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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
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: email@example.com Website: http://www.imp.lodz.pl
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
. Anticarcinogenic effects of glucosinolate breakdown products John D. . . . . . . . . . . . . Simone G. . . . 179 . . . . . . . . . . . . . . . . . . . . . Hayes. . . . . . 160 3.6. . . . . . . . . . . Kelleher. . . . . Combined action of different dietary compounds preventive of cancer Theo M. Eggleston . . Michael O. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 3. Probiotics and prebiotics — potential anticarcinogenic food components Joseph Rafter . . . . . . de Kok. . . . . . . . . . . . . . . . . . . . . . . . .3. . . Sotiroudis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 3. . . . . . . . . . . Kyrtopoulos . . .4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ian M. . . . . . . . 170 Annex ECNIS participants . . . . Anticarcinogenic compounds of olive oil and related biomarkers Theodore G. . . . . Soterios A. . . . . . . . . . .5. van Breda .3. . . .
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.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. that of reviewing the mechanisms of action of some of the most promising anticarcinogenic compounds. Vitamins and selenium Although the measurement of vitamins in body fluids is a classical area for nutritional biomarker research. and bioactive compounds. although plantbased foods appear to be the richest source. 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. Biomarkers are a key tool for assessing nutritional status and dietary exposure to specific substances. only certain selected topics. These compounds are found in foods of many different types. which is a very complex field indeed in view of the large number of cancer diseases. Many compounds contained in the diet have been proposed as having anticarcinogenic properties. on the other. food components and food preparation methods involved. vitamins and selenium on one hand. The important links found between use of a substance as a biomarker and its mechanisms of action led to a further aim. Since this is a vast area. especially those of relevance for the partners of the NoE ECNIS and its contacts. The present review focuses on two major groups of food components. pathogenetic mechanisms. The degree to which these compounds are present in plant tissue can vary markedly and be dependent on the plant variety. The ECNIS project aims at addressing important problems in this field and identifying knowledge gaps that exist. Current research . Nutritional biomarkers are essential for studies of the links between dietary intake and the risk of cancer. as outlined below. the growth conditions and other factors. some of them mainly reflecting recent dietary intake and others indicating more the individual’s long-term nutritional status. which to a large extent reflects the kinetics of metabolism and transport. the confounding factors affecting a given biomarker vary appreciably for different dietary components as well as in terms of the body fluid or tissue considered suitable for sampling. and pool sizes of the substance in question. particular progress has been made here recently. Markers differ considerably in their characteristics. Particular attention is directed at identifying dietary factors that modulate environmental and lifestyle factors related to cancer risk. Also. are considered in detail.
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. are controversial. but there is a need of developing simpler methodologies. strong research efforts in this area should be encouraged. at present it is impossible to assess the quantitative involvement of oxidative stress in the origin of cancer. The measurement of vitamin D metabolites presents a number of problems including the occurrence of vitamin D in different forms. The latter has the advantage of possessing lower detection limits. Concerning the functions of vitamin A.Dietary vitamins. polyphenols. In contrast. including cancer. The determination of vitamin D status is still a challenging task. but they are not useful as yet for large screenings. as determined by isoprostanes and by inflammatory markers. 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. The effects that have been postulated of high levels of vitamin E supplementation on exercise-induced lipid oxidation. Biomarkers of vitamins A. C and E are an important part of the diet-cancer field. and since metabolites of vitamin D have important biological effects. Simpler tests using fluorescence or immunological techniques have a high level of accuracy and precision. can readily be determined in plasma by automated colorimetric assays or by HPLC. Serum retinol is still the most reliable indicator for evaluating vitamin A deficiency. Vitamin E intervention has not been shown to decrease the oxidation of plasma proteins. the variations in measurement between different assays and different laboratories. both HPLC and GC-MS methodology have high precision and accuracy for its detection in plasma. there are six investigations that have reported beneficial effects . No studies are currently available on the relationship between lipid or protein oxidation markers and chronic disease. There is limited evidence that plasma ascorbate is a functional antioxidant. HPLC methods are those which are most sensitive. such damage is strongly implicated in the aetiology of cancer. epidemio-logic variations in vitamin D status and its determinants. it appears to have only limited capacity as a direct antioxidant in vivo. Regarding vitamin E. clear short-term pro-oxidant effects on lipid oxidation have been observed following the infusion of high-dose vitamin C into the bloodstream. Vitamin C. Regarding intervention studies using antioxidant supplementation. and that the intake of high doses of vitamin C can be used to decrease oxidative stress. probably higher accuracy as well as the possibility it to determine ascorbate and dehydroascorbate simultaneously. but colorimetric assays have a much higher throughput. The handling and storage of plasma for vitamin C determination has a strong impact on its accuracy. and the problems in defining the appropriate threshold for vitamin D sufficiency. 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. which is water-soluble. Since DNA mutations are a crucial step in carcinogenesis and elevated levels of oxidative DNA lesions have been noted in many tumours. The antioxidant vitamins have been studied very much in relation to risk of cancer. There is limited evidence that of a protective effect of vitamin E supplementation at levels of 200-2000 IU/d as judged from its effects on markers of lipid oxidation. as assessed by currently available markers of lipid or protein oxidation in humans.
flavonoids exhibit a wide range of potentially beneficial activities in terms of cancer prevention. 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. the cell cycle. There is also a need of clarifying the mechanistic role of selenium in the etiology of cancer. Flavonoids can also induce cell cycle arrest by modulating key components of cell cycle regulation. release of cytochrome c and the activation of caspases. whereas there are 13 studies that have reported a null effect. Several large human intervention studies have indicated that selenium supplementation can prevent the development of several different forms of cancer (prostate. Like other types of dietary chemopreventive agents. It is important to explore the possibility of using other selenoproteins as biomarkers too. including cyclins. These are usually divided up into blocking and suppressing activities. Most studies have involved healthy individuals. It is also important to determine whether there is an increased risk of certain forms of cancer after selenium supplementation. colon). mitochondrial membrane depolarisation. primarily glutathione peroxidase. and the induction of apoptosis. lung. The blocking activities include antioxidant effects and the modulation of drugmetabolising enzymes and multidrug resistant genes. but the few well-controlled studies that have reported realistic appearing levels of oxidative DNA damage in leucocytes isolated from oxidatively stressed subjects do not lend much support to the notion that such a population benefits more from antioxidant supplementation than a normal study population would. methylated selenocompounds having been found to have particularly strong chemoprotective effects. Several epidemiologic case-control studies have indicated a protective role of selenium in helping prevent the development of cancer. The suppressing activities include the inhibition of signalling pathways responsible for cell proliferation and survival. Both organic and inorganic forms of selenium have been evaluated. cyclin-dependent kinases and inhibitors. epigallo- . The optimal type and dose of selenium supplements needs to also be defined. DNA repair and signal transduction. mainly through intrinsic pathways involving members of the Bcl family. In these studies.Executive summary 7 of it on oxidative DNA damage. Further studies are underway. Experimental studies have shown that selenium compounds affect cell growth. The relationship of low selenium status and selenium supplementation to cancer disease is another important segment of focus in the diet-cancer field. use has been made of several biomarkers such as the concentration of total selenium in different body fluids as well as different selenoproteins. 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. Some recent mechanistic findings on the flavonoids apigenin. 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.
as well as squalene and β-sitosterol. which also applies to analyses of these compounds in urine. It is unclear whether the formation of thiocyanates. 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. resveratrol. In addition. such as isoflavones and lignans. 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. is undesirable in terms of chemoprevention of cancer. a number of recent studies on the bioavailability of certain minor but important olive oil components (polyphenols. The bioavailability of different compounds varies markedly and for many compounds information on this point is scarce. From an analytical point of view. xanthohumol and the novel flavonol tricin have been summarised. 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. certain phenolic compounds (tyrosol. promotion and progression of multistage carcinogenesis. it is difficult to relate responses of cells in culture to particular concentrations of phytochemicals to the situation in vivo. An especially interesting matter here is that intake of olive oil can increase the bioavailability of anticarcinogenic compounds. They also appear to influence apoptotic responses to chemotherapeutic agents. It is also unclear whether the activity of the epithiospecifier protein (ESP). relatively little is known of the pharmacokinetic properties of glucosinolate breakdown products in humans. Also. cyano-epithioalkanes and oxazolidine-2-thiones from glucosinolates. 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. The more recently developed metabolomic techniques may offer new possibilities in the future. nitriles. squalene and β-sitosterol) are reviewed. the availability of immunoassays is of great value. Mammalian cells display marked dose responsiveness to phytochemicals. but further study regarding this will be needed. hydroxytyrosol. To clarify the matter. its becoming increasingly clear that it is their breakdown products that can influence the initiation and progression of carcinogenesis. These are areas in need of further investigation. It has also been pointed out that their efficacy is dependent on their bioavailability. Without such information. These include tocopherol and carotenoid antioxidants. lignans. polyphenols. at low doses of phytochemicals cytoprotective adaptive responses . genistein. which occurs at the expense of their forming isothiocyanates. the use of polyphenols as biomarkers of dietary intake has attracted considerable interest. is undesirable. Hydrolytic and clean-up steps are usually needed. secoiridoids and lignans). which reduces the formation of isothiocyanates from glucosinolates. These compounds have a number of different mechanisms of action. The anticancerogenic properties of the glucosinolates have received a great deal of attention. For some compounds. quercetin. but their utilisation in this way is hampered by several factors.Dietary vitamins. the chalone. 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.
Different strains of the bacteria may possibly target different mechanisms. The development of ideas regarding this has also been stimulated by findings that dietary supplements of only a single compound may have negative effects. An 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. whereas at higher doses cell cycle arrest and apoptosis occur. growth arrest or apoptosis is to occur are determined. The strongest evidence for the anticancer effects these can have comes from animal studies. it appears that many combinations of complementary modes of action may be involved. promising and as deserving of closer scrutiny. 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. This can well contribute to cancer prevention. As has been indicated here. 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 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. 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. The synergistic effects of dietary phytochemicals need to be investigated further. Many healthful diet effects are attributed to probiotic bacteria. evidence from human studies still being limited. and green tea flavonoids can also show increased activity when present together with other phytochemicals. More work needs to be done to identify the specific strains and strain characteristics responsible for particular antitumour effects and the mediating mechanisms involved. combinations of green tea flavonoids are more active than single compounds of this sort. .Executive summary 9 being activated. It is presently unclear how these different types of responses are coordinated by the cell and how matters of whether adaptation. Identification of the mechanisms that control such outcomes would be very useful. Even with the above reservations and the limited number of human studies available in mind. In some systems. It is important here to have insight into recent developments in analytical technology and thorough knowledge of biomarker kinetics. Several possible mechanisms may explain how lactic acid bacteria can protect against tumour development in the colon. 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. one can regard the use of lactic cultures for human cancer suppression as interesting. Although our understanding of the molecular mechanisms behind the observed combinational effects is still limited. Much hope is attached in this connection to the powerful techniques available today within the field of nutritional genomics. An understanding of the mechanisms involved is a necessary basis for the development and validation of new biomarkers of nutritional status.
