and immunoglobulin synthesis. NK cells and macrophages. micronutrients and phytochemicals [81. These metabolites that are synthetised in the cell can be involved in the cell’s metabolic pathways and destroy the integrity of tumour cells or induce apoptosis in these cells [51. Case-control studies tend to reflect the short-term Se concentrations in the organism. due to the large amounts of unsaturated fatty acids present in their structure. Insufficient dietary intake of selenium results in many types of defects of the immune processes. however. such as in connection with antibody production and specific cell immunity [77]. Low plasma selenium concentrations are thought to be associated with increased morbidity and mortality from cancer. Low Se concentrations in the tissues and in human body fluids also appear to be associated with numerous changes in the immune system. and impairment of the body’s ability to reject implants and to destroy neoplastic tumours [75. Se can stimulate the expression of IL-2 receptors found on activated T lymphocytes and on NK cells [79].76]. stimulate the expression of cytokine receptors and the proliferation of lymphocytes [73. such as suppression of the host immune response to bacterial and viral infections. including nutrients. An inverse association between selenium and risk of cancer has been reported both in case-control studies and in followup cohort studies. Research over the last decade points to a significant protective role of Se in preventing the development of malignant neoplasms. Edyta Reszka. a statistically significant increase in morbidity from oesophagus and stomach cancer was noted in a population with low levels of selenium. methylselenol).74].82]. To date. In a Chinese study. the results of epidemiologic studies . 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. Selenium compounds can activate cytotoxic cells.80]. such as the Se level associated with the dietary pattern at the time of sampling in cases and controls. reduction in the activity of T lymphocytes. Wojciech Wàsowicz 100 Effects of selenium on immune functions Certain anticancer properties of the selenocompounds may be associated with their effects on cellular immunity.

60) after adjustments for smoking status were made [90]. when the study population was divided up according to Se level.). The significant.24–0. whereas no association between selenium level and risk of lung cancer was found in two studies of non-European populations [88.72 (95% CI: 0.20. 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. 95% CI: 0. indicated selenium to have a certain protective effect. dosedependent protective activity of Se was documented in Finnish and Dutch studies [86.87] in which Se in both serum and toenail samples was analyzed. Some authors have reported there to be an association between cancer risk and Se status. those with high toenail Se levels were found to have a particularly high relative risk of lung cancer (RR: 4. representing a daily Se intake of 55 mg. the mean value obtained for groups showing a high basic concentration of Se was RR = 0. 11 of which had a prospective design.00 (95% CI: 0.57–0.10) for the serum Se level.33. In most of the reports.61–1. 95% CI: 0. only the findings for the two Finnish populations (RR = 0.54–34. . The findings for three major forms of cancer are summarized below. 95% CI: 0.50. to 1.45–1.17–0. In a metaanalysis of 14 epidemiologic studies.81) revealed a statistically significant protective effect of high Se concentrations.77–1. Interestingly.14. whereas others have obtained null results [84. These finding confirm the assumption that the Se level in the toenails can be used as a marker of long-term Se concentration [91]. whereas the value for groups with a low basic level of Se was RR = 0.44) and for the Dutch population (RR = 0.22). but only in populations with a low mean Se level. In the control group of non-cancer patients the mean serum Se concentration was 100 ng/ml blood serum. The protective effect of Se in connection with lung cancer could also be noted in studies examining Se concentration in the toenails. 95% CI: 0.85].97) (Table 2.86 (95% CI: 0.89]. undertaken by Zhou et al [91].09–0. the risk of lung cancer at high Se concentrations was found to be RR= 0.30–0.94 and RR = 0.30) in studies of Se intake based on use of a questionnaire.87) when toenail Se was used as a marker of the concentration of Se in the body.Vitamins and selenium: Anticancerogenic activity of selenium 101 have been rather inconsistent.46 (95% CI: 0. The total RR values for lung cancer in patients showing high Se levels ranged from 0.16).41.80 (95% CI: 0. It is notable that of the women investigated within the Nurses Health Study. Of the studies considered in the meta-analysis.14. the protective activity of Se could be clearly discerned when the reference group consisted of subjects showing the lowest level of this trace element.74 (95% CI: 0.). A review of epidemiologic studies of lung cancer and of the selenium concentration found in biological material.58–1. to 0.

19) 0.6 (15.41–1.102 Table 2.20 (0.108 ppm) 515 (0.08 0.166) µg/g) 250 (0.70–2. c data from 91 individuals.17–0.02) 1. [93] NCC Serum Knekt et al.206) µg/g male.30 (0. [96] Male Female China The Netherlands CC Diet All China — 25 — 1.3) µg/l F) Male All Male Female USA Male Finland 5—8 3.2 (24. b data from 189 sets.308) µg/g) 250 (0.20) 63 (NA) 290 (36.20 (0.56 (0.0 (13. 112.50 (0.14.27–1.41–2. [90] NCC Toenail Hartman et al. low Se) 0.547 (0. [88] NCC Serum Garland et al.54–34.134) mg/kg) China 4—5 108 (46.0) µg/day) 870 (64.5) µg/l)b 145 (57.10 (16.52 0.49 NA Comstock et al.48 >0.98 (0. Epidemiological studies of the selenium level in the body and risk of lung cancer (according to Zhuo et al.47–1.76 (0.65 (0.17 NA Knekt et al.006 Hu et al.3 317 (0.7 (18. cases (mean Se level) No.0) µg/day) 0.77–1.9) µg/l)d 216 (45 µg/l) 47 (0.1 (2.126) µg/g male. Wojciech Wàsowicz a Nested case-control.40) 4.44)e 0. [98] CC Diet Jolanta Gromadziƒska. 0.537 (0.33 (0.5) µg/l) USA 15—18 258 (0.6) µg/l) 356 (117.15) 0.88) 0. f male subjects randomized in the 5th year.43) P for dose-response trend 0.60) 0.001 0. [95] NCC Toenail Van den Brandt et al.8 (16.550 (0.07 µg/day) 0.0 (16. [89] NCC Serum Kabuto et al.897 (0. g data from 370 individuals.80 (33.5 47 (0.529 (0.09–0.11 Kromhout et al.575 (0.01 0.7) µg/l)b 153 (61.94) 1. [86] NCC Serum Ratnasinghe et al.1 (19. Edyta Reszka.537 (0.110 ppm) Study Se index Gender Population Years of follow up No.129) mg/kg) Japan 11—13 77 (113.30–0. d data from 177 individuals. controls (mean Se level) RR (95% CI) (high vs.109) µg/g female) 227 (33.2 (2.37–1. [94] NCC Serum 0.3) µg/l)c Finland 8—12 153 (57.811 (0.41) f 0.46 0.61 (0.60–2.36) 0. [91]) Author All All All M. .66 (0.2) µg/day) 290 (39.20 (0. e male subjects randomized in the earliest in the trial. [87] cohort Toenail All The Netherlands 3.0) µg/l USA 5—14 356 (119.12) µg/day 227 (33. [92] NCCa Serum Goodman et al.81) 0. 2459 (0. [97] cohort Diet Zhou et al.20–1.5 µg/l) Finland 16—19 77 (53.080) µg/g female)g 0.0 µg/l) 120 (119.41 (0.

3 years’ follow-up period. 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]. high Se concentration in the toenails was associated with an appreciably lower risk of prostate cancer [101]. however. A high prediagnostic Se level was found. was 0.15.25–0.39) in case-control studies.74 (95% CI: 0. Also. In the Netherlands Cohort Study. 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. In the Health Professionals Follow-Up Study.49 (95% CI: 0. especially men who had a PSA concentration equal to or higher than 4 ng/ml [106].16. In two large case-control studies of male populations in Great Britain and Canada. Several studies have shown that selenium can be of specific help in combatting prostate cancer [99].103]. In a US case-control study of the relation between the risk of colonic malignant or benign tumour and the serum Se level.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 2.61–1. 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. 95%CI: 0. colorectal cancer patients with low Se concentrations were found to have a significantly lower survival time . in which the Se concentration in the toenails served as a measure of long-term Se intake.84) in cohort studies and 0. although a protective effect of a high Se level (fifth quintile) was found (OR = 0. 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.). 95% CI: 0. 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 = [100].9) [105]. defined as the average of the 1st and 4th quintiles or the 1st and 3rd quartiles. In addition. in the Physicians’ Health Study. no protective effect of higher Se levels was found in either of the groups investigated [108]. indicating that the intake of selenium was able to reduce the risk of prostate cancer [107]. the odds ratio (OR) for the development of cancer that was associated with a high level of Se intake was 0. 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].85). the Se level in the toenails was not found to be associated to any marked degree with the level of risk of prostate cancer [102.3–0. A lower Se concentration in the serum was found to be associated with a stronger tendency to be afflicted with a colorectal tumour [109].17–0. which involved a 6.72 (95% CI: 0. to be associated (at a 13-year follow up) with a significantly reduced risk of prostate cancer. The findings of this review showed that the pooled RR of prostate cancer for a particular Se intake.61-0.