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. Sweden Dietary assessment methodology and the development and validation of biomarkers of nutritional status are key scientific fields within nutritional science. that role that various food components and food preparation methods play in connection with this. Lund. the pathogenetic mechanisms involved. The major aim of this review is to summarize the state-of-the-art of biomarkers that can be applied to various anticarcinogenic food components and to highlight different knowledge gaps. A number of textbooks and reviews are available within the field of nutritional biomarkers [see e. Introductory overview and future prospects Björn Åkesson Biomedical Nutrition. their physiological basis and the use of biomarkers in studies of different types. . This is a very complex field in view of the large number of different types of cancer. 1–6] dealing with the basic theories and methodology. Table 1.g. Various properties of biomarkers are summarised in Table 1. A main concern is to identify dietary factors that modulate environmental and lifestyle factors that can affect risk of cancer and which in the long run can also provide support for the development of functional foods. markers of nutritional status Markers of differing kinetics (response times) Markers of nutrients vs.1. They have expanded markedly with the advent of new analytical technology and the emergence of scientific findings showing that hitherto neglected food components may have interesting and important health effects.1.1. Lund University Hospital. Lund University and Department of Clinical Nutrition. Some major issues regarding nutritional biomarkers Markers of dietary intake vs. The ECNIS project aims at addressing important problems in this field and identifying various knowledge gaps.2. A further aim is to examine and explore the mechanisms responsible for the action for different promising anticarcinogenic compounds. A brief overview of food components that appear to have an anticarcinogenic effect is provided in Appendix 1. Introduction 1. Pure and Applied Biochemistry.1.
are in routine daily use in clinical laboratories. such as the retinol-binding protein and ceruloplasmin. . The amount of a substance occurring in the erythrocytes. Various diet-related compounds in the plasma also differ considerably in turnover times. interactions between dietary or endogenous components or xenobiotics. 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. 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. Also proteins with a transport function as well as other functions are used as biomarkers. the gut flora. the main function of some markers being that of serving as indicators of malnutrition. Biomarkers show temporal variation.Björn Åkesson 12 Dietary components as biomarkers Many biomarkers involve the measurement of a nutrient or some other dietary component in the blood. The level of a biomarker often varies as well with age. diurnal variations. In addition. since the erythrocyte cells have a lifetime of about 120 days . whereas others are performed only in a few specialised research laboratories. Some methods. 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 . and inflammatory and other diseases can influence the levels of different biomarkers . . urine or hair. gender and gene polymorphism (as outlined below). such as the analysis of cobalamine and folate in plasma. One such example is olive oil discussed in this volume by Sotiroudis and Kyrtopoulos. for example zinc and copper to superoxide dismutase activity and selenium to the activity of glutathione peroxidase and other selenoproteins. often reflects much more the long-term intake if it than the plasma level does. 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. variations in metabolism or in excretory pathways. different nutrient-dependent physiological tests can be employed . 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. 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. trace elements are coupled to the levels of enzyme activities. plasma. Also. A large number of confounding factors such as hormones. The selection of the type of sampling to carry out depends very much on the components to be studied and the sampling facilities available. and they may not respond to overexposure of nutrients at all.
Also. thus strengthening dietary associations. As summarised by Hunter . buccal mucosa. Their use may increase. as exemplified in the present volume by Linseisen and his colleagues in connection with vitamin D and polyphenols. For this reason. 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. and 4) eventually provide the basis for gene-based screening tests”. faeces. or for the calibration of the dietary assessment methods . Although the use of the biomarker concept for these purposes is very attractive. This topic is only taken up briefly in the present volume. it should be borne in mind that there are very few (if any) “ideal” biomarkers that have been validated for studies of different types. methods for measuring nutrients or nutrient-dependent variables in cells in the blood (mainly leucocytes). An overview of this matter is provided in . 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. although epidemiological findings on the association between the selenium level and cancer are reviewed by Gromadziƒska et al. however. Folate has served as a prototype here for demonstrating how genetic make-up can influence the nutrient status of an individual. Biomarkers in studies of that type have been used either alone. urine. The two contributions mentioned also illustrate the difficulty in defining valid cut-off points. adipose tissue or biopies from other organs have been developed. 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].12]. These matters are outside the scope of the present review. 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. 3) aid in distinguishing causal components of complex dietary mixtures. information on the interactions between dietary intake and gene polymorphisms “can serve to 1) define susceptible subpopulations. A number of statistical methods for the correction and evaluation of such data are available . The assessment of dietary intake in epidemiological studies has been a major use of biochemical markers of nutritional status. together with an assessment of dietary intake by a food frequency questionnaire or by some other methods.Introductory overview and future prospects 13 Although several of the biomarkers mentioned here are widely used. There are particularly good reasons for using the 25-hydroxy vitamin D as a biomarker in extended studies in the future. Some of these samplings require much skill and effort and can only be used in small studies. if the analytical methodology improves so that smaller amounts of sample suffice. 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. . 2) help establish causality of food and nutrient associations in epidemiology studies.
17]. the existence of a renal threshold or other factors. Biomarkers are also used in long-term human intervention studies in which there are clinical endpoints. a meta-analysis showing there to be an increase in mortality in studies of antioxidant supplementation by β-carotene. To the surprise of the scientific community it was found that some antioxidants. . a matter reviewed recently [16. adenomatous polyps. usually responds to an increase in the supply of them. de Kok and their colleagues in this volume. Other aspects of cancer-related biomarkers have been the subject of a previous review conducted within the ECNIS project . It was also stated that “in four trials (three with unclear/inadequate methodology).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 of this category include tumours and the mortality in animal models. vitamin A and/or vitamin E . certain metabolites and various effects on DNA and its metabolism . Studies in which nutrient antioxidants or combinations of those are employed are of special relevance for various topics taken up in the present volume. especially β-carotene. A possible further use to which such biomarkers could be put would be to substantiate ‘anticancer ’ claims regarding various food components. had negative effects. The content or profile in the blood of β-carotene. work that is as reviewed by Manson. The potential preventive effect of selenium should be studied in adequate randomised trials” . selenium and polyenoic fatty acids for example. cell proliferation and differentiation. In the present volume Loft et al. α-tocopherol. Hayes. as well as precancerous lesions. although for other nutrients and their biomarkers there may be little or no response due to homeostatic regulation of their plasma levels. Biomarkers for the assessment of dietary effects on carcinogenesis Biomarkers used as a surrogate endpoint in studies of the effect that dietary manipulation has at different stages of carcinogenesis are another type of biomarkers of importance in the diet-cancer field. the criteria to be employed for the evaluation of efficacy in the future being proposed [14. C and E on biomarkers of oxidative stress. apoptosis. selenium showed significant beneficial effect on the incidence of gastrointestinal cancer. gut-lumen enzymes and carcinogen-metabolising enzymes. The results of the first generation of nutritional intervention studies concerned with prevention of cancer were summarized recently . and Dragsted summarises the effects of vitamins A. review the evidence that individual antioxidants as well as for antioxidant-rich diets affecting oxidative DNA damage.15]. such enzymes as cyclo-oxygenase 2. 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.
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 . 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 .
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 .
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.
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.2. but plant-based foods appear to be their richest source.2. Plant tissues can also vary enormously in the store of such compounds. Table 1. Different plant families harbor different mixtures of compounds of this sort.3. Overview of possible anticarcinogenic food components Per Mercke Biomedical Nutrition.). for example. This information was collected with use of a PubMed search conducted in 2005. The criteria used for the selection were their relevance as adjudged from their title. 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. These protective compounds are found basically in all types of food. depending on the plant variety and the growth conditions. Lund University. a list of putative anticarcinogenic food components has been generated (Table 1. emphasis on recent reviews. To provide an overview of the cancer-protective compounds at which special attention is directed in the literature and assemble various criteria used in selecting such compounds.Per Mercke 18 Appendix 1. Sweden There is a vast range of dietary compounds proclaimed to have anti-carcinogenic properties as investigated by the scientific community. 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. .
 Pre. Sanderson et al. Rafter et al. Rafter et al.Overview of possible anticarcinogenic food components 19 Table 1. methionine) . Milner . Maruvada and Srivastava . Vlastos et al.  Branca et al. Greenwald . choline. Loft and Paulsen . . . . . . Rafter et al. . Milner . Vlastos et al. Ferguson . . Gill and Rowland . Vlastos et al. Crews et al. . Seifried et al. . Sharma and Farmer . Ferguson . Greenwald . Kelloff et al. Ferguson . . Mayne . . Ferguson . . Mayne . . Vitamin E Vitamin D Vitamin C B vitamins (Folic acid cobalamine. Granado et al. Wild et al.  Branca et al. Mayne . Maruvada and Srivastava . . . Sanderson et al. Ferguson . Kelloff et al. . Sharma and Farmer . . . Gill and Rowland . Sanderson et al. Loft and Paulsen . Vlastos et al. Crews et al. .  Branca et al. Seifried et al. Greenwald . Rafter . Kelloff et al. . . Seifried et al. Ferguson . . 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. . Turini and DuBois  Branca et al. Ferguson . . Milner . . Rafter et al. . . Wild et al. . Mason . Vlastos et al. Manson et al. . Sanderson et al.and probiotics Butyrate Vitamins Vitamin A including retinol and retinoic acids α. Wild et al. Milner . Loft and Paulsen . Manson et al. Turini and DuBois . Wagner et al. Kelloff et al. . Ferguson . Kelloff et al. . . Milner . Greenwald .  Branca et al. . Ferguson  Starch and resistant starch Fibre/non-starch polysaccaride Vegetables Branca et al. Konety and Getzenberg . . Sharma and Farmer .and β-Carotene Lycopene Lutein Zeaxanthin β-Cryptoxanthin Carotenoids Orange vegetables Tomatoes Green vegetables Branca et al. Bowen et al. Branca et al.  Branca et al. Gill and Rowland . riboflavin.3. Turini and DuBois  Branca et al. . Manson et al. Manson . . . pyridoxine. Wild et al. .
Ferguson . Manson et al. broccoli. Ferguson . Sharma and Farmer . Manson . .Per Mercke 20 Table 1. onion Radish. . . Wild et al. Milner . . Sanderson et al. . . Atkinson and Bingham . . Milner . Wild et al. Branca et al. . garlic. broad beans. . . . Rafter et al. Maruvada and Srivastava . Sharma and Farmer  Branca et al. Turini and DuBois  Maruvada and Srivastava . . Kelloff et al. soybeans) Branca et al. Ferguson . . . Rafter et al. tomatoes. Seifried et al. Duthie and Crozier . berries. . horse-radish. Sanderson et al. Ferguson . potatoes. Mayne . An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected — cont. . Ferguson . Manson . . Zhang  Ferguson . . Branca et al. kale. Manson et al. Sharma and Farmer  Flavonoid polyphenolics Catechins Catechin Tea. . Saleem et al. pulses (millet. .  Bianchini and Vainio . Rafter et al. Sanderson et al. . sorghum. chocolate Epicatechin Epigallocatechin (EGC) Epigallocatechin gallate Caffeic acid Ferulic acid Ellagic acid Genistein Cereals. chives Citrus fruits. Manson  Phenolic acids Isoflavones Adlercreutz . Kelloff et al. . endive Citrus fruits Branca et al.3. Seifried et al. . Manson et al. Greenwald . . Manson . Gill and Rowland . . Greenwald . Seifried et al. Manson . Manson et al. Manson  Crews et al. Rafter et al. . Crews et al. Ferguson . Milner . Kelloff et al. Wiseman  . . 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. Gill and Rowland . scallions. Seifried et al.  Allium compounds Onions.3’-Diindolylmethane Indole-3-acetonitrile Diallyl sulphide Allylmethyl trisulphide Tangeretin Apigenin Nobiletin Rutin Quercetin Kaempferol Taxifolin Cruciferous vegetables Bianchini and Vainio . . Manson . Manson  Iron Iodine Isothiocyanates Benzyl isothiocyanate Cruciferous vegetables Phenethyl isothiocyanate Sulphoraphane Oltipraz (synthetic) Cruciferous vegetables Sulphones Dithiolthiones Glucosinolates Indole-3-carbinol 3. .  Ferguson . Sanderson et al. Kelloff et al. squash. Kelloff et al. Kelloff et al. .
4. . et al.166:257–75. Turmeric Branca et al. Mathers JC. Milner JA. Nair J. Mutat Res 1999. Farmer PB. Pannemans D. Incorporating basic nutrition science into health interventions for cancer prevention. Gescher A. Kelloff et al.3. cola. Greenwald P. Branca F. 7.25 Suppl:29–36. Cancer risk factors for selecting cohorts for large-scale chemoprevention trials. Br J Nutr 2001. Verhagen H.  Other non-flavonoid phenolics Lignans Olive oil Adlercreutz . . Gill CI. Steward WP. An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected — cont. pecans. Eur J Nutr 2004. Manson . Class of compound Methylxanthines Example of bioactive substance Caffeine Theophylline Theobromine Limonene Perillyl alcohol Geraniol Menthol Carvone Hydroxytyrosol Curcumin Resveratrol Food source Tea. 10. Sanderson P. J Cell Biochem 1996. Pool-Zobel B. walnuts. Johnson IT. dietary fat and antioxidants. raspberries. 2. Rechkemmer G. 9. Rafter J. strawberries. Pool-Zobel B. 8. Bartsch H. sis: potential role of lipid peroxidation. black-berries. J Nutr 2003. Rafter et al. Owen RW Exocyclic DNA adducts as oxidative stress markers in colon carcinogene. et al. Br J Nutr 2002. Prospects for cancer prevention. Primary prevention: phytoprevention and chemoprevention of colorectal cancer. 6. Rowland IR.86 Suppl 1:S55–S92. Turini ME. Wiseman  Grapes. Cereals. 5. Recent Results Cancer Res 2005. Govers M. Br J Nutr 2004.43 Suppl 2:II47–84. Downes CS. . DuBois RN. Innovative agents in cancer prevention. Rafter JJ.428:329–38. Br J Nutr 2002. Biomarkers in disease and health.Overview of possible anticarcinogenic food components 21 Table 1.88 Suppl 1:S73–87. Ferguson LR.91:315–23. 3. cacao Manson  Reference Monoterpenes Citrus fruits (peel) Ferguson . Scientific basis of biomarkers and benefits of functional foods for reduction of disease risk: cancer. . Martel P. McGlynn AP. coffee. Powers HJ.383:915–21.16:811–40. Bartsch et al. Hanley AB. 11.133 Suppl 1:3820S–6S. Diet and cancer: assessing the risk. PASSCLAIM — dietrelated cancer. olive oil References (selected from the PubMed search described above) 1. Emerging dietrelated surrogate end points for colorectal cancer: UK Food Standards Agency diet and colonic health workshop report. Hematol Oncol Clin North Am 2002. Manson MM.88 Suppl 2:S219–24. Biol Chem 2002.
J Steroid Biochem Mol Biol 2002. 25. J Mol Med 1996. Konety BR.130 Suppl:467S–71S. Duthie G. Poulsen HE. 23. J Nutr 2003. Boone CW. et al. Vlastos AT. Mason JB. Mukhtar H. Mayne ST.90:487–502. Woods JA. et al. Mutat Res 2004. 2002.86 Suppl 1:S5–35.83:113–8. Br J Nutr 2001. 19.29:95–106. 24. A critical evaluation of the application of biomarkers in epidemiological studies on diet and health. Br J Nutr 2001. Wilson L. Br J Nutr 2003. Kelloff GJ.47:13–23. 27. Elmadfa I. 21.Per Mercke 22 12. O'Brien NM. Tea beverage in chemoprevention of prostate cancer: a mini-review. Breast Cancer Res. Wild CP.4:1–4. Bianchini F. Lubet RA. Clin Cancer Res 2004. 18.63:4295–8. Alink G. Atkinson C. 22. Crews H. Granado F. Olmedilla B. Vainio H. 26. 32. Braesco V. Cancer risk and oxidative DNA damage in man.555:173–90. 31. 13. Andersen R. 20. Milner JA.74:297–312. Kamal-Eldin A.48:169–88.133 Suppl 3:941S–7S. Greenwald P. Crowell JA.10:4901–12. Malone WA. 30. McDonald SS. 17. Ghosh L. Blanco I. Sharma RA. Bowen P. Cancer prevention — the potential for diet to modulate molecular signalling. Biomarkers of nutrient exposure and status in one-carbon (methyl) metabolism. Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis. Stacewicz-Sapuntzakis M. Duncan C. Cancer Res 2003. Phytoestrogens and breast cancer. Biomarkers and their use in cervical cancer chemoprevention.86 Suppl 1:S37–53. Nutr Cancer 2003. Drug Metab Rev 2004.36:655–67. Vitamin D and prostate cancer. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Exp Biol Med (Maywood) 2002. Steele VE. Follen M. J Nutr 2004. Mammographic breast density as a biomarker of effects of isoflavones on the female breast. Saleem M. The antioxidant conundrum in cancer. Progress in cancer chemoprevention: development of diet-derived chemopreventive agents.9:11–8. Schottenfeld D. Anderson DE. Manson MM. Seifried HE.227:886–93. Zhang Y. Maiani G. 29.3:447–51. J Nutr 2000. Siddiqui IA. Curr Opin Clin Nutr Metab Care 2000. Loft S. Andersson C. Crit Rev Oncol Hematol 2003. 28. Adhami VM. Wagner KH. Adlercreutz H. Maruvada P.46:261–73.134 Suppl:1640S–5S. Isothiocyanates in cancer prevention. Biological relevance of adduct detection to the chemoprevention of cancer. Chen L. Biomarkers for cancer diagnosis: implications for nutritional research. Getzenberg RH. Holst B. Trends Mol Med 2003. Urol Clin North Am 2002. Srivastava S. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. Sharifi R. .133 Suppl 3:933S–40S. 14. Plant-derived phenolic antioxidants. et al. 15. A critical assessment of some biomarker approaches linked with dietary intake. Crozier A. Nutritional and clinical relevance of lutein in human health. Gamma-tocopherol — an underestimated vitamin? Ann Nutr Metab 2004. Bingham SA. Farmer PB. 16. J Nutr 2003.
Molecular epidemiological approaches to the study of the genotoxic effects of urban air pollution. Nutr Rev 1999. Hollman PC. 47. Kyrtopoulos SA. Expert Opin Investig Drugs 2000. Public Health Nutr 2002. Jubb AM.9:1829–40. Who's afraid of n-6 polyunsaturated fatty acids? Methodological considerations for assessing whether they are harmful. Rivlin RS. Future directions. Tahir A. Quirke P. Carroll PR. Am J Clin Nutr 1997. J Pathol 2001. Berry EM.65 Suppl 4:1240S–5S. Mutat Res 1999. Antioxidant vitamins: evidence from biomarkers in humans. observational versus experimental human studies. Nutr Metab Cardiovasc Dis 2001. 39. . Frei B. 34. Epidemiology and prevention of colorectal cancer.Overview of possible anticarcinogenic food components 23 33. Eur J Cancer Prev 1997. Methylation and colorectal cancer. Int J Vitam Nutr Res 2003. 41. 37.6:147–51.5:977–84. Surg Clin North Am 2002. Establishing the significance and optimal intake of dietary antioxidants: the biomarker concept. Limburg PJ. low-dose carcinogenesis and dietary exposures. Prentice RL.20:465–73. Arts IC. Moyad MA. Nutrition and cancer prevention: new insights into the role of phytochemicals. Kaaks RJ. Hawk ET. Neuhouser M. Bibl Nutr Dieta 2001. 35. Moyad MA. McCall MR. 44. A novel PSA reducer and preventive/treatment option for prostate cancer? Urol Clin North Am 2002. Wang CY.492:255–62. Assessment of nutritional status and fluid deficits in advanced cancer. Viner JL. 43. part I: time to redirect our attention? Urol Clin North Am 2004. 36. Bell SM. Kampman E.11:181–8. 46. Sugar E. 40. The therapeutic potential of phytoestrogens. Ocke MC. Plant foods versus compounds in carcinogenesis. 42.55:46–67. Christie R. Patterson R.31:289–300.428:91–8. Lifestyle/dietary supplement partial androgen suppression and/or estrogen manipulation. Lifestyle recommendations to prevent prostate cancer. Research strategies and the use of nutrient biomarkers in studies of diet and chronic disease.195:111–34. Wiseman H.29:115–24. Adv Exp Med Biol 2001. Am J Hosp Palliat Care 2003. Mahmoud FA. Georgiadis P. Biomarkers. Sarhill N. 45. 38.82:905–41. Biochemical markers as additional measurements in dietary validity studies: application of the method of triads with examples from the European Prospective Investigation into Cancer and Nutrition. Vineis P.73:70–8.57:104–13. Halliwell B.