622 µg/g) 13 586 (0.42—2. [89] NCC Yoshizawa et al. [103] Case-control Nails Jolanta Gromadziƒska.9) 0.13) 1.0 µg/l) The Netherlands 6. [104] (2000) Toenail USA (JapaneseAmericans) USA UK — 300 (0.104 Table 2.48—0.3—0.77 µg/g) 233 (0.54—1. b data from 51 sets.10) 0. [100] NCC Helzlsouer et al.8) µg/l)b Study Se indicator Population Years of follow up No. low Se) 1.3 (14.6) µg/l) 456 (114.79 µg/g) USA 5 181 (0.090) µg/g) NCC Van den Brandt et al.39 (0.69 0.78 (0.5 (0.96 µg/g) USA 5—14 235 (114.73—2.707 0.12 Knekt et al.24 (0.05 0.38 (0.018) µg/g) — 249 (128.69 (0.108 (0.106 (0. Edyta Reszka.00 (0. [105] Case-control Serum Li et al.4) µg/l)b 46 (58.82 µg/g) 181 (0.8 (19.6 (19.84) 0.85) P for dose-response trend 0.008 0.547 (0.15.581 Allen et al.60) 0. cases (mean Se level) No.02 (0.3 (20.530 (0. [94] NCCa Goodman et al.16 0.02 Nomura et al.126) µg/g) 249 (131.6 µg/l) 0.611 µg/g) 0. .17—0. [101] Cohort 1211 (0.65—1.3 540 (0. controls (mean Se level) RR (95% CI) (high vs. [106] NCC Plasma 577 (0. Epidemiological studies of the selenium level in the body and risk of prostate cancer Author Serum Serum Toenail Toenail USA 1—6 117 (0.40) 1. Wojciech Wàsowicz a Nested case-control.4) µg/l) Finland 8—12 46 (59.18—0.018) µg/g) 300 (0.99) 0.

146) µg/g) 47 (0.3 (18.7 (0.575 (0.92) 0.273 0. low Se) P for dose-response trend Knekt et al. [94] NCCa Serum Knekt et al.092) µg/g) Rectal 76 (0.44—1.7) µg/l) 276 (130.547 (0.3 Colon 112 1246 (0. controls (mean Se level) RR (95% CI) (high vs.578 (0. c data from 59 sets.863 (0.22) 0.30) 1.45) 0.2) µg/l)b 0.131 0. [90] NCC Toenail Van den Brandt Cohort Toenail Vitamins and selenium: Anticancerogenic activity of selenium et al. [111] a Nested case-control.50 NA Study Se indicator Gender Population Years of follow up No.Table 2.3 (17.106)) Rectal 36 (0.12 0. b data from 32 sets.88—4.5 89 (0.7) µg/l)c 48 (65.43—1. [112] NCC Serum Nelson et al.42—2.82 (0.10 (0.5 (19.843 (0.186) µg/g) 2.91 (0.724 0.643 0.41—2.560 (0.58) 0. 105 .18—5.8) µg/l) Finland 8—12 48 (64.75) 0. cases (mean Se level) No. [94] NCC Serum Wallace et al.3) µg/l)c Finland 8—12 29 (63. [111] Van den Brandt Cohort Toenail et al.00) 0.9) µg/l)b 1. [108] Case Serum -control Female Male (0. Epidemiological studies of the selenium level in the body and risk of and colorectal cancer Author Male Female 1.535 (0.0 (18.01 (0.76 (0.326 Garland et al.108)) 1.733 (0.0 (18.59—4.16.091)) The Netherlands 3.123) µg/g) The Netherlands 3.3 (15.65) 29 (64.04 (0.77 (0.9) All All USA — 25 (138 µg/l) 138 (134 µg/l) USA 1—4 276 (131.41—1.5—5.3 Colon 121 1209 USA 3.593 (0.58 (0.411) µg/g) Female (0.

genetic susceptibility. The potential role of dietary Se in the early prevention of colorectal neoplasms would need to be confirmed. there are some reports of a significant inverse relationship between blood Se levels and the prevalence of adenomatous polyps [117. In a Canadian case-control study.118]. The majority of studies on relations between selenium level and occurrence of colorectal cancer have yielded null results for both males and females. possibly due in part to the rather low proportion of women in the study population [94]. A low level of selenium concentration in the body was shown to be associated with a higher risk of lung. Summary Results of epidemiologic and laboratory studies have indicated selenium to have a protective role in counteracting or preventing the development of cancer. although this result was only statistically significant in the case of the Polyp Prevention Study (OR: 0.42. a significant inverse association was obtained between toenail Se level and risk of colon cancer (OR = 0. A similar result was obtained in a 3. a variety of confounding factors such as geographical location. 95%CI: 0.57. Wojciech Wàsowicz 106 and a lower cumulative cancer-related survival rate than such patients with higher Se concentrations did [109]. especially in males. breast cancer was not found to be influenced by the selenium level [114–116]. to be associated with an elevated risk of lung and prostate cancer.93) [102]. and the Polyp Prevention Study.34–0.3-year follow-up study of a Dutch cohort in which toenail Se level was used as an indicator [111]. environmental exposure. gender. a pooled analysis of data from three clinical trials of colorectal adenoma was conducted: the Wheat Bran Fiber Trial. There is a need of clarifying the role of Se . No inverse association of Se level and cancer risk was found for lung cancer in females. After adjustment for age. The selenium level was measured in blood specimens of 1763 trial participants.95) [113]. To sum up. However. however. However. 95% CI: 0. gender.19–0. and the like need to be taken into account. a number of epidemiologic studies show a low Se level. 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. no significant association being found between risk of cancer and toenail Se status. as found in the Polyp Prevention Study [113]. 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. age. and the preventive role of Se regarding cancer of this type. would need further verification as well. In contrast to prostate cancer. smoking status and study site.112]. selenoprotein P (SeP) concentration or glutathione peroxidase activity in the plasma [110]. Edyta Reszka. To gain further insight into the anti-carcinogenic potential of selenium.Jolanta Gromadziƒska. However. 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. Colorectal cancer risk was also analyzed in a 41-month follow-up of the Nurses’ Health Study [90]. prostate or colorectal cancer. the Polyp Prevention Trial.

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

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

↑cleavage of caspases 3. p16. ↑Bax. COX-2. p27. ↓IκBα degradation and phosphorylation. ↑p21. ↑APAF-1. ↑cyt c release. apoptosis HER2-overexpressing breast tumor cells ↓PI3K & Akt activity. ↑IκBα . ↓IKKα actvity.8.Bioactive components in foods: Anticarcinogenic effects of flavonoids 117 Table 3. ↓NF-κB-DNA-binding. ↑HER2 degradation Apoptosis [22] A549 lung cancer cells in vitro ↓Akt. ↓ colony formation [19] PC-3 prostate cancer cells ↓p50. ↓p65. LNCaP. ↓TNFα activation of NF-κB ↓Bcl2.9 and cIAP-2 ↓fatty acid synthase activity Apoptosis [16] [17] Growth inhibition and apoptosis ↑p53. altered Bax:Bcl2 ratio. ↓VEGF. ↑p21. NOS-2. VEGF Apoptosis [20] DU145 prostate cancer cells ↓cyclin D1/2 & E. ↓VEGF and in nude mice OVCAR-3 and A2780/CP70 ovarian cancer cells ↓Akt.7. ↑p27 Apoptosis. ↑cleavage of caspase 3 [18] Apoptosis (p53-dependent) ↓Her2. ↓cyclin D1/D3 & cdk4. Chemopreventive suppressing effects of apigenin Tissue/cell type Jurkat T cells Mechanism ↓chymotrypsin-like activity of 20S and 26S proteosomes. ↓p70S6K1. ↓TNF a-induced NF-κB activation Apoptosis. ↓Bcl2. p18. SK-N-BE neuroblastoma cells Breast cancer cells ↑ROS. ↑DFF-45 cleavage. ↓Akt. ↑cleavage of caspase 3. cyclin D1. ↓PI3K-HER2 docking. [26] .↓HIF-1α. PC-3. ↓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. ↑Bax. HT-29 colon cancer cells ↓CK2. LAN-5. ↓CDK2/4/6.1.↓HIF-1α. G1 arrest. MMP9. ↑IκBα Apoptosis Effect Reference [15] PWR-1E. DU145 prostate tumour cells Breast and prostate cancer cells NUB-7. ↓NF-κB p50 & p65 [21] Growth inhibition. ↓HER2/neu phosphorylation. ↓p70S6K1. ↑p53.

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

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

Safe SH Flavonoids as aryl hydrocarbon receptor agonists/antagonists: effects of structure and cell-context. 12. Tunstall RG.63:131–42. Morrissey C. 14. Mut Res 2005. Boots AW. Ambudkar SV. Mol Cancer Ther 2005. Prostate 2005. Inhibition of P-glycoprotein function and expression by kaempferol and quercetin. Hladky SB. 10.4:1287–92. Hudson EA. 7. Steward WP et al. Khantamat O. VanAlstyne PC. Verhoeven. Kuhn DJ. Heiss E. Induction of caspase dependent. 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. Mut Res 2004. Pintha K. Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells. 4 and 5 (ABCC1. Dietary flavonoids as proteosome inhibitors in human leukemia cells. Lavoi JF. 18. de Pascual-Tereasa S. Dou QP. 8. Brit J Cancer 2004. 11. Klimo K. et al. Greaves P. 15. 4 and 5). Hollman PCH. O’Leary KA. Swinnen JV.17:86–95. Landis-PiWowar KR. Shukla S. Greaves P. Way TD. Env Health Persp 2003. Knauft J. Spengler B. Brusselmans K.91:1364–71.Margaret M. Qin CH.69:1421–32. Degradation of Her2/neu by apigenin induces apoptosis through cytochrome c release and caspase 3 activation in HER2/Neu overexpressing breast cancer cells. Daniel KG. Verschoyle RD. Gerhauser C. The rice bran constituent tricin potently inhibits cyclooxygenase enzymes and interferes with intestinal carcinogenesis in Apcmin mice. p53-mediated apoptosis by apigenin in human neuroblastoma. Bao YP. Manson MM. Steward WP. Birt DR.551:245–54. Torkin R. Gamal-Eldeen A. Kleinjans JCS. 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. 19.48:106–14. J Chemotherapy 2005. Kao MC. Cancer preventive activity of xanthohumol. Febs J 2005. Chen D. Barrand MA. Limtrakul P.280:5636–45. Williamson G. Chen S. O’Neill A. Gupta S. Platton S. et al. a natural product derived from hop. Fitzpatrick JM. 9. 17. Cai H. .4:1–11. Zhang S.1:959–69. Effect of flavonoids and vitamin E on cyclooxygenase-2 (COX-2) transcription. Al-Fayez M. Yeger H. Wilms LC. Stewart JW. Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity. Wang WQ. Febs Lett 2005. J Biol Chem 2005. 16. O’Brien NM. Kaplan DR. Nutr Cancer 2004. Chen MS. Lin JK. Manson 120 6. Calcagno SB. Modulatory effects of plant phenols on human multi-drug resistance proteins 1. Neumann I. Cai H. Vrolix R. Wu CP. 13. Christoffel V. Irons KA. Biochem Pharmacol 2005. 20.272:4725–40. Watson RWG.10:3169–78. Protection by quercitin and quercitin-rich fruit juice against induction of oxidative DNA damage and formation of BPDE-DNA adducts in human lymphocytes.579:145–52.582:155–62. Mol Cancer Ther 2002. Clin Cancer Res 2004. Mol Cancer Ther 2005.111:1877–82. Individual and interactive effects of apigenin analogs on G2/M cell cycle arrest in human colon carcinoma cell lines. Alt A.