Copenhagen. menadione and phylloquinones. these techniques took over and today retinol can be determined in serum routinely by direct. Due to worldwide concern for vitamin A deficiency (VAD). With the advent of high performance liquid chromatography (HPLC). the retinol binding protein (RBP) [1.1. Denmark Since antioxidant vitamins can affect an organism’s capacity for defence against reactive oxygen species. 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. which is advantageous from a collection standpoint. 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.2. xanthophylls. 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.2]. the development of fast and simple methods for the determination of vitamin A status has long been given a high priority.[3. 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]. such as pro-vitamin A carotenoids. has also been demonstrated . biological markers of the dietary exposure to these vitamins is of importance. . Vitamins and selenium 2. The direct-phase methods can also typically measure a large number of other lipid-soluble vitamin isomers in the same run. except for markers of DNA damage. The structure of vitamin A and pro-vitamin A carotenoids is shown in Figure 2. This chapter is concerned with such markers.1.4] or reversed-phase [5–7] liquid chromatography . C and E Lars O. Dragsted University of Copenhagen. tocopherols and tocotrienols. which are dealt with elsewhere in this volume. Biomarkers of exposure to and effects of vitamins A. although reversed-phase methods able to separate certain of the retinol isomers have been published . 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. a technique that was later superseded by various chromatographic and fluorescent techniques. The possibility of using dried blood spots.
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    
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 . 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 . 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 . 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 .
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 , 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.
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 . 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 , 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 . 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 . 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 .
Recovery is better than 98% with . There was a significant increase in the concentration of dehydroascorbate over time at low total vitamin C concentrations . around 0. Structures of ascorbate. allowing high sample handling rates to be achieved through automation . which can be photometrically measured by use of manual or automated equipment.1% of the total plasma vitamin C in non-smokers when the sample has been handled carefully. C and E 29 However. The HPLC methods for detecting plasma ascorbate electrochemically give results similar to those using UV detection . erythorbate. the stereoisomer of ascorbate.27–28]. since treatment with dithiotreitol is known to reduce dehydroascorbate to ascorbate.8% under the same conditions in the case of smokers . The colorimetric and fluorometric methods are generally based on redox-reactions. with ascorbate and dihydroascorbate leading to the formation of a chromophore or a fluorophore. but a few of them use assay blanks produced by adding ascorbate oxidase to the sample. A method for the simultaneous detection of ascorbate and uric acid by means of capillary zone electrophoresis (CZE) has also been described . Results in determining plasma ascorbate in this way correlate well with those obtained by use of chromatographic methods . and about 1. can be determined simultaneously [25. creating greater sensitivity with retention of speed. Fig. Postcolumn derivatization can be used to reduce dihydroascorbate so that it can be determined by use of an electrochemical detector. 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 . the ascorbyl radical and dihydroascorbate. Most of these methods are quite unspecific .Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. Some of these methodologies are very fast. this procedure cannot be recommended if both compounds are to be determined separately. Dehydroascorbate is not readily determined by use of this approach. possibly reflecting higher leakage of haem in this group. its true concentration seems to be very low.2. If dehydroascorbate is physiologically present. together constituting vitamin C. 2.
3. Typical performance of different methods for determination of plasma vitamin C Test method Reversed phase HPLC Capillary electrophoresis Automated colorimetry ND — not determined. which led to relatively larger relative errors being registered at low concentrations . In a European study of laboratories carrying out population surveillance. ex vivo LDL oxidation. Dragsted 30 use of the HPLC and CZE methods and good linearity is obtained even at low ascorbate concentrations. and since there is no generally accepted assay procedure . also within the same laboratory.3.3% < 5% CV% DHAA interday < 6% ND ND Limit of detection 0.39] 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.1 mg/l 0. 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 . to detect malondialdehyde. . This marker is highly controversial since it is variable both within and between laboratories. following HPLC separation. irrespective of the concentration. since it may partially measure aldehydes deriving from endogenous metabolism.5 mg/l 3 mg/l Speed (samples/day] 40–50 40–50 500 Reference   [38. Only randomised study designs are included in this review. Table 2. These flaws have caused some journals to generally regard the method as being invalid as a lipid oxidation biomarker . plasma lipid hydroperoxides. The performance of different methods for the measurement of vitamin C in plasma is summarized in Table 2. In interlaboratory comparisons. The most commonly used marker is determination of TBARS.Lars O. antioxidant capacity markers. 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. In another study an interlaboratory difference of about 15% was observed. The assays employed to this end here are the following: plasma thiobarbituric acid reactive compounds (TBARS). 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. either directly or online. depending on the method employed for hydrolysis. CV% AA interday < 4% 1. 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 %. with or without calibration. and plasma or urinary isoprostanes. whereas the intralaboratory variation was about 2 µmol/l.
given for 3 months. 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. in which isolated LDL is exposed to copper chloride or to a semistable radical such as AAPH.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. no change in whole plasma ex vivo oxidation was observed in this group . The interday CV for this latter assay is less than 10%. or a placebo in a parallel design. by a decrease in plasma MDA concentration . given in a normal formulation. No differences between groups in plasma malondialdehyde concentrations were observed either before or after supplementation . In addition. in which 19 smokers participated for 4 weeks following a 2-week . Overall it appears that the intake of vitamin C does not consistently affect plasma MDA but that significant increases may be observed following the infusion of large. Another controversial marker used by many laboratories is the ex vivo LDL oxidation assay. randomised double-blind crossover study the effect of vitamin C supplementation (six weeks. 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 . 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. as compared with placebo treatment. 25 males were exposed to vitamin C (500 or 1000 mg/d) and/or to exercise. 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 . vitamin C plus 182 mg/d dl-α-tocopherol. 30 mg beta-carotene and 100 µg organic selenium. 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 . Oxidative stress induced by Zn deficiency was found to respond to 250 mg/d vitamin C. In a smaller parallel study of vitamin C supplementation (1 g/d) versus placebo. acute doses. In a larger study involving 56 smokers. In a third study of this sort. No effect of either treatment on plasma MDA was observed . The method is disputed because the outcome depends on the antioxidants present in the LDL and these may be lost during LDL isolation. 182 mg/d dl-α-tocopherol alone. In another counterbalanced design. In another. The lag-time to oxidation and/or the slope of the oxidation curve are used as end points. In a group of 48 middle-aged male and female participants in a 36-month intervention study receiving 500mg/d of either vitamin C. 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 . but MDA to be unaffected by a slow-release formulation as compared with placebo treatment . 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. the intake of 500 mg/d of vitamin C was found not to affect MDA as determined by HPLC . the oxidative formation of conjugated dienes being followed spectrophotometrically at 234 nm . 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 .
At the same time. 60 mg or 6 g of vitamin C was administered daily during 14 day-periods separated . 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 . Overall there seems to be no consistent effect of vitamin C supplementation on ex vivo LDL oxidation kinetics. the availability of a photometer with exact filters or one equipped with a narrow grid being required. receiving only 60 mg vitamin C plus iron per day. in which 0 mg. However. No effect on LDL oxidation lag-times of 500 mg/d vitamin C supplementation for 4 weeks as compared with placebo was observed in 30 type II diabetics . A borderline effect on LDL oxidation was observed in a similar study with only 18 participants after a shorter period of 12 weeks . the size of the CVs depending on the exact wavelength used for determining the absorbance. In a subsequent study of 30 young smokers. These methods generally have interday CVs of less than 10% for repeated measurements of the same sample. especially for whole plasma. no effect was observed after 8 weeks supplementation by 1 g/d of vitamin C . usually one leading to a change in visual or UV absorption of the test matrix [52–55].Lars O. When applied to plasma samples some authors accordingly report poor correlation between methods  whereas others observed a high degree of correlation between some of the most commonly applied methods. 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 . The oxidizability of LDL is inherently an antioxidant capacity assay for this particular compartment. In another 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. There are important differences between the methods which call for caution when they are interpreted . They are all ex vivo oxidation systems composed of some oxidising system and some relatively simple marker of plasma oxidation.59]. 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 without a control group. 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). no such increase was observed despite changes in plasma ascorbate . or placebo. there was a significant increase in ex vivo LDL oxidation lagtime in the vitamin-supplemented group . decreased LDL oxidation or antibodies to oxidised LDL . Dragsted 32 ascorbate depletion period (≤ 30 mg/d). in a cross-over intervention study involving 48 non-smoking men and women. A variety of antioxidant capacity markers exist for other blood compartments. 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 . 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 .
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 . 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 . 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 . 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 . 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 . 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) . 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 . 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 . 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 . 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 . 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 . Improvements have included a combination of solidphase extraction cartridges and HPLC [76,77], two sequential solid-phase extractions  and most recently a single anion exchange cartridge . The derivatization of the compounds is most often done by use of pentafluorobenzyl bromide followed by a silylation step employing bis-(trimethylsilyl) trifluoroacetamide . 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 . 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 . 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 . 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 . 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 . 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  [42,43]    [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.
Using the same method for protein carbonyls. Vitamin C was found to decrease the exercise-induced increase in protein carbonyls in a dose-dependent manner . Following protein isolation by a rapid sizeexclusion chromatographic step and complete acid hydrolysis. 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. involving seven divers. 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 . including 150 mg/d vitamin C. the observed effect of vitamin C on this specific marker may reflect enzyme leakage from the cartilage rather than prooxidative stress. No effect of the supplement on protein carbonyls was observed . None of the volunteers had suboptimal vitamin C levels before intervention. A third method employed for assessment of protein oxidation takes advantage of the reaction of protein aldehydes with fluorescein and reduction of the product by cyanoborohydride to form a stable adduct. No effect of fruit. Another line of evidence comes from studies of dietary fruit and vegetable depletion. In a very small study. the influence of taking 500 mg/d versus 1 g/d of vitamin C for 2 weeks prior to 30 min of hard exercise was evaluated in 12 males using a counterbalance design. indicating that the depletion of ascorbate might have an antioxidant effect. whereas no change in the total plasma sulfhydryls was detected . A marginal increase in plasma protein AAS was observed (Dragsted. . 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 . fluorescence or APCI-MS detection . The decrease was confined to individuals with a suboptimal intake of vitamin C. The level of protein carbonyls in the immunoglobulins was found to decrease after both 10 and 15 weeks of supplementation. whereas no change was observed after supplementation of the known nutrients from the fruits and vegetables. unpublished data). 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. Since vitamin C is a cofactor for the lysine oxidases that cross-bind cartilage in the body. Use of this approach was found in one study to produce a significant time-dependent decrease in AAS during a 24-day period of fruit and vegetable depletion in the diet. A 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. showing a rapid depletion of plasma ascorbate and a concomitant decrease in AAS. 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. The main disadvantage is the longer time required for analysis and the higher technical demands. the fluoresceinamine adduct could be detected by HPLC through the use of UV. Dragsted 36 To assess oxidative stress by use of the simplest version of this assay.Lars O.