Young CYF. Flavonoid quercetin. Fenton JI.279:4479–89. Mol Carcinogenesis 2004. Chang JY. Calderwood SK. Apigenin induces apoptosis through proteosomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway. Yang CH. Carcinogenesis 2005. Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K and HDM2/p53. Hu XW Shi XL. Zazrivcova K. 34. 35.52:273–9. Zheng JZ. Lee PPH. Eivemark S. Gupta S. 5. Zhou Q. 25. Anticancer Res 2005. Quercetin. Cao ZX. Knudson A. silymarin.328:227–34.6-dichloro-ribifuranosylbenzimidazoleand apigenin-induced sensitization of colon cancer cells to TNF-alpha-mediated apoptosis. Liu HF. Fang J. FASEB J 2005. The chemopreventive bioflavonoid apigenin modulates signal transduction pathways in keratinocyte and colon carcinoma cell lines. Moussavi M. 30. Quercetin decreases the expression of ErbB2 and ERbB3 proteins in HT-29 human colon cancer cells. Salh B. Muga SJ. curcumin. Lin YS. Hahm ER. Eng C. Lui LZ. induced apoptosis in human myeloid leukemia cells and their resistant variants. Wargovich MJ. Human Mol Genetics 2005. J Nutr 2003. Farah M. 23. 27. Biochem Biophys Res Comm 2005.26:1450–6. Lin JK. Gong AY. Waite KA. Kao MC. Jiang BH. Kim WK. Parhar K. Targeting of focal adhesion kinase by flavonoids and small interfering RNAs reduces tumor cell migration ability. Sulikova M. a potent inhibitor against beta-catenin/Tcf signaling in SW480 colon cancer cells.20:835–49. Shukla S. Van Dross R. Flavonoids promote cell migration in nontumorigenic colon epithelial cells differing in APC genotype: implications of matrix metalloproteinase activity. Kang NE. Huang YT. Sinden MR. 29. Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells. Carcinogenesis 2005. Cho HJ et al. J Nutr Biochem 2005. 33. Am J Physiol 2003. Int J Hyperthermia 2004. Fang J.16:155–62. Volate SR. Park S. The 70 kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer. Phytoestrogen exposure elevates PTEN levels. Mol Pharmacol 2005. Lee LT. J Biol Chem 2004. 26.14:1457–63. 36. Bang MH. Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells. Kuo PC. Pelling JC.26:793–801. Neoplasma 2005. Modulation of aberrant crypt foci and apoptosis by dietary herbal supplements (quercetin. 28. Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells. Duraj J. Jiang BH. Kim HK. 31. Stevenson MA. Jones EL.Bioactive components in foods: Anticarcinogenic effects of flavonoids 121 21. Way TD. Lee MT. Jung KC.25:2017–25.48:182–8.279:55875–85. Hord NG. Reed E. Xue Y. Apigenin inhibits expression of vascular endo. Chao JI. .133:3800S–4S. 39:114–24. Nutr Cancer 2004. Park CH. 32.285:G919–28. Davenport DM. but not apigenin or luteolin. J Biol Chem 2004.68:635–43. 22. thelial growth factor and angiogenesis in human lung cancer cells. 24. Sedlak J. Kim ES. Zhao MJ. Bodo J.19:342–53. ginseng and rutin). Yuan HQ.

Christman JK. 39. Ai N.11:7033–41. Clin Cancer Res 2005. Zhang SZ. Manson 122 37. . Morris ME.Margaret M.63:7563–70. Yang XN. Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Fang MZ. Reversal of hypermethylation and reactivation of p16 ink4a.135:609–14. et al. Fang MZ.21:1263–73. Cancer Res 2003. Sun Y. Romero C. Chen D. J Nutr 2005. Mertens-Talcott SU. RARβ and MGMT genes by genistein and other isoflavones from soy. Combined effects of multiple flavonoids on breast cancer resistance protein (ABCG2)-mediated transport. Lu H. 38. Percival SS. 40. Sun Y. Yang CS. Talcott ST. Hou Z. Bomser JA. Pharmaceutical Res 2004. Wang Y. Ellagic acid potentiates the effect of quercetin on p21 (waf1/cip1) p53 and MAP-kinases without affecting intracellular generation of reactive oxygen species in vitro.

for example). The use of biomarkers for such compounds should overcome some of the methodological problems in nutritional epidemiology just referred to. flavanols. Germany Introduction The primary aim of analytical cancer epidemiology is to detect associations between exposure variables and disease endpoints. however. measurement error being independent of that contained in the corresponding questionnaire data. anthocyanins and proanthocyanins. lignans. needs to be demonstrated in advance of their use in epidemiological and etiological studies [1]. and components for which bioavailability is low. Even more problematic. German Cancer Research Centre. Estimates of dietary intake estimations are hampered by incomplete or missing data in food composition tables. The basic flavonoid structure and various examples of them are given in Figure 3.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 123 3.1. the sugar moiety of the compound in question and its further metabolism by the gut microflora. for example. Isoflavones and gallic acid are polyphenols that are absorbed to the highest extent. from imprecision. Current evidence from bioavailability studies suggests that the bioavailability varies from about 0. Polyphenols Polyphenols are provided by plant-derived foods. 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. followed .2. Nutritional epidemiology deals with dietary factors that are difficult to assess. Biomarkers of dietary polyphenol intake for studying diet-cancer relationships Jakob Linseisen and Sabine Rohrmann Division of Clinical Epidemiology. including beverages. these including the flavonols. Dietary measurements tend to suffer. however. phenolic acids consist of two main subgroups. whereas others are limited to only a few kinds of food (such as apigenin found in parsley and celery). flavanones. The analytical data obtained are objective and more precise. is the fact that the polyphenols markedly differ from one another in their bioavailability and intestinal metabolism. reaching plasma concentrations of 0–4 µmol/l at an intake of 50 mg aglycone equivalents [2]. particularly concerning dietary components provided by only certain kinds of foods.3 to 43% (based on urinary excretion) of the dose administered. flavones. Heidelberg. Flavonoids are classified in several different sub-groups. such as various secondary plant products. Some of the polyphenols are found in a rather wide variety of foods (kaempferol contained in many vegetables. Bioavailability is determined by different factors. Long-term dietary habits are usually explored in epidemiological studies by use of food frequency questionnaires (FFQ). The validity and reproducibility of nutritional biomarker measurements. the hydroxybenzoic and the hydroxycinnamic acids. isoflavones.

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

such as plasma. Accordingly. serum. Some techniques such as liquid chromatography-mass spectrometry (LC-MS). However. Hydrolysis (acidic or enzymatic) is frequently used to simplify the analytical procedure. Thus.3. The availability of antibodies for isoflavones and lignans has enabled the antibodybased assays with a high degree of sensitivity to be developed [6]. with the option of time-resolved measurement. or urine samples. . frequent use is made of enzymic hydrolysis by means of sulfatase and glucuronidase unless a study aims at determining the exact metabolites. Thus far.). the flavonoids are usually glycosylated and the phenolic acids are ester-bound. solid phase extraction (SPE)-columns provide the most convenient solution for removing from a sample any matrix compounds that would disturb the analysis. Phenolic acids undergo further metabolism and degradation. flavonoids mainly undergo modification by means of phase II enzymes leading to methylated. Hydrolysis In foods. For the flavonoids. do require only minor sample-preparation steps. the clean-up procedures for this type of samples may be more complicated than required for many food systems. HPLC has become the method of choice. sulfated and glucuronidated compounds. LC-MS) to characterise clusters of polyphenolic compounds that can potentially be used as biomarkers. the fluorescence emitted is recorded by means of a plate reader. mass spectrometric techniques need to be used. although a part of them is excreted unmodified. For the structural characterization of compounds. a single mass-selective detector often fails to fulfil the requirements for sensitivity.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 [4.5]. Clean-up procedures For human specimens. new developments include the creation of HPLC-ESI-MS-MS systems and similarly coupled devices. Identification of compounds by means of mass fragmentation is used as a gold standard. For the purpose of quantification. Separation and detection systems The two major separation techniques for the quantification of polyphenolics are HPLC and GC. and the phenolic acids are genarally quantified by means of GC after derivatisation. the respective aglycones and acids being subsequently detected and quantified. Although in principle these methods can be applied to human specimens. the scientific literature contains no report on use of the metabonomics techniques (NMR. In biological specimens. although the use of LC-MS is becoming increasingly common. combined with different detection systems (Table 3.