HPLC has the advantage of having lower detection limits. 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. and possibly possessing higher accuracy. including the susceptibility of erythrocytes . 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. Various functional tests have also been suggested. Conclusions Total plasma vitamin C can readily be determined by automated colorimetric assays or HPLC. of which RRR-α-tocopherol has the highest vitamin E activity.3. however. γ. Evidence for the prevention of stress-induced carbonyl formation by increasing the intake of vitamin C is controversial and needs further confirmation.and delta-tocopherols as well as the tocotrienols. vitamin C and chronic disease. none of the four S-forms of α-tocopherol and none of β-. whereas the colorimetric assays have much higher throughput. 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. permitting simultaneous determination of ascorbate and dehydroascorbate. 1 IU corresponding to 1. This is still controversial. but in humans vitamin E status is preferably determined as the plasma.49 mg RRR-α-tocopherol. that of the formation of plasma isoprostanes. no difference in the total vitamin E content of LDL was observed . The vitamin E status in rats and possibly in other rodents can be determined as a function of all the active isomers. gamma. The handling and storage of plasma for vitamin C determination has a strong effect on accuracy. beta-. 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. There are no studies currently available on the relationship between lipid or protein oxidation markers. Clear short-term pro-oxidant effects on lipid oxidation were observed following high-dose vitamin C infusion into the bloodstream. The relative vitamin E activity of the various tocopherols and tocotrienols in the rat together with their structures are shown in Figure 2. Since only the four R-forms of α-tocopherol (d-α-tocopherol) are recognised by the human vitamin E transporter in the body. Vitamin E Biomarkers for vitamin E in body fluids Vitamin E is a collective term for alpha-.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. serum or erythrocyte concentration of d-α-tocopherol. The reference for the international unit (IU) is 1 mg of all-rac-α-tocopheryl acetate. since in a study comparing the delivery of RRR-α-tocopherol and all-rac-α-tocopherol to lipoproteins in humans following 8 weeks of supplementation by 1600 mg/d of either product.and δ-tocopherols or of the tocotrienols are thought to have any vitamin E activity in humans as opposed to the rat.
Also. Fig. Vitamin E has a physiological transporter in the human that only binds d-α-tocopherol. In one study no correlation was found between results of the erythrocyte membrane stability test and plasma or erythrocyte vitamin E concentrations .Lars O. 2. since these functional tests may be lipid oxidation markers. Redrawn from . or the exhalation of breath pentane . and deletion of this transporter leads to neurological symptoms . . Structures of tocopherols and tocotrienols and their relative potency as vitamin E in the rat (TE. Dragsted 38 to lysis following an in vitro challenge with hydrogen peroxide [88–90]. dl-α-tocopherol equivalents).3. it seems more appropriate to evaluate the relationship of vitamin E supplementation to currently used lipid oxidation markers.
lipid hydroperoxides. giving a 900 IU dose of vitamin E was found to be no better than a placebo in decreasing MDA . No change in plasma MDA either within or between the two groups could be shown . antioxidant capacity markers. In a trial of lipid oxidation induced in 18 untrained men by three sessions of resistance exercises. given in a normal formulation. ex vivo LDL oxidation. 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). In a three parallel groups of 16 middle-aged male and female participants. 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 . C and E 39 Vitamin E and markers of lipid oxidation This review concerns only randomised. MDA was found to increase significantly following daily doses of 200 mg vitamin E combined with 250 mg ascorbate. In a randomised 2-months intervention study of 59 healthy smoking males. a significant decrease in plasma MDA was found in the vitamin-supplemented group as determined by HPLC . or isoprostanes (see desciption in the section on vitamin C above). no effect on the plasma MDA was detected . 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 involving 80 men showing an increase in plasma lipid oxidation from exposure to either olive oil or menhaden oil. . a reduction in plasma MDA occurred in the group assigned vitamin supplements . 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 . 30 mg beta-carotene and 100 µg organic selenium. 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. there was a significant decrease in erythrocyte MDA in the vitamin-supplemented group . a decrease in serum MDA was found in the group given vitamin E . In a double-blind. In 49 diabetic patients given either 504 mg/d d-α-tocopherol or a placebo for a 6-month period a decrease in ex vivo induced TBARS in the erythrocyte membranes was found for the supplemented group . controlled intervention study. 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 study of 49 HIV patients randomised to supplementation with a placebo or with 800 IU/d vitamin E and 1000 mg/d vitamin C for a 3-month period. In another study. no effect on exerciseinduced oxidative stress could be shown by serum MDA .Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. In a subsequent study of 24 diabetic children by use of the same design. In another study. 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. 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. who were given either 500 mg/d of vitamin C together with 182 mg/d dl-α-tocopherol. but to be unaffected by a slow-release formulation as compared with placebo treatment .
no difference was observed in LDL-MDA. In a study without a parallel control group. Non-induced lipoprotein oxidation ex vivo was found to be delayed in the group given the vitamin supplement. In a double-blind controlled intervention study of 56 patients with congestive heart failure. 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 . 900 mg/d vitamin C and 18 mg/d beta-carotene for 6 months were compared with those of a placebo. including IL-6 . or LDL-hydroperoxides induced ex vivo . this together with 182 mg/d dl-α-tocopherol. the effects of giving mixed supplements of 200 mg/d vitamin E. 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 . 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 . no effect on plasma antioxidant capacity was observed at 12 or at 36 months . In 49 diabetic patients given 504 mg/d d-α-tocopherol or a placebo for 6 months. 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. the treatment with vitamin E was found to significantly increase the level of plasma F2-isoprostanes as well as of several markers of inflammation. 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. In a trial involving 80 men who had an increase in plasma lipid oxidation following exposure to olive oil or to menhaden oil. 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. In a double-blind controlled intervention study of 56 patients with congestive heart failure. In a blinded intervention study of 33 triathletes participating in a world championship. . or placebo.Lars O. 182 mg/d dl-α-tocopherol alone. who were given 800 IU/d vitamin E or a placebo for 2 months prior to the race. Dragsted 40 182 mg/d dl-α-tocopherol alone. A significant change in these groups in whole plasma ex vivo oxidation at 36 weeks was likewise observed . and the strength of this effect was correlated with the level of plasma 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 dose-response study of 40 healthy men assigned doses of 60–1200 IU of vitamin E for an 8-week period. LDL-dienes. a 1000 IU/d vitamin E supplement for a 10 d period was found to reduce the exhalation of breath pentane . 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 . vitamin E given at doses higher than 200 IU/d was found to affect the kinetics of ex vivo oxidation of LDL . or a placebo in a parallel design. In a group of 45 randomised healthy males and females. 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 .
In an intervention study of 46 healthy smokers given 0. no effect on the plasma F2-isoprostane level of taking 1000 IU/d of vitamin E was found for 20 patients with endothelial dysfunction . irrespective of the treatment . Running as such was found to increase the plasma isoprostane level. particularly in the males. no effect of either vitamin treatment was observed. 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. 300. In a study without a control group. the adding of vitamin E was found to have no further effect on the urinary excretion of F2-isoprostanes . No other significant differences were observed between the two groups . In a study of the combined effects of giving either vitamin C (500 mg/d) together with d-α-tocopherol (400 mg/d). 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 . In a double-blind controlled intervention study of 56 congestive heart failure patients. In a small non-blind study of cirrhotic patients. simvastatin together with 600 mg/d vitamin E or a placebo for 2 months. 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 . This was not observed for the young men. or a placebo during the 6 weeks prior to the race. 800. In another double-blind placebo-controlled trial with a crossover design. but this was not changed by the vitamin supplements. groups of 5 healthy individuals received either 0. 400. no effect on the excretion of F2-isoprostane could be observed for any of these interventions . 600 or 1200 IU/d of vitamin E for 3 weeks. 200. No effect on urinary excretion of F2-isoprostanes was observed . The inflammatory markers were also affected by running. No significant change in the plasma F2-isoprostane level could be detected by GC-MS at any time point. In a dose-response study. In a group of 33 sclerosis patients. which may have caused the difference. followed by 8 weeks of washout. 1200 or 2000 IU/d of vitamin E for a period of 8 weeks. 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.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. In 43 hypercholesterolemic patients randomised to taking simvastatin. 21 ultramarathon runners were given either a combination of 300 mg/d d-α-tocopherol and 1000 mg/d vitamin C. 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 . 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 . or a placebo during a 28 d period on the plasma isoprostane concentrations induced by exercise. . vitamin C (500 mg/d) together with d-α-tocopherol (290 mg/d) and d-γ-tocopherol (130 mg/d). In a third study of the effects of vitamin E on oxidative damage induced by exercise. C and E 41 In another study. The study design could not control for period effects. and the vitamin supplement was found to prevent this . the rest being given a placebo. 10 of them were given 500 mg/d and 10 of them 1000 mg/d vitamin E for a period of 3 weeks.