(according to [7]).0 27. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al.1) 5.3 (0.3 (0.21) Range 0.56 (1.04 1.76–3.7) 7.5 42.0) 6.5 (0.0) 3–24. Phenolic acids Flavonoids.26–4.8–29.6) 4.Jakob Linseisen.50–2.5 15.46–4.4–8.1 (0.27) 2.3 3. Most of the studies concerned only one or some few compounds within a given subclass of polyphenols. Phenolic acids Phenolic acids Flavonoids.0) 19.9 2.8) 8. Table 3.50 Urinary excretion2 (% of intake) Mean(SEM) Range Elimination half-life (h) Mean (SEM) 5. Sabine Rohrmann 126 Table 3.21–0.8–7.7–9. Kinetic data from the experiments in question are summarized in Table 3. GC — gas chromatography.0–9.6 4.3 0.8 (0.4–5.0–55. Relatively low plasma concentrations were obtained in each case.26 42. Phenolic acids Flavonoids.0) 21. Phenolic acids Isoflavones.4 . Bioavailability studies and other short-term interventional studies A report was published vey recently summarizing all scientific studies (n = 97) that has been conducted thus far on the bioavailability of polyphenols [7].4.88 (0.87 8.6) 5.0–6.4.1 6.6 (4.8) 8.9 (1.6) 4.5 (0.92 (0.25) 1.0 7.2 1.36–3.52) 1.4–62. [7]) Tmax (h) Mean (SEM) Range Daidzin Daidzein Genistin Genistein Glycitin Hesperidin 6.3) 8.1 (0.2 Cmax (µmol/l) Mean(SEM) 1.0 3.6 6. Differences between the various compounds and of classes of polyphenol are striking nevertheless. even after the administration of polyphenol preparations or polyphenol-rich food corresponding to 50 mg aglycone equivalents.0–5.14 0.5 (0.3 (3. Phenolic acids Flavonoids.57 (0.3 5. LC — liquid chromatography. 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.84 (0.38) 0.0 5. lignans GC LC Immunoassays Mass spectrometry Mass spectrometry Time-resolved fluorescence HPLC — high-performance liquid chromatography.8 4.7 7.46 (0.4–9.9 (12.00 0.3.7–10.00) 1.8) Range 3.6 (1.

Usually represent 24-h urine samples.3–2.70 37.5 17.1 (3.2–15.03) 4.51–3.57) 0.6–3.6–5.3) 18. The magnitude of the variation in the plasma polyphenol concentrations found between free-living subjects following their usual diet habits was .09) 1.7–2.6 (0.45–1.7 0.0 2.50 0.1 (0.10 (0.008–0.7–4.4) 2.10 0.6 (17.0 19.1–1. Tmax (h) Mean (SEM) Range Naringin Quercetingluc. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al.7 27.0 1.1) 1.7) 11.0 4.03 (0.8–28. EGCG — epigallocatechin gallate.52 0.5 (0.7 5. flavanones or isoflavones) as biomarkers of polyphenol intake [9–16].96 (0.0 1.5 (0.4.3 0.4 (0.7) 1.02) 0.5–2.5 0.03) 2.17) 2.1–55.5 0. although one study in particular failed to support this conclusion [12].4) 2.1 (0.01) 0.5 (5.1 2.40 (0.09–1.09–0.7–2.4) Range 1.50 (0. Rutin (Epi)catechin EGC EGCG Gallic acid Chlorogenic acid Caffeic acid Ferulic acid Anthocyanins Proanthocyanidins 1 2 Cmax (µmol/l) Mean(SEM) 0.2) 1.3 0.004–5.6) 3.20 0.2) 10.0 1.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 127 Table 3.4–39.00 (0.57–4.6) 2.80 0.0 0.1–4.1 0.8 (0.8 (3.3 (0. EGC — epigallocatechin.4 0. The authors examined evidence relating to the effects of these polyphenols had on biomarkers used to test various pathophysiologically relevant hypotheses in vivo.3–9.46 (0. 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].26) 0.2 0.2) 3. [7]) — cont.3 (0.2) 0.1–61.9 (2.2 1.1–30.06 (0.31–6. 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.3) 1.70 Urinary excretion2 (% of intake) Mean(SEM) 8.1 1.40) 0.5–2.1) 2.30–2.9 (8.6 0.5) Range 1. Tmax — time to reach Cmax.03 All the data were converted so as to correspond to a supply of 50 mg aglycone equivalent.9–28.1 1.5–2.38 0.5 (0.2) 1.02 (0.0) 36.3) 0.20 (0.0 4. 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.5 (1.06) 0.33) 1.4 (0.3–1. AUC — area under the plasma concentration-time curve.5 0.6 0.13–1.9 4.35 10.1) 1.12 (0.0 0.001–0.1) 1.7 (1.26 Range 0.8 2.3) 6.45) 0.4 (0.5–5.07–1.03 0.1) 11.03–0.7 (0.5 (0.1 Elimination half-life (h) Mean (SEM) 2.0 (0.0–0.3 (0.

a precondition most likely to be fulfilled by compounds such as kaempferol that are widely distributed in plant foods.8 (15.9 372. 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. Due to the short half-life time of most polyphenols.1 (94.1 6849. (nmol/l) Max.8) 108.8 1121.75.0 6103.0a 0.4 104468.0a 0.9) 140.4 2904.3) 652.6 413.1 (41.5.3 (67.6 2542.0a 0.5) 908.4 59.1 1357.7 1348. 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. and if the bioavailability of the compound is not particularly low.Jakob Linseisen.0a 0.4 159.2 (17. 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 68.3 388.0a 0.).8 82.0a 0.9 563.2) 1031.0a 0. close to of steady-state plasma concentrations of them can only be achieved if the substance in question is consumed regularly.0 0.3 644.7 (15. and that the correlations are much lower when intake calculations are based on data obtained either three or seven days prior to blood sampling. participating in a cross-sectional study (according to Bolarinwa and Linseisen [16].6) 477.9 7103. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet. the intra-individual variation also being high [14].0a 0.2) 260.5 114.6) 2160.8 26.8 0.1 53.9) 0. Table 3.0a 78.9 4528.5 Mean (SD) Min.0 (3077.0 742.7 561.9 2641.6 460.0a 58.7 (48.3 (42.0 478.7 (78.6 (275.8 .6 (254.5.6 339.6) 6990.1 (203.8 (77.8 158.4) 107.1) 520.9) 267.3 (153.0a 48. Sabine Rohrmann 128 found to be rather high (Table 3.3 1253. 91.7 2290.4) 10.2 0.1 (2.7) 277.0a 19.0a 0.5) 1304.

122. . In the following. the correlation coefficients for selected flavonols and flavanones being between 0.6). several studies on associations with the risk of cardiovascular diseases and with cancer at different sites. little use has been made of biomarker measurements. except as regards the intake of phytoestrogens (isoflavones and lignans). participating in a cross-sectional study (according to Bolarinwa and Linseisen [16] — cont.8 (80.3 14.4) 9. (nmol/l) Max.2 Mean (SD) Min. Only few studies concerning the use of biomarkers of polyphenol intake were found to be available (Table 3.1 (24.0a 0. some of which appear very promising. One study reported correlation coefficients of between 0. The urinary polyphenol excretion rates show a high degree of variability.7) 545. naringenin.7 1356.0a 533. 50(32).Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 129 Table 3.8) 157.2 639. just as was indicated already for the plasma concentrations.1 (40.0 108.1) 37.38 [13].5.5 204.9) 126.8 5.3) 0.28 and 0. are taken up. respectively.9) 31. In large-scale epidemiologic (etiological) studies of disease-related effects of dietary polyphenol levels. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet.8 Below the detection limit. Nielsen and coworkers [13] reported average (SD) concentrations of quercetin.7 151. Compound Naringenin Luteolin Genistein Hesperetin Kaempferol Apigenin Isorhamnetin a Median 79.8 52. although this has not been investigated tested extensively.3 (1.0a 0. Their work provided the basis for the estimation of dietary flavonol and flavone intake. the dietary intake estimates being based on FFQ data [11].0 (7. To give an example of the polyphenol concentrations found in 24-h urine samples of subjects on a habitual diet. Hertog and colleagues were the first to analyze commonly consumed foods in terms of their flavonol and flavone content by means of HPLC.1 2555. Plasma and urinary polyphenol concentrations are not expected to reflect long-term or habitual dietary intake.1 27.6 36. phloretin. 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.74 for plasma isoflavone concentrations. 701(659). 76(110).0a 0. kaempferol.2 (4.0a 0.24 and 0.7 388.5 (21.0a 0.0a 0. and total flavonoids as being 25(23). 1638(1316) µg/24 h.

isoflavones Urine. Urine. [23] Pietinen et al. Epidemiological studies of the risk of breast cancer risk in which biomarkers of dietary isoflavone and mammalian lignans intake were employed. Flavonoid Urine. isoflavones. [25] Hulten et al.Jakob Linseisen. [27] Cohort: nested CCS 97/187 114/219 Serum.6. . [24] den Tonkelaar et al. isoflavones Urine. enabling the attainment of the requirement of sufficient statistical power. cases/ controls 250/250 Specimen. Sabine Rohrmann 130 Table 3.7. total phenols Breast NS Sun et al.) may be the ready availability of appropriate immunoassays suitable for measurements on large numbers of samples. 2002 [17] Type CCS N. enterolactone Serum. enterolactone Result Significant inverse association NS Significant inverse association Significant inverse association Significant inverse association Significant inverse association NS 248/492 Plasma. [21] Zheng et al. [26] Type CCS CCS CCS CCS CCS CCS Cohort: nested CCS Cohort: nested CCS N. [18] Murkies et al. [17] Piller et al. enterolactone for highest and lowest categories Significant positive association Grace et al. tea polyphenols Gastric and esophageal Significant inverse association CCS — case-control study. enterolactone Urine. Table 3. Epidemiologic (observational) studies of the risk of cancer in which biomarkers of dietary polyphenol intake (other than that of isoflavones and lignans) were employed Author Dai et al. lignans Plasma. NS — not significant. NS — not significant. enterolactone Urine. lignans Higher risk when isoflavone estimates are higher CCS — case-control study. 2002 [19] Cohort 232/772 Urine. 1999 [18] CCS 60/60 Urine. cases/ controls 144/144 60/60 18/20 250/250 220/237 194/208 88/268 Specimen. isoflavones. citrus flavonoids Cancer site Breast Result NS Zheng et al. genistein.7. equol. [22] Dai et al. One of the major reasons for the frequent use of biomarkers of phytoestrogen intake in epidemiological studies (see Table 3. Author Ingram et al. Phytoestrogen Urine.

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

Flavonoids in human urine as biomarkers for intake of fruits and vegetables. Kaaks R. Aro A. J Chromatogr B Analyt Technol Biomed Life Sci 2005.81 Suppl 1:243S–55S. Am J Clin Nutr 2005. Zock PL. Bioavailability and bioefficacy of polyphenols in humans. Bolarinwa A.. Hollman PC. IARC Scientific Publications No. Lyon: IARC. 13. 10.38:771–85. et al. Kelly IE.79:727–47. Br J Nutr 2001. Steroids 2000. Br J Nutr 2004. Bioavailability and bioefficacy of polyphenols in humans. Pharmacokinetics and metabolism of dietary flavonoids in humans. 4. Plasma concentrations of the flavonoids hesperetin. Beecher GR. 16.823:143–51. Appleby PN. Uehara M.86:415–21. Buysman MN. Williamson G. Meyboom S. Cancer Epidemiol Biomarkers Prev 2002. 3. Linseisen J. 5. Erlund I. . I. 2. Rantala M. Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake. Prediction of dietary flavonol consumption from fasting plasma concentration or urinary excretion. 11. Riboli E. Kleemola P. Blake A. Manach C.48:577–99. Measurement of food flavonoids by high-performance liquid chromatography: A review. 1997. and diets high or low in fruit and vegetables. Lapcik O. Phenolic acids in foods: an overview of analytical methodology. Verkasalo PK.Jakob Linseisen. 142. Lean ME. 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. Eur J Clin Nutr 2002. Scalbert A. Morand C. Crozier A. Ritchie MR. Eur J Clin Nutr 2000. et al. Stumpf K. Key TJ. Deighton N. Kesaniemi YA. 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). Remesy C. Sabine Rohrmann 132 References 1. Al-Maharik N. 7. Sinha R. de Vries JH. Morand C. In: Toniolo P et al. 9. 12. Noroozi M. van Staveren WA. Williamson G. Review of 93 intervention studies. 14. Application of Biomarkers in Cancer Epidemiology.68:60–5. Biochemical markers of dietary intake. Freese R.41:203–9. Alfthan G. Plasma and urinary phyto-oestrogens as biomarkers of intake: validation by duplicate diet analysis. Silaste ML. Fasting plasma concentrations of selected flavonoids as markers of their ordinary dietary intake. Eur J Nutr 2002. J Agric Food Chem 2000. Mutanen M. Polyphenols: food sources and bioavailability. Hampl R. II. 8.54:143–9. Manach C. Linseisen J. Remesy C. Am J Clin Nutr 2004. Review of 97 bioavailability studies. editors. 6. Burns J. J Agric Food Chem 2003. Free Radic Res 2004. Donovan JL. naringenin and quercetin in human subjects following their habitual diets. Am J Clin Nutr 1998.91:447–57. Wang GJ. Wolfram G. Robbins RJ. Manach C.56:891–8. Nielsen SE. Allen NE. Time-resolved fluoroimmunoassay of plasma daidzein and genistein. Scalbert A. Merken HM. Morton MS. Manach C.51:2866–87.11:459–66. Radtke J. Jimenez L. Adlercreutz H.65:339–48. Davey G. 15.81 Suppl 1:230S–42S. Am J Clin Nutr 2005. Cummings JH.

Stumpf K. 20. Serum enterolactone and risk of breast cancer: a case-control study in eastern Finland. Kataja V. Eur J Nutr 2002. Veer PV.10:223–8. Burger HG. Menopause 2000. Manach C. Gonthiera MP. Briganti EM. 21. Morand C. High-throughput profiling of dietary polyphenols and their metabolites by HPLC-ESI-MS-MS in human urine. Cancer Epidemiol Biomarkers Prev 1999. Wahlqvist ML.22:241–3. Mulligan AA. Shu XO. . Ross RK. et al. Ito H.41:168–76. Dai Q. An incident casereferent study on plasma enterolactone and breast cancer risk. Shu XO. Dalais FS. et al. et al. Den Tonkelaar I. Hebert JR. Hallmans G. Sun CL. Sanders K. Botting NP.13:698–708. Urinary excretion of phytoestrogens and risk of breast cancer among Chinese women in Shanghai.11:815–21. Yang CS. Winkvist A. Phytoestrogens and breast cancer in postmenopausal women: a case control study. Arts CJ. Luben RN. Eur J Cancer Prev 2006. Hulten K.10:339–44. Zheng W.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 133 17. 18.7:289–96. 25. Lee MJ. Franke AA. Gao YT. 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. Cancer Epidemiol Biomarkers Prev 2001. Adlercreutz H.350:990–4. Cancer Epidemiol Biomarkers Prev 2004. China. et al. et al. Healy DL. Piller R. 27. Biofactors 2004. 22. Jin F. et al. Murkies A. Lancet 1997. 23.15:225–32. Kolybaba M. 24. 26. Uusitupa M. Taylor JI. Carcinogenesis 2002.8:35–40. Custer LJ. Johansson R. Lopez D. Urinary phytoestrogens and postmenopausal breast cancer risk. Linseisen J. Grace PB. Ingram D. Remesy C.23:1497–503. Dai Q. Chang-Claude J. Wen WQ. Mannisto S. Urinary excretion of isoflavonoids and the risk of breast cancer. Mennen L. 19. Adlercreutz H. Adlercreutz H. Plasma enterolactone and genistein and the risk of premenopausal breast cancer. Yuan JM. Keinan-Boker L. Urinary tea polyphenols in relation to gastric and esophageal cancers: a prospective study of men in Shanghai. Pietinen P. Low YL. Cancer Epidemiol Biomarkers Prev 2002. Custer LJ. Jin F. Cancer Epidemiol Biomarkers Prev 2001. Case-control study of phyto-oestrogens and breast cancer. Thijssen JH. Lenner P.

This indicates olive oil phenolics to maintain their antioxidant activities in vivo. 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. a biomarker of in vivo lipid peroxidation processes. Greece Olive oil is an important ingredient of the Mediterranean diet. considered as putative nutritional biomarkers in relation to cancer the incidence of cancer. Sotiroudis and Soterios A. oleuropein.8. A brief overview is presented of recent findings concerning the bioavailability of certain minor but important olive oil minor components (polyphenols. squalene and β-sitosterol). tyrosol. a number of simple and bound phenolics (tyrosol. was observed. Anticarcinogenic compounds of olive oil and related biomarkers Theodore G.2]. a statistically significant negative correlation has been found between homovanillyl alcohol (HValc. that have been thoroughly studied.. Thus.). the aglycone of ligstroside.2. Kyrtopoulos 134 3. The National Hellenic Research Foundation. two secoiridoids (dialdehydes related to oleuropein and ligstroside but lacking the carboxymethyl group at C4). When olive oil samples containing increasing amounts of a phenolic extract of olive oil were administered to human volunteers. a dose-dependent decrease in the urinary excretion of the F2-isoprostane 8-iso-PGF2α. A plethora of minor constituents in olive oil have been identified as being effective agents mitigating against the initiation. Athens. Kyrtopoulos Institute of Biological Research and Biotechnology.3. the excretion of both HValc and HVA also being significantly correlated with the dose of administered hydroxytyrosol. promotion and progression of multistage carcinogenesis. and a peak containing the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol [1–3] (Table 3. Figure 3. Soterios A. hydroxytyrosol. the triterpene hydrocarbon squalene and the phytosterol β-sitosterol [1–3]. Sotiroudis.Theodore G. lignans. are dose-dependently absorbed in humans after the ingestion of realistic doses of virgin olive oil. These include tocopherol and carotenoid antioxidants. which represent major antioxidants in olive oil. Phenolic compounds HPLC chromatography of the methanol extract of virgin olive oil reveals seven major polyphenol peaks corresponding to hydroxytyrosol. Oleuropein and its metabolites tyrosol and hydroxytyrosol. together with homovanillic acid — HVA) and isoprostane excretion. The occurrence of these constituents also calls for the development of specific nutritional biomarkers that reflect the nutritional status of these dietary constituents with respect to their intake or metabolism and that can provide information useful for nutritional epidemiology regarding the effects of disease processes that can occur [4]. a major metabolite of hydroxytyrosol. Epidemiological studies demonstrate rather conclusively that populations within Europe consuming this diet have a particularly low incidence of a number of common cancers [1. In addition. HValc in urine reflects the in vivo . secoiridoids and lignans).