Analytical methods. Craft NE. Swanson D. 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. 2nd ed.127]. p. Sperm counts and sperm motility are thought to be partially influenced by oxidative stress.131:1626S–30S. alpha-tocopherol and carotenoids (lutein. New York: Raven Press Ltd. alpha. 1994. Goodman DS. 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 . No evidence was presented that this effect was related to antioxidation. 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. Bui MH. Vitamin E deficiency is also known to cause semen abnormalities and infertility in rats. 3. Jakobsen J. Kalina RE. . In two randomised studies. Bawa AB. all-translycopene. Roberts AB. 4. 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. 179–209.Lars O. editors. chemistry. Simple determination of retinol. 5.154:201–10. In one study. rapid fluorometric determination of retinol in serum.Appl 1994. J Nutr 2001. and medicine. References 1. Cancer Lett 2000. 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. Innovative approaches to vitamin A assessment. Dragsted 42 Effects on protein oxidation In a study without a control group. The retinoids: biology. Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat.654:129–33. Invest Ophthalmol 1975. Furr HC. Olson JA. Conclusions In conclusion. A new. Daneshvar B. each with 30 men. Lauridsen ST.14:125–30. 2. In: Sporn MB. but this parameter was not assessed in the other study [126. J Chromatogr B Biomed. these proposed functional tests for vitamin E must be regarded as obsolete.and beta-carotenes) in human plasma by isocratic liquid chromatography. Since plasma vitamin E levels are not always correlated with the stability of the erythrocyte membrane or with the exhalation of breath pentane. Breinholt V. Futterman S. present day HPLC and GC-MS methodology has high precision and accuracy in the detection of vitamin E analogues in plasma. little effect of taking 600–800 mg/d of vitamin E for 2–3 months was shown.
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oxidative DNA damage needs to occur at a sufficiently high frequency to exceed the cell’s capacity for DNA repair. Ryszard Oliƒski3 1 2 3 University of Copenhagen. which can be produced both in the biochemical utilization of oxygen and as a by-product of O2 metabolism in the mitochondria. Not only is DNA mutation a crucial step in carcinogenesis. Denmark University of Leicester. though not generally classed as carcinogenic agents. are human carcinogens. together with their mutagenicity in mammalian cells. Marcus S. that metabolites of atmospheric oxygen. When mutations accumulate. In order to produce mutations. which are implicated in the development of cancer. this can be due to the individual’s overall lifetime exposure to endogenous and exogenous agents that damage the DNA. Nicolaus Copernicus University.Vitamins and selenium: Antioxidant vitamins and cancer risk 51 2. nevertheless.8-dihydroguanine (8-oxoGua).2. ROS. It is quite possible. can damage cellular molecules of different types — in particular proteins. Rafa∏ Ró˝alski3. Oxidative mechanisms have been shown to have a potential role in the initiation. One of the most widely studied lesions of this sort is 8-oxo-7. corresponding to about 2000 oxidative modifications of guanine per cell daily. increasing attention has been directed at oxidative DNA damage in relation to cancer. Poland The role of oxidative DNA damage in the development of cancer Cancer is a disease of the genes. promotion and malignant conversion (progression) stages of carcinogenesis. has been documented in aging cells and tissues. unless repaired prior to DNA replication. has led to the proposal that they be used as intermediate markers . Food and beverages frequently contain carcinogens. This. Peter Møller1. An increase in somatic mutations.and purine-derived lesions being formed in the DNA (as reviewed in ). It is of interest to note in this context that a urinary excretion study showed the average 8-oxoGua and 8-oxodG (7-hydro-8-oxo-2’-deoxyguanosine) excretion in the urine of healthy subjects to be some 2. The hydroxyl radicals that ROS creates result in a large number of pyrimidine. but the large numbers of oxidative DNA lesions observed in many tumours strongly suggests that such damage is frequently a cause of cancer . such as in the cooking of meat. Some of these modified DNA bases have considerable potential for damaging the integrity of the genome (reviewed in [2. so-called reactive oxygen species (ROS). The presence of 8-oxoGua residues in DNA.3]). lipids and nucleic acids.5 nmol per kg per day. Since the cumulative risk of cancer increases at a rate corresponding to the fourth power of age and is associated with accumulated DNA damage. and partly carcinogens that occur naturally in the products. Antioxidant vitamins and cancer risk: is oxidative DNA damage a relevant biomarker? Steffen Loft1. however. UK Collegium Medicum in Bydgoszcz. The presence of 8-oxoGua in the cells can thus result in point mutations. Lesions such as 8-oxodG are used as biomarkers of oxidative stress. partly those generated in processing. leads to GC→TA transversions . Cook2.
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. . regarding the question of ‘cause or consequence’. In addition. 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 nuclei of the undifferentiated. it has been argued that the mere presence of 8oxodG in DNA is unlikely to be a necessary and sufficient condition for tumour formation. connected with the spurious oxidation of DNA during sample preparation. or (ii) there being reduced DNA repair . Again. At the same time. Elevated ROS levels may activate transcription factors and the corresponding genes being permanently activated. but also the damage must be within a coding region of the DNA. In order for a mutation to come about. the levels of damage having no causal role in carcinogenesis. Oxidative DNA damage may be an epiphenomenon in relation to the ongoing pathophysiological process. Numerous studies have been concerned with the relationship between the level of oxidative DNA damage and cancer (see  for a review). proliferating stem cells have to be affected.2 lesions per 106 unaltered guanines and that reports of values outside this range should be viewed skeptically . Rafa∏ Ró˝alski. Ultimately. This raises a number of different issues: 1. the analytical procedures in current use are unable to quantify the lesion levels found in the most important ‘target’ cells. 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 . although their suitability for this has yet to be verified in prospective studies of cancer risk. Although studies that indicate this support the hypothesis that oxidative DNA damage can be an important risk factor for carcinogenesis. 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. Since tissue samples from both tumours and normal cells represent a heterogeneous mixture of differentiated and undifferentiated cells (the former being likely to predominate). such as a high level either of metabolism or of cell turnover. For DNA mutations to arise through oxidative damage.Steffen Loft. creates a selection pressure for the malignant phenotype seen in the case of cancer. Ryszard Oliƒski 52 of a potential disease endpoint such as that of cancer. not only must the DNA of the target cells be affected. Peter Møller. This. An elevated level may be due to some well-established characteristic of the tumour. Issues of this sort have to be addressed before a causal link between oxidative DNA damage and cancer can be established. 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. 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. Cook. Marcus S. 2.3–4. there are serious problems in the chromatographic measurement of 8-oxodG. together with the increase in DNA damage that occurs. 3.
large-scale intervention studies have failed to demonstrate use of antioxidant vitamins to reduce the risk of cancer . 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. used for the detection of DNA strand breaks (SB). but that it is impossible at present to determine to what extent oxidative stress is directly involved in carcinogenesis. Although these events may come about by way of different mechanisms. At the same time. supplementation trials would appear to represent the most relevant way of exploring antioxidant effects. Intuitively. One should bear in mind. although sampling is usually restricted to use of such surrogate tissues as white blood cells (WBC) and urine. nevertheless. 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. . It is very difficult. It is reasonable to assume that agents that decrease oxidative DNA damage should also decrease the subsequent development of cancer. Subsequently. this soon being followed by the performance of antioxidant trials in which urinary 8-oxodG excretion served as the key biomarker.12]. altered gene expression and mutations are necessary elements in the process of carcinogenesis. since full development of the disease in response to exposure to a carcinogen may take 20–40 years. A plethora of descriptive epidemiological studies have concerned a possible protective effect of a diet rich in fruits and vegetables . These antioxidants are effective free-radical scavengers and should protect the DNA from oxidative damage. At the beginning of the 1990s. C and E or phenolic compounds. Reliable detection of urinary 8-oxodG excretion became possible at about the same time . oxidants are involved in each case. Nevertheless. 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. the comet assay. the mode of action of dietary micronutrients is complex and is far from being fully understood. that DNA damage. Both experimental and epidemiological data indicate vitamin C to protect against both stomach and oesophageal cancer . to establish directly that the DNA lesion responsible for a carcinogenic process is the lesion present in a tumor many cell generations later.Vitamins and selenium: Antioxidant vitamins and cancer risk 53 Summing up. 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). have become by far the most popular assays for use in conjunction with antioxidant intervention trials. therefore. however. as the possibility of detecting 7-hydro-8-oxo-2’-deoxyguanosine (8-oxodG) appeared. the first of many antioxidant supplementation trials dealing with this lesion in WBC was carried out . Dietary antioxidants as inhibitors of oxidative DNA damage and as a factor decreasing cancer risk There is widely believed to be a link between diet and the incidence of cancer. respectively.
Rafa∏ Ró˝alski. however. 280 mg vitamin E.Steffen Loft. Ryszard Oliƒski 54 Many studies suffer. Peter Møller. 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. Table 2. but an increase in excretion during the washout period 14 30 Vitamin C (500 mg) for 3 wk 30 MF (NS) 24 Six groups receiving combinations of vitaminsd for 2 mo Vitamin C (1 g) and vitamin E (0.6 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 . Marcus S. from use of a non-optimum design.6. 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. and 25 mg β-carotene) for 20 wk Multi-vitamin (250 vitamin C. Table 2. 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. Cook. 200 IU α-tocopherol. but a decline in the course of the trial in both groups 32 MF (NS) 32±11 No effect compared with baseline. 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.6.
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. No effect on the 24-h urinary excretion of 8-oxodG (HPLC). Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont. but a decline in all the groups 21—45 No effect on FPG sites (Comet) in WBC 19 20 18 MF (NR) 14 MF (NS) 10 MF (NS) 10 M (NS) 15 26—54 A decrease at the ENDOIII and FPG (Comet) sites 22 Brussels sprouts (300 g) for 1 wk NR No effect on the 24-h urinary excretion of 8-oxodG (HPLC) A decrease in the 24-h urinary excretion of 8-oxodG (HPLC) in the active group 40 Brussels sprouts (300 g) for 12 d NR 41 Fruit juice (480 ml)j for 4 d 11 M (NR) 21±1 A decrease in the 12-h urinary excretion of 8-oxodG (ELISA) in the active group 57 MF (6S) 19—52 No effect on ENDOIII and FPG sites (Comet) in WBC 26 Parallel study of blackcurrant juice or anthocyanin drink (475—1000 ml) for 3 wk Green tea or black tea (4 cups) for 1—4 mo 18 120 MF (S) 18—79 A decrease in the excretion of 8-oxodG (ELISA) in spot urine samples in the green tea group after 4 mo.6. but not earlier 68 MF (13S) 18—45 A decrease in the 12-h urinary excretion of 8oxodG (HPLC) 27.Vitamins and selenium: Antioxidant vitamins and cancer risk 55 Table 2. 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. 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.