It was observed in this respect.Bioactive components in foods: Anticarcinogenic compounds of olive oil and related biomarkers 135 concentration of hydroxytyrosol [5–11].71 [3] 103–205 [3] 27. a biomarker of the intake of tomato-rich food and hypothesized to be responsible for reducing the risk of various .8. Structures of certain phenolic compounds detected in olive oil. Concentrations of the major phenolic compounds found in virgin olive oil Compounds Tyrosol Hydroxytyrosol Oleuropein aglycone Total secoiridoids Lignans References [2] and [3].53 [2].75 [3] 14. Recent findings suggest that olive oil may also affect the bioavailability of other food bioactive components with a chemopreventive potential. Table 3. oleuropein aglycone (B). An HPLC method for the simultaneous determination of oleuropein and of its metabolites hydroxytyrosol and tyrosol in human plasma has been developed [13. 1. Hydroxytyrosol (A). 3.14]. After the ingestion of olive oil of low phenolic content plasma glutathione peroxidase activity was found to decrease postprandially.42 [2].65–4. 38–65 [3] Fig.2.83–4.72 [2] 41. but this was not observed after the intake of olive oils of moderate to high phenolic content [12]. mg/kg 27. that the concentration in human plasma of lycopene.45 [2]. 2. (+)-pinoresinol (C).

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

Bioactive components in foods: Anticarcinogenic compounds of olive oil and related biomarkers

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.

1. Sotiroudis TG, Kyrtopoulos SA, Xenakis A, Sotiroudis GT. Chemopreventive potential of minor components of olive oil against cancer. Ital J Food Sci 2003;15:169–85. 2. Owen RW, Haubner R, Wurtele G, Hull WE, Spiegelhalder B, Bartsch H. Olives and olive oil in cancer prevention. Eur J Cancer Prev 2004;13:319–26. 3. Criado MN, Morello JR, Motilva MJ, Romero MP. Effect of growing area on pigment and phenolic fractions of virgin olive oils of the Arbequina variety in Spain. J Am Oil Chem Soc 2004;81:633–40.

Theodore G. Sotiroudis, Soterios A. Kyrtopoulos

4. Maruvada P, Srivastava S. Biomarkers for cancer diagnosis: Implications for nutritional research. J Nutr 2004;134:1640S–5S. 5. Visioli F, Caruso D, Galli C, Viappiani S, Galli G, Sala A. Olive oil rich in in natural catecholic phenols decrease isoprostane excretion in humans. Biochem Biophys Res Commun 2000;278:797–9. 6. Visioli F, Galli C, Bornet F, Mattei A, Patelli R, Galli G, et al. Olive oil phenolics are dosedependently absorbed in humans. FEBS Lett 2000;468:159–60. 7. Miro-Casas E, Farre Albaladejo M, Covas MI, Rodriguez JO, Menoyo Colomer E, Lamuela Raventos RM, et al. Capillary gas chromatography-mass spectrometry quantitative determination of hydroxytyrosol and tyrosol in human urine after olive oil intake. Anal Biochem 2001;294:63–72. 8. Caruso D, Visioli F, Patelli R, Galli C, Galli G. Urinary excretion of olive oil phenols and their metabolites in humans. Metabolism 2001;50:1426–8. 9. Visioli F, Galli C, Galli G, Caruso D. Biological activities and metabolic fate of olive oil phenols. Eur J Lip Sci Technol 2002;104:677–84. 10. Miro-Casas E, Covas MI, Fito M, Fare-Albadalejo M, Marrugat J, de la Torre R. Tyrosol and hydroxytyrosol are absorbed from moderate and sustained doses of virgin olive oil in humans. Eur J Clin Nutr 2003;57:186–90. 11. Casas EM, Albadalego MF, Planells MIC, Colomer MF, Raventos RML, Fornell RD. Tyrosol bioavailability in humans after ingestion of virgin olive oil. Clin Chem 2001;47:341–3. 12. Weinbrenner T, Fito M, Farre AM, Saez GT, Rijken P, Tormos C, et al. Bioavailability of phenolic compounds from olive oil and oxidative/antioxidant status at postprandial state in healthy humans. Drugs Exp Clin Res 2004;30:207–12. 13. Tsarbopoulos A, Gikas E, Papadopoulos N, Aligiannis N, Kafatos A. Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography. J Chromatogr B 2003;785:157–64. 14. Grizis C, Atta-Politou J, Koupparis MA. Simultaneous determination of oleuropein and tyrosol in plasma using high performance liquid chromatography with UV detection. J Liquid Chromatogr Rel Technol 2003;26:599–616. 15. Fielding JM, Rowley KG, Kooper P, O’Dea K. Increases in plasma lycopene concentration after consumption of tomatoes cooked with olive oil. Asia Pac J Clin Nutr 2005;14:131–6. 16. Lee A, Thurnham DI, Chopra M. Consumption of tomato products with olive oil, but not sunflower oil increases the antioxidant activity of plasma. Free Rad Biol Med 2000;29:1051–5. 17. Peeters PHM, Keinan-Boker L, van der Schouw YT, Grobbee DE. Phytoestrogens and breast cancer risk — Review of the epidemiological evidence. Breast Cancer Res Treatment 2003;77:171–83. 18. Webb AL, McCullough ML. Dietary lignans: Potential role in cancer prevention. Nutr Cancer 2005;51:117–31. 19. Jansen GHE, Arts ICW, Nielen MWF, Muller M, Hollman PCH, Keijer J. Uptake and metabolism of enterolactone and enterodiol by human colon epithelial cells. Arch Biochem Biophys 2005;435:74–82. 20. Kuijsten A, Arts JCW, Vree TB, Hollman PCH. Pharmacokinetics of enterolignans in healthy men and women consuming a single dose of secoisolariciresinol diglucoside. J Nutr 2005;135:795–801.

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21. McCann SE, Muti P, Vito D, Edge SB, Trevisan M, Freudenheim JL. Dietary lignan intakes and risk of pre- and postmenopausal breast cancer. Int J Cancer 2004;111:440–3. 22. Nenadis N, Tsimidou M. Determination of squalene in olive oil using fractional crystallization for sample preparation. J Am Oil Chem Soc 2002;79:257–9. 23. Relas H, Gylling H, Rajaratnam RA, Miettinen TA. Postprandial retinyl palmitate and squalene metabolism is age dependent. J Gerontol Ser A 2000;55:B515–21. 24. Ketomaki A, Gylling H, Siimes MA, Vuorio A, Miettinen TA. Squalene and noncholesterol sterols in serum and lipoproteins of children with and without familial hypercholesterolemia. Ped Res 2003;53:648–53. 25. Saudek CD, Frier BM, Liu GCK. Plasma squalene:lipoprotein distribution and kinetic analysis. J Lipid Res 1978;19:827–35. 26. Newmark HL. Squalene, olive oil, and cancer risk:A review and hypothesis. Cancer Epidemiol Biomarkers Prev 1997;6:1101–3. 27. Smith TJ. Squalene:potential chemopreventive agent. Expert Opinion Investig Drugs 2000;9:1841–8. 28. Li JH, Awad AB, Fink CS, Wu YWB, Trevisan M, Muti P. Measurement variability of plasma beta-sitosterol and campesterol, two new biomarkers for cancer prevention. Eur J Cancer Prev 2001;10:245–9. 29. Bouic PJD. The role of phytosterols and phytosterolins in immune modulation: a review of the past 10 years. Curr Opinion Clin Nutr Metab Care 2001;4:471–5.

the olefinic glucosinolates sinigrin. The glucosinolate content is primarily under genetic control. though it can be influenced by environmental factors [16. namely. Cruciferous vegetables uniquely contain glucosinolates at approximately 20 µmol/g dry mass of vegetable [8. Brussels sprouts. such as broccoli. The types of neoplastic disease in man that these vegetables appear to protect against include colorectal cancer [4]. Subsequently. valine. Michael O. catalysed by members of the cytochrome P450 (CYP) 79 family [19]. kale. tyrosine and tryptophan [2]. leucine. Each cruciferous vegetable contains a mixture of glucosinolates that varies according to the strain of the plant [8.12–15]. The task of establishing a link between the ingestion of particular glucosinolates and their possible health benefits is not straightforward. cauliflower.17]. phenylalanine.John D. Eggleston2 1 2 Biomedical Research Centre.11]. Claverton Down. is associated with a reduced incidence of cancer [1. Glucosinolates are substituted β-thioglucoside N-hydroxysulfates. Bath BA2 7AY. As shown in Figure 3. glucobrassicanapin and progoitrin. Ian M. and the aromatic glucosinolate gluconasturtiin (Table 3. though certain broccoli . the synthesis of glucosinolates proceeds through the conversion of elongated amino acids to their oxime derivatives. This endeavour is simplified to some extent by the fact that relatively few glucosinolates are present in the human diet. principally valine. cabbage. Ninewells Hospital and Medical School. gluconapin. lung cancer [5]. this variable elongation of amino acid side chains entails repetitive additions of methyl groups through a series of transamination. University of Bath. 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 [10.4. Furthermore. used in their biosynthesis. University of Dundee.3. the oxime is metabolised to a thiohydroximate. The most common of these are the methylsulfinylalkyl glucosinolates glucoiberin and glucoraphanin.. alanine. Over 115 naturally occurring glucosinolates have been identified. Kelleher. condensation. Glucoraphanin has been reported to be abundant in broccoli [9]. Anticarcinogenic effects of glucosinolate breakdown products John D. greater health benefit may be obtained from raw as opposed to cooked vegetables [3]. Kelleher1 and Ian M. Michael O. methionine. Hayes. isoleucine. isomerisation and decarboxylation reactions [18]. Dundee DD1 9SY Department of Pharmacy and Pharmacology. formed by the plant from any one of eight amino acids. Eggleston 140 3. Hayes1. Feeding experiments in animals have also suggested broccoli can protect against liver cancer [7]. which is in turn conjugated with glucuronic acid to form a desulfoglucosinolate before finally being sulfated to yield the glucosinolate [2].) [9. United Kingdom Glucosinolates and their association with cancer chemoprevention Epidemiological studies have revealed that regular consumption of cruciferous vegetables.20]. and possibly prostate cancer [6].2].9. swede and turnip.9]. Much of the diversity amongst glucosinolates arises from the addition of different sized alkyl groups to the side chain of those amino acids. phenylalanine and methionine.

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. Table 3. cauliflower and kale [9]. . The aromatic glucosinolate gluconasturtiin is present in watercress. and whilst not abundant it can elicit distinct pharmacological effects. Brussels sprouts and cauliflower [9. Sinigrin has been reported to be the predominant glucosinolate in Brussels sprouts. The R group is derived from the original amino acid and is highly variable.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 141 strains also contain substantial amounts of glucoiberin [21].3. 3. Substantial amounts of progoitrin are present in many cruciferous vegetables [9]. cabbage.9. The indolyl glucosinolate glucobrassicin is present in Savoy cabbage.22]. gluconapin is also found in high levels in Brussels sprouts [9]. Synthesis of glucosinolates.

4A. or during chewing whilst eating. during food preparation. Upon wounding of the vegetable. Hayes. and 3. myrosinase activity may be present in human colonic microflora. but rather it appears to be due to certain of their breakdown products. Epithiospecifier protein (ESP) in the presence of Fe2+ ions interacts with myrosinase to promote the transfer of the sulfur to the alkenyl group from the S-Glucose of the terminally unsaturated glucosinolate. the pH and the presence of ferrous ions (Figure 3. thiocyanates. EC 3. .4B. nitriles. 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. for example during harvesting. Hydrolysis of these phytochemicals is catalysed by myrosinase (β-thioglucoside glucohydrolase. Michael O. Ian M.2. Hydrolysis of glucosinolates. suggesting that glucosinolates can be hydrolysed in the gastrointestinal tract during digestion of food [24.4A. during freeze-thawing. myrosinase is released from the “myrosin” cells and is able to hydrolyse glucosinolates within the damaged plant. 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].3.). 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. Fig. The outcome of the reaction with myrosinase depends on the nature of the aglycone.147). 3. resulting in the formation of an epithioalkane. In addition.25]. Kelleher.John D. as well as the reaction temperature. Eggleston 142 Production of isothiocyanates. At high or neutral pH the formation of isothiocyanates is favoured while at low pH the formation of nitriles is favoured.

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

3. Likewise. Production of indoles from glucosinolates The indolyl glucosinolates glucobrassicin and neoglucobrassicin are synthesised by the plant from tryptophan. If the aglycone generated by myrosinase is from a glucosinolate with a side chain lacking a double bond.5. Two cDNAs for ESP have recently been cloned from Arabidopsis and broccoli and the purified proteins characterized following their heterologous expression in E.5-epithiopentane [31].3-epithiopropane [29]. this reaction probably proceeds Fig.36]. Kelleher. Production of indoles from glucosinolates.34]. At neutral pH.5. coli [35. it can be hydrolysed by myrosinase to crambene (1-cyano-2-hydroxy-3-butene) [33.31]. Examples of these include progoitrin. A few glucosinolates produce thiocyanates though the mechanism involved is unclear [23]. Ian M. Upon hydrolysis by myrosinase. but rather gives rise to indole-3-carbinol and a thiocyanate ion (Figure 3. those aglycones from glucosinolates that contain β-hydroxylated sidechains form oxazolidine-2-thiones. the sulfur atom may be lost and a nitrile formed [37–39]. . 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. glucoconringiin and gluconapoleiferin [2].John D. and is diminished by heating [40]. glucobrassicanapin can be hydrolysed to 1-cyano-4. Hayes. The best studied of these is glucobrassicin. Eggleston 144 myrosinase and ESP convert sinigrin to 1-cyano-2. is also converted in the presence of myrosinase. a (2R)-hydroxy-3-butenyl glucosinolate. Progoitrin.). 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). as a consequence of spontaneous cyclization. Gluconapin can similarly be converted by the combined actions of myrosinase and ESP to 1-cyano-3.4-epithiobutane [30. Michael O. This reaction may involve ESP.

). cell cycle arrest. Induction of gene expression mediated by Nrf2 Isothiocyanates increase the expression of antioxidant and detoxication proteins. whereas activation of cell cycle arrest and apoptosis occurs at higher levels of phytochemical [43.3∂-diindolylmethane. At acidic pH. Structures of indoles produced from glucosinolates — 3. It can also combine with ascorbic acid to form ascorbigen [42] (Figure 3. and oxazolidine-2-thiones. These include induction of antioxidant and detoxifying genes. inhibition of CYP enzyme activity. There is a dose-dependency in these responses: generally. Thus. indolo [3.44]. and stimulation of apoptosis. In the acidic environment of the stomach.46]. when compared with the data about thiocyanates. both in vivo and in vitro [45. such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1). indole-3-carbinol condenses to form various compounds including indolo[3. 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. A challenge in evaluating the literature arises from the emphasis placed on certain glucosinolate breakdown products and the dearth of data relating to others. Agents such as ITC that increase GST and NQO1 . cyanoepithioalkanes.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 145 through a Lossen rearrangement generating an unstable ITC intermediate [22].3’-Diindolylmethane (DIM). 3. hydrogen sulfide and elemental sulfur [22]. nitriles.6. both of which have potent pharmacological effects [41]. Chemopreventive mechanisms stimulated by glucosinolate hydrolysis products In view of the diverse spectrum of chemicals generated from glucosinolates by the actions of myrosinase and ESP. Fig. hydrolysis of glucobrassicin yields indole-3-acetonitrile. A major problem exists in interpreting experiments utilizing vegetable extracts because of uncertainty about attributing biological effects to specific phytochemicals. induction of cytoprotective genes and inhibition of CYP activity occurs at relatively low concentrations of phytochemical. there is a relative abundance of information about isothiocyanates and indoles.2-b]carbazole and 3.2-b] carbazole (ICZ) and ascorbigen (ASG).6.

7. Sulforaphane. benzyl-ITC and phenethyl-ITC.55–58]. Fig. 2) Phenethyl-ITC. Structures of isothiocyanates which induce NQO1: 1) Allyl-ITC. GST and NQO1 in the gastrointestinal tract in an Nrf2-dependent fashion [21]. 7-methylsulfinylheptyl-ITC. Ian M. Michael O. 3-methylsulfinylpropyl-ITC. Through this characteristic.54. glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits. 4) 3-methylsulfinylpropyl-ITC. Many of these genes are induced by sulforaphane in vivo in an Nrf2-dependent fashion in the stomach.John D. as far as is known. ferritin. The battery of genes regulated by Nrf2 includes those encoding aldo-keto reductase (AKR). leading to its inhibition and inability to serve as a substrate adaptor . thioredoxin. metallothionein. as does the rat GSTP1 gene (called GPEI) [52]. small intestine and liver of rodents [53. It is thought that ITCs possess the ability to induce ARE-driven gene expression because they are thiol-active [59]. Examination of nrf2–/– and wild-type mice. thioredoxin reductase. 3.49] (Figure 3. NQO1. induce NQO1 in the mouse Hepa-1c1c7 hepatoma cell line [48. heme oxygenase 1. Hayes. are sometimes called mono-functional inducers [47]. 6) 8-methylsulfinyloctyl-ITC. GST. 5) 7-methylsulfinylheptyl-ITC. suggests AREs are present in the promoters of at least 100 genes [53–55]. 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].). all genes that are induced by ITCs contain an ARE in their promoters and are regulated by Nrf2. The mouse nqo1 gene contains a functional antioxidant response element (ARE) in its 5∂-upstream region [51]. Indeed. 3) Benzyl-ITC. Eggleston 146 enzymes without increasing arylhydrocarbon hydroxylase activity. Many of these compounds induce GST P1-1 in the rat liver RL-34 cells [50]. multidrug resistance-associated protein. Kelleher. carboxyl esterase. ITCs modify cysteine residues in the Cullin 3:Rbx1 E3 ubiquitin ligase substrate adaptor Keap1. microsomal epoxide hydrolase. 8-methylsulfinyloctyl-ITC.7. catalysed by the phase I drug-metabolising enzyme cytochrome P450 (CYP) 1A1. UDP-glucuronosyl transferase. Importantly. allyl-ITC. feeding broccoli seed to mice increased the levels of GCLC.

69]. allyl-ITC does not increase Nrf2 stability [64] and there may therefore be other factors involved in enzyme induction by these phytochemicals. and thereby generate reactive oxygen species.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 147 required for the ubiquitination of Nrf2 under homeostatic conditions [60–62]. Amongst genes for drugmetabolising enzymes. this seems unlikely [72]. rat UGT1A1. Although indole-3-acetonitrile has not been studied in the Nrf2 knockout mouse. western and northern blotting that CYP1A1 is inducible by indolo[3. However. mouse. but not CYP1A1. Surprisingly. indicating that AhR ligands could lead to an increased production of Nrf2 mRNA.3∂-diindolylmethane. and there is abundant evidence from enzyme assays. Treatment of cells with benzyl-ITC causes a rapid increase in the level of reactive oxygen species.64].3∂-diindolylmethane [2]. The promoters of other genes including rat and human NQO1. This cytochrome has O-deethylase activity towards ethoxyresorufin. Indole-3-carbinol is a modest inducer of Nrf2-dependent ARE-driven gene expression in the liver and small intestine of mice [52. rat GSTA2. By comparison with sulforaphane. a fact that implies it works through Nrf2. exposure of RL-34 cells or HepG2 cells to sulforaphane causes stabilisation and rapid accumulation of Nrf2 [63. this may not necessarily lead to an increase in Nrf2 protein levels. indole-3-carbinol or ascorbigen [43.3∂-diindolylmethane can increase the stability of Nrf2 protein is not known. The indole-containing glucobrassicin breakdown products can activate gene expression by several mechanisms.2-b]carbazole is a much more potent inducing agent than 3. Interestingly. rat ALDH-3. 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. Whether indolo[3. and it is therefore anticipated that activation of AhR by glucobrassicin-derived indoles will influence significantly the metabolism of xenobiotics (for a review. . cranbene was found to be an approximately equally potent inducer in the rat [66].2-b]carbazole and 3. and this may also contribute to gene induction [46]. and rat UGT1A6 contain an XRE. rat and human CYP1A1 are prototypic XRE-regulated genes.2-b]carbazole and 3.2-b]carbazole and 3. as does the mouse BAX gene. see [70]).3∂-diindolylmethane. it is a good inducer of GST enzyme activity in mouse liver and small intestine [68]. the mouse nrf2 gene promoter also contains an XRE [71]. The identity of this metabolite is not known. 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 has been found that administration of cranbene to Fischer 344 rats causes an elevation in hepatic GST and NQO1 enzyme activities. suggesting it is a mono-functional inducer [65]. Examination of the dose of indole required to double XRE-driven reporter gene expression shows that indolo[3.67]. Consistent with this view. but unless they are metabolised by CYPs. 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.