12].2 mg). Supplement consisting of β-carotene (6 mg). Tablets containing β-carotene (12 mg).Steffen Loft. a protective effect could be shown after only 20 weeks of supplementation but no effect after 10 weeks (or 6 months of supplementation) [13. Ryszard Oliƒski 56 Table 2. orange. whereas tocopherols and carotenoids exert their effects somewhat longer. spinach.16]. and zinc (30 mg).6. α-carotene (1. Tablets containing micronutrients similar to those of 3 fruits and 3 vegetables (containing 107 mg vitamin C and 83 mg vitamin E). the question of whether multiple vitamin supplementation provide better protection against oxidative DNA damage than a single dose does cannot be answered unambiguously. 4 mg β-carotene). and pomegranate extract (5 mg). beet. This suggests that the protective effect of a single antioxidant is relatively short.5 mg). Rafa∏ Ró˝alski. Supplement given per day Subjectsa Age (yr)b Effect Ref Soy milk. selenium (167 mg). selenium (100 mg). The vegetable capsules were made from carrots. capsules (containing 90 mg vitamin C. 3) 500 mg slow-release vitamin C. being given rye crisp . Age is shown as range or as mean ± standard deviation.14]. Among studies in which multiple doses of single antioxidants are given. lycopene (4.12]. 150 mg vitamin E. cranberry and peach.4 mg). 18 IU vitamin E. Containing vitamin A (240 mg). 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. or a carotenoid-rich diet.4 mg). 4) 90 mg coenzyme Q10 in oil. The fruit capsules were made from juiced apple. respectively [15. and tomato. vitamin E (1. lutein (4. 60 mg vitamin E. vitamin C (500 mg). 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. Consisted of β-carotene (20050 IU). there are fewer that reveal protective effects than those that report only negative results [9. Accordingly. Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont. and papika carotenoids (2.44 mg) per day. papaya. N-acetyl-1-cysteine (181 mg). The daily dose consisted of 200 mg vitamin C. possibly because of differences in the respective rates of bioavailability and elimination. parsley. however. The effect of a single dose of vitamin C seems to disappear after several hours. Peter Møller. pineapple. 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). Cook. bixin (11. lycopene (100 mg). lutein (500 mg). broccoli. rice milk. vitamin C (219 mg). Consisted of tablets containing vitamin antioxidants (400 mg vitamin C.4 mg β-carotene. 6) placebo. kale. and powder or extract of fruits and berries). tocopherols (650 IU). In two well-designed studies of the effects of administering a combination of antioxidant vitamins. vitamin E (400 IU). The six groups received daily supplements of 1) 200 mg vitamin E. and 15 mg β-carotene. 2. catechin (13. vitamin C (330 mg).5 mg/10 MJ). Smokers (S) and nonsmokers (NS) are indicated in brackets. Marcus S. 5) 30 mg coenzyme Q10 granulate. Consisted of 1000 mg extract/kg body weight in meat patties (total phenolics 23. Also. cabbage.2 mg).7 mg). β-carotene (399 mg). 2) 500 mg plain-release vitamin C.
no appreciable difference was present in terms of duration of the intervention period. The other. a cross-over study of male smokers. 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. there in fact being a tendency in the group of subjects drinking black currant juice for the number of FPG sites to increase . 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. whereas 13 studies reported null effect. was negative with respect to effects on the ENDOIII and FPG sites . assessment of the DNA damage occurring showed the levels of ENDOIII to be reduced . An overall summary of the studies showed that six investigations reported beneficial effect of antioxidant supplementation.Vitamins and selenium: Antioxidant vitamins and cancer risk 57 bread (76. 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. The one investigation. unintentional intoxication of the gastrointestinal tract (some of those in the active groups complained of nausea. For 24 studies of the effects of antioxidant supplementation on the urinary excretion of 8-oxodG in which a controlled design was employed [20. 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. . Two studies concerned the effects of an intervention of providing vegetables and fruits. Anthocyanines have low bioavailability and the dose provided here was rather high. The only study showing consistent effects involving more than one endpoint was a study of the effects of kiwi fruit supplementation (1–3 kiwi fruits/d for 3 wk).23–44]. and their effects in the gastrointestinal tract are easier to comprehend.or FPG-sensitive sites. showed no effect on the ENDOIII sites of ingesting 500 g/d of such food for 3 wk . 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. for example). which showed the numbers of ENDOIII and FPG sensitive sites to be reduced . There is little support for the notion that ingestion of antioxidant-rich foods is associated with lower spontaneous level of oxidative DNA damage in WBC than intake of single antioxidants. or to have any effect on the ENDOIII sites . 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. It can be speculated that the subjects suffered a slight. number of subjects. or power to detect a 50% change between these studies reporting beneficial effects and those reporting no effects.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.
38.44. bixin (11. In the subsequent study.39]. 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 . tea. On the other hand. 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 . the Brussels sprouts supplementation was found to lower the urinary excretion of 8-oxodG . whereas this intervention was found to have a pronounced period effect . Investigations of the effects of natural food products are distributed about equally between studies reporting beneficial and those reporting null effects.5 mg). cabbage. In the first study.45]. A positive effect of vegetable juice consumption on 8-oxodG excretion was also observed in subjects enrolled in a soccer summer training camp . kale.Steffen Loft.e. there being a tendency for only the male subjects to benefit from ingestion of Brussels sprouts.7 mg).41]. spinach. fruits. cranberry. Rafa∏ Ró˝alski. Peter Møller. beet. Further studies showed multi-vitamin tablet supplementation to provide no beneficial effect either in normal subjects [20. In four additional 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. parsley. the effect was less clear.34. pineapple. 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 comparable study involving supplementation of a mixture of carotenoids (daily intake: α-carotene (1. Marcus S. Mixed results concerning the effects of a dietary supplementation of Brussels sprouts (300 g/d) were obtained in two studies [40. orange.4 mg). neither supplementation of vitamin C or vitamin E or a combination of them was found to have any effect in healthy subjects [24. In the one study a beneficial effect was found for drinking 300 ml/d for a week . No effect on the urinary excretion of 8-oxodG was found for the ingestion of capsules containing juices and powders of fruit (apple.2 mg)) revealed a statistically significant difference between the active and the placebo group in the delta values obtained (i. in which both sexes were included.32. 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. whereas in one of the other two studies . 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 . that involved only male subjects.37] or in subjects undergoing cold-weather field training at a moderate altitude [33. lutein (4. and peach) and of vegetables (carrot. the difference between results at the end of supplementation and at baseline) but no difference between the two groups at baseline .0 mg). lycopene (4.28. β-carotene (6.35. broccoli. papaya. and vegetables. A number of studies have involved supplying antioxidants in the form of berries. Cook. Three additional studies in this area concerned the effect of drinking green tea [see 27.36]. Taking capsules containing extracts of fruits and berries and eating diets rich in carotenoids were both found to lower the excretion of 8-oxodG . and paprika carotenoids (2.31. and tomato) .4 mg).35].
5. adjustment of the data for a number of variables. including baseline 8-oxodG levels. could increase the likelihood of detecting a protective effect. . 2) the putative active constituents (isoflavones) were not measured in the plasma. Under some circumstances. 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 . 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.Vitamins and selenium: Antioxidant vitamins and cancer risk 59 no effect of ingesting green tea extract in meat patties for 3 wk was obtained . 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.45]. therefore. It is possible. Interestingly. and 3) the alterations in the urinary concentration could not be assessed due to insufficient information . that the presence of oxidative stress. which leads to iron overload. drinking soya hypocotyl tea was found to be associated with a lower urinary excretion of 8-oxodG. It is possible that a preventive effect of the vitamins can be only be seen when their basal levels are very low. It is worthy of note in this context that presumably healthy men may have the hereditary disease idiopathic haemochromathosis. revealed a statistically significant positive effect of drinking green for 4 months [27. Indeed. antioxidant vitamins may have biological activities. although it should be emphasized that the fate of the antioxidants in this investigations was inconclusive since 1) the plasma concentration of carotenoids decreased. that are separate from their direct antioxidant effects . 2. Paradoxically. 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. The absorption of non-haem iron has also been found to be affected by ascorbic acid . such that iron catalytic to free-radical reactions (a so-called labile iron pool — LIP) is present in the blood plasma ). In still a further study. a positive correlation has been shown between LIP and the oxidatively modified nucleoside in human lymphocytes . Although the unadjusted data of the third study indicated no beneficial effect of drinking green tea. 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. which might fail to be recognized. 3. 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. Experimental data suggest that supplementation of vitamin C to iron-overload subjects may increase the oxidative DNA damage that occurs . such as those of regulating changes in gene expression. 4.
such as those involving bulky DNA adducts. hundreds of subjects would be needed. whereas the protective effect of antioxidants may be more easily detected in subjects who are suffering from oxidative stress. Although single studies may possess considerable power due to specific aspects of their design such as use of repeated measurements. however. to obtain significant results. It should also be borne in mind that the majority of studies involve healthy individuals. There is a tendency for supplementation with use of antioxidant-rich food to decrease the urinary excretion of 8-oxodG. yet the use of WBC as a surrogate tissue may not be particularly well suited for detection of this effect.Steffen Loft. It should be noted. The most likely effect ratio in healthy subjects involves a difference of less than 10%. There is an obvious need for controlled antioxidant intervention studies encompassing subjects who are oxidatively stressed and in whom oxidative DNA damage is measured by enzymic or chromatographic methods. Cook. Also the chemopreventive effect of antioxidants on non-lymphatic tissue. Hopefully. At the same time. particularly as regards the need of proper investigative designs and of the validity biomarkers. which are of pivotal importance for such studies. no firm conclusions can be reached on the basis of antioxidant intervention studies. Marcus S. most studies encompassing far fewer subjects than this. which means that. such studies will benefit from the lessons learned from antioxidant intervention studies. Peter Møller. . should be addressed more thoroughly before the idea of antioxidants having clearly beneficial effects is abandoned. more attention should probably be devoted to alternative chemopreventive mechanisms such as the upregulation of DNA repair systems. many of the studies performed are of only limited value due to methodological problems related to the study design and the assays. which has been only sparsely investigated. which is not the case with use of single antioxidants. which is considered to be the least effective form of antioxidant supplementation. 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. it is easy to understand the ingestion of antioxidants being associated with lower levels of oxidative DNA damage. On an experimental basis. although there may be a beneficial effect in the first few hours after ingestion. WBC studies provide little support for the idea that long-term antioxidant supplementation lowers the basic level of oxidative DNA damage. In the future. that most of the studies reported have used supplements of vitamin E. Rafa∏ Ró˝alski. At present. This can be interpreted as antioxidants having an overall beneficial effect on the body as a whole. 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. and to other types of DNA damage. a major problem is that many of these studies have too few subjects. and many of them lack the power of detecting 50% differences between two separate groups. 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.
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to lipids. Such matrix effects can markedly diminish the validity of assays carried out .25-(OH)2D or calcitriol) occurs primarily in the kidney (1α-hydroxylase). strong efforts have been made to simplify them for routine use .8]. Since the time of first assays in 1971. One of the major problems in measuring 25-(OH)D lies in the molecule itself. As compared with 25-(OH)D.Jakob Linseisen. e. is rather short (24 h in the blood circulation). or cholecalciferol. In addition. Because of its hydrophobic nature. 25-(OH)D2 and 25-(OH)D3.25-(OH)2D whereas 25-(OH)D has only limited biological activity [1.25-dihydroxycholecalciferol (1. which is the major circulating metabolite. Heidelberg. vitamin D (cholecalciferol). the half-life time of its precursor. Although such measurement is very accurate. There is overall agreement that 25-(OH)D is the appropriate biomarker to measure vitamin D-status in humans.). Because of its serum half-life time of about three weeks . The biologically most active vitamin D metabolite is 1. it has to be performed by experienced personnel. First of all it is a very hydrophobic compound and secondly it exists in two forms. this metabolite is a good indicator of the vitamin D-stores obtained both from exposure to sunlight and ingestion of vitamin D .2]. Vitamin D is metabolised by hepatic 25-hydroxylase into 25-hydroxycholecalciferol (25-(OH)D). Determination of 25-(OH)D As reflected in the recent scientific literature. Considerable interest has developed in assays for measuring 25-(OH)D. the determination of circulating 25-(OH)D is not an easy task . Plasma 1. is synthesized in the skin. Its precursor.g. Extraction from the matrix (serum or plasma) is thus required for most analytical procedures (see Table 2. In the blood vitamin D and its metabolites are bound to the vitamin D binding protein. is also not regarded as a suitable biomarker of vitamin D status . Further hydroxylation to 1. the circulating 1. since its conversion from 25-(OH)D is also tightly regulated. Measurement of serum 25-hydroxycholecalciferol as marker of vitamin D status Jakob Linseisen and Sascha Abbas DKFZ. Measurement by means of HPLC is considered by most to be the gold standard here [6. with an even shorter serum half-life time of 4–6 h.25-(OH)2D.25-(OH)2D level does not provide valid information on the individual’s vitamin D status . 25-(OH)D measurements are quite vulnerable to matrix effects. Cancer Epidemiology. 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. arising from the irradiation of ergosterol). requires expensive equipment (HPLC) and is more time-consuming than other . 7-dehydrocholesterol.7. its thus being more strongly affected by very recent sun exposure or dietary intake of vitamin D .3. Germany Vitamin D3. is converted by UV light from the sun (UVB 290–315 nm) into previtamin D3 which slowly isomerizes to vitamin D3. Sascha Abbas 64 2.
7.plasma nostics or urine 500 µL 8—312 2. Radioimmunoassays (RIA).2 8. HPLC — high-performance liquid chromatography. as well as being fast. however.0 5. 25 (OH2) D.5—300 18 6. ELISA — enzyme-linked immunosorbent assay.25-dihydroxyvitamin D. 50 µL Extraction detection (nmol/l) Acetonitrile Sensitivity Intraassay Interassay Assay time** 2 h. Sample type Manufacturer and volume* RIA. requires luminometer Comments (nmol/l) CV (%) CV (%) ≤6 <8 <12 RIA. RIA — radioimmunoassay. There are important differences in the performance of the various detection methods. IDK Serum 500 µL 17. 0 min ELISA. CV — coefficient of variation. or plasma Nichols 20 µL Institute Diagnostics HPLC. Serum. 0 min ChemilumiSerum nescence. and are thus more frequently used.5 7.3—250 2. Table 2.25 (OH2)D The primary antibody is the vitamin D binding protein from serum Fully automated. enzyme immunoassays (EIA) and chemiluminescence assays (CLPBA) are easier to handle.4 9 11 5 h.2 75 min Acetonitrile and C18 cartridge extraction Acetonitrile 15—150 4. no yield determination required No radioactive waste. ** Does not include extraction. CPB — competitive protein binding.7. Commercially available 25-hydroxyvitamin D assays  Range of Test principle.4 20 min The laboratory must have an HPLC unit with a silica column CPB. Immundiag. 0 min ELISA.5 9. 24. summarizes some of the performance characteristics of most of the assays that are available commercially.9 3 h.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 65 methods. counting or microplate reading times. . IDS Serum or plasma 50 µL Two-step reagent extraction 4—400 ≤5 5. no yield determination required Calibrators and controls in the serum matrix. 10 min Uses vitamin D binding protein * Represents the initial starting volume of the sample. Biomedica Proprietary extraction reagent Unknown 6. 10 min Calibrators and controls in the serum matrix.9 14 1 h. 100% cross-reactivity with 24. 24. DiaSorin Serum or plasma. Table 2. IDS Serum or plasma 50 µL Serum or plasma 50 µL None 6—360 ≤5 <6 <9 3 h.6 11.
1 to 35. Sascha Abbas 66 Validity of different assays Only few studies have compared the different commercially available assays [9–13].4. Binkley et al. On the basis of their results. Mean (SEM) results for the serum 25-(OH)D concentrations for 10 subjects as estimated by six different laboratories (for abbreviations see Table 2.5.  recommended the use of RIA techniques for 25-(OH)D measurement until other methodologies have been developed further. RIA — radioimmunoassay. 2.8. the serum of 10 subjects was sent to six different laboratories.Jakob Linseisen. .6 ng/ml (p < 0. Table 2. HPLC — high-performance liquid chromatography. Binkley et al. lists the methods and the normal range for 25-(OH)D measurement in the laboratories that participated. Table 2.  reported recently not only immense variation between different assays but also variability between different laboratories in using a given assay. The means varied widely between laboratories from 17.4.8. 25-(OH)D assay methodology and the normal range employed in different laboratories  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.). After spiking of the serum samples with a defined quantity of 25-(OH)D. and the proportion of subjects below an arbitrary threshold of insufficiency (32 ng/ml) varied between 17 and 90%. Fig.).).8. The mean serum 25-(OH)D concentrations differed 2-fold between laboratories (Figure 2.005) . a broad range (17–95%) of the expected concentration was found by the different laboratories  (Figure 2. In their study.
13].Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 67 Fig. According to the latest DEQAS (Vitamin D External Quality Assessment Scheme) report. 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)D2.8). Patients. the authors of the report state that the validity of the 25-(OH)D results that are obtained will justifiably continue to be questioned. 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) . 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 . especially those in the US. However. There has been discussion regarding the importance of the assays detecting 25-(OH)D2 as well as 25-(OH)D3. finding discordant serum 25-(OH)D-values.6 ng/ml (p < 0.4 to 44. 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). The means varied widely between laboratories from 27. Figure 2. These give the impression that only method 6 exceeded the limit. Mean (SEM) results for the 25-(OH)D concentrations in the serum samples of 10 subjects spiked with the 25-(OH)D standard. . In accordance with these findings.005) from 7. 59% of the participating laboratories were found to have met the target [12. 2.6. This suggests that the contribution of 25-(OH)D2 to the overall 25-(OH)D supply is less important than had been previously thought. The increment was less than anticipated and varied between laboratories (p < 0.5. However. as estimated by five different laboratories (for abbreviations see Table 2. 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 . Each of the laboratories was given five serum samples quarterly. the greatest increment being detected by HPLC .7 to 18 ng/ml. Another study compared the DiaSorin RIA assay with the Nichols chemiluminescence assay. several authors have called for international standardization of the vitamin D assays that are available and that are affordable for practicing clinicians. however. Both assays also underestimated the 25-(OH)D-concentration as compared with the HPLC-method . shows the latest results of this external quality control project.05).
. but there may also be a significant proportion of the population that exhibits asymptomatic subclinical vitamin D insufficiency. Accuracy of 25-(OH)D methods used by the different DEQAS participants . method 3. dietary intake. Sascha Abbas 68 Fig. that set the cutoff points at much lower levels. especially the home-bound elderly. HPLC. competitive protein-binding assay. epidemiological variability needs to also be taken into account. A summary of studies on vitamin D status in the elderly is given elsewhere . race. Each column shows the mean deviation (% bias) and SE from the target value (ALTM) during the period of 2000–2004. season. Populations at risk include nursing home residents and the elderly. and the composition of the population being studied.Jakob Linseisen. around 25–40 nmol/L [1. IDS EIA.6. due to the serious health risks encountered at levels less than this . In addition to the high degree of uncertainty that the divergence of the results for different techniques and different laboratories creates. Nichols automated chemiluminescence assay. DiaSorin RIA. Recent reports suggest European and North American populations to have a higher degree of insufficiency than had previously been thought [15. method 4. suggested cut-offs for insufficiency range from 10 to 30 ng/ml (25–80 nmol/l). Apart from the analytical problems. 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. IDS RIA. age. In a recent publication. It is important to note that “normal” is not defined as the median population level. Hollis defined 25-(OH)D levels below 80 nmol/L as being deficient. method 6.18–23]. the serum 25-(OH)D concentrations in different populations vary with latitude. In epidemiologic studies. method 5.18].16]. There is growing evidence that there is not only a specific seasonal decline in serum 25-(OH)D during the winter months. method 2.15. as shown by the seasonal variation in vitamin D status that occurs [17. This is in contrast with most of the published studies. 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 1. The method means of samples known to contain 25-(OH)D2 were excluded. 2.
J Clin Endocrinol Metab 1978.89–90:611–4. The measurement of vitamin D: analytical aspects. 4. Cowgill CS. 5. Plum L. 3. vitamin D3). Zhou XY. DeLuca HF. Potts JT. et al. Overview of Vitamin D Measurement and Methodologies. Krueger D. Hansen KE.40:546–51. Clin Chem 2000. Wilz DR. J Clin Endocrinol Metab 1971. Immunodiagnostic Systems (IDS). Review SeriesVolume 2. Hollis BW. DeLuca HF.63:656–60.46:1657–61. J Clin Endocrinol Metab 1986. Caldas AE. References 1. McGuiness M. Myles M.89:3149–51. Endres D. Comparison of commercially available (125)I-based RIA methods for the determination of circulating 25-hydroxyvitamin D. May 2005. Belsey R. Ann Clin Biochem 2004.135:317–22. Lake E. Issues of methodology. J Nutr 2005. Epidemiologic studies in particular can contribute knowledge concerning the association between vitamin D insufficiency and the risk of disease. epidemiologic variation in vitamin D status and its determinants. 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. Lips P.46:756–65.25-(OH)2-vitamin D3 in healthy adults. Nonetheless. Hollis BW. This makes strong scientific research efforts needed. Hart GR. .Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 69 Conclusions