Within the cell. Inhibition of histone deacetylase by isothiocyanates The isothiocyanate sulforaphane has been shown to inhibit histone deacetylase (HDAC) [83. sulfotransferase and UGT1 [74]. other cytochromes are inducible. 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. Most importantly.76]. Eggleston 148 In addition to induction of CYP1A1 by indoles. such as growth arrest and DNA damage (GADD) GADD34. recognition of this possibility has lead to considerable recent interest in the ability of HDAC inhibitors to act as both chemopreventive and chemotherapeutic agents. prior treatment of the LS-174 cells with both indolo[3. NF-IL6 and the GADD proteins are regulated through XREs and whether the process is mediated by AhR.79]. GADD45 and GADD153 [75.3∂-diindolylmethane can induce expression of the transcription factors ATF3. It is not however clear whether the genes for ATF3. p21 is induced by indole-3-carbinole [77]. Inhibition of carcinogen activation by glucosinolate breakdown products Certain isothiocyanates can block the activation of several carcinogens to their ultimate carcinogenic forms. and this protection was greater than was achieved by either phytochemical alone [43]. GST T1-1. This function is likely to alter gene expression substantially. Furthermore. Inhibition of CYP can also be achieved by sulforaphane [80. c-Jun. Kelleher. as are the drug metabolising enzymes AKR. Each of the histone proteins contains an evolutionary conserved amino tail protruding from . 3. c-Jun and NF-IL6 as well as genes involved in cell growth. In the case of the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. 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. This viewpoint is probably an oversimplification and does not take into account the multiple changes in gene expression that indoles affect. Bonnesen et al [43] have reported that treatment of human colon LS-174 cells with indolo[3.2-b]carbazole before exposure to benzo[a]pyrene provides a small measure of protection against DNA damage as measured by the comet assay. such as CYP1B1 and CYP19 [73]. Indeed. the basic structural unit of chromatin. Ian M. Also. the ITCs with highest lipophilicity and low reactivity of their NCS group had the greatest ability to inhibit lung tumorigenesis [82].2-b]carbazole and sulforaphane before exposure to benzo[a]pyrene was found to confer substantial protection against genotoxicity.84].81]. Michael O. Hayes. 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. DNA is tightly coiled around an octamer of histone proteins in a structure known as a nucleosome.John D.

allowing transcription factors access to the regulatory regions of genes. The tail is also subject to many post-translational modifications including acetylation. mediated through activation of checkpoint kinase 2 (Chk2) [84]. removal of acetyl groups causes the tail to wrap tightly around the DNA thereby preventing interaction with the transcription machinery. In human PC-3 prostate cancer cells. and was accompanied by its translocation from the nucleus to the cytoplasm [92].dependent fashion [87]. The addition and removal of the acetyl groups is carried out by HAT and HDAC. Thus the ability of sulforaphane. the majority of the studies into mechanisms by which this class of chemical inhibit cell growth have focussed on sulforaphane and phenethyl-ITC.94]. 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]. 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. The link between inhibition of HDAC activity and the resultant increase in transcription of tumour suppressor genes has been reported for p21 [84.85].92]. respectively. sulforaphane has been found to cause a G2/M phase delay with an increase in apoptotic cell fraction in a timeand dose. apoptosis and differentiation [89]. by deacetylation of histones associated with the p53 gene. Inhibition of HDAC may prevent the removal of acetyl moieties from histones thus allowing transcription of the tumour suppressor genes. In pre-cancerous and cancerous cells. this was associated with a higher level of apoptosis and lower proliferation in animals on the ITC containing diet [90]. to affect chemoprevention via its ability to alter chromatin structure is of acute importance to the welfare of the cell. human embryonic kidney cells [83] and human colorectal cancer cells [83]. In addition. Cell proliferation by ITCs may also be achieved by distrupting cytoskeletal structure and tubulin polymerisation [93. p53 [86] and Bax [84. along with other dietary HDAC inhibitors such as diallyl disulfide [88]. mammalian HDAC is capable of downregulating p53 function. Equally. which can determine the accessibility of the DNA to transcription factors. Relocation of Cdc25C was found to be controlled by its phosphorylation at Ser-216. 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. resulting in a reduction in its transcriptional activity.85]. tumour suppressor genes are associated with deacetylated histones resulting in the inactivation of these genes. Conversely. The loss of Cdc25C was reported to be due to proteasomal activity. For example.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 149 the nucleosome. However. 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. .

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

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

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evaluation of the effects of synergistically acting phytochemicals is needed. 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. 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. In the research conducted.5.10. Isolated and purified compounds. de Kok. 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. which are believed to have cancer-preventive effects. The combinations of phytochemicals that natural foods contain can reduce the risk of cancer by affecting different overlapping and complementary mechanisms. . 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.2]. de Kok and Simone G. evidence that bioactive compounds act synergistically is reviewed. and the like. summarizes various mechanisms by which phytochemicals can modulate the risk of cancer [1. vegetables. and their potential health-promoting effects evaluated extensively. Evaluating the effects of phytochemicals that act synergistically Carcinogenesis is an extremely complex multistep process in which numerous molecular mechanisms play an important role. both in vitro and in vivo. 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. Some studies show no reduced incidence of cancer as a result of taking supplements of vitamin C [3] or β-carotene [4]. whole grains.Theo M. van Breda 160 3. Combined action of different dietary compounds preventive of cancer Theo M. in contrast. Cancer-preventive dietary compounds may interfere at a variety of different levels with these processes. Although relatively high doses of single bioactive agents may show potent anticarcinogenic effects. In this chapter. One of the key problems of research in this field. In addition to characterizing the chemopreventive effects of individual compounds. van Breda Department of Health Risk Analysis and Toxicology. however.6]. are far from certain. therefore. The intended positive effects of an increased intake of β-carotene or vitamin C as dietary supplements. Maastricht. University of Maastricht. in contrast. Table 3. The Netherlands Introduction The relatively consistent epidemiological finding that the consumption of whole foods of different types such as fruits. numerous bioactive compounds have been isolated and identified. and there are even reports of increased occurrence of lung cancer in smokers receiving dietary β-carotene supplements [5. 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. Simone G.

10.11.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 161 Table 3. 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. Epicatechin was found to promote the occurrence . another green tea polyphenol. [2]. one without a galloyl moiety. [7] reported that the effects of tritium-labelled epigallocatechin gallate (EGCG) being incorporated into human lung cancer cells were enhanced by epicatechin (EC). presents a selection of relevant studies concerning such synergistic effects. Suganuma et al. Table 3. 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.

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

being due instead to the effects of the complex mixture involved. 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. and epicatechin gallate (ECG) — and it was not found to be improved further by the addition of other compounds [11]. but under some circumstances substrates may be activated to become mutagens. [27] Van Breda et al. both on the inhibition of cell growth and the induction of apoptosis.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 163 Table 3. peas and unions) Up.and down-regulation of genes involved in carcinogenesis. the activity levels of caspases 3.24] Different types of vegetables (cauliflower. Since the cytochrome P450 (CYP) enzymes are responsible for the metabolism of many environmental carcinogens. catalase was found to block the synergistic effects of EC and EGCG. carrots. 8 and 9 became elevated. Various gastric cell lines were shown to differ in their susceptibility to EGCG treatment. Interestingly. Williams et al. EC alone had virtually no effect on carcinomic cell growth or induction of apoptosis. carcinogens or cytotoxic substances [9]. 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]. Synergistic effects of green tea catechins. were also found in gastric carcinoma cells [8]. [10] demonstrated that complex green tea extracts exert mixed agonist/antagonist activity on the Ah-receptors. Enzyme induction usually enhances detoxification. suggesting that the reactive oxidative species and production of hydrogen peroxide play a part in the synergistic mechanisms here [8]. whereas others are inducible by xenobiotic compounds or by phytochemicals. Some of the cytochrome P450 genes are expressed constitutively. EGCG. 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. [25] Jørgensen et al. indicating them to be involved in catechin-induced apoptosis. but it had a significant synergistic effect on the induction of apoptosis when combined with other catechins. epigallocatechin (EGC). 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). [23.11. The optimal degree of synergy was achieved by a combination of the four major tea catechins — EC. After this combined treatment. Selection of studies of the synergistic effects of mixtures of polyphenols and of combinations of these with other types of phytochemicals — cont. whereas EGCG acts as a strict AhR antagonist. interpreted mainly in preventive terms No indication of specific synergetic mechanisms * SPC — Soy Phytochemical Concentrate. the modulation of their expression and activity by phytochemicals is a potential mechanism by which the risk of cancer can be reduced. .

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

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

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

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

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

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

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

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

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

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

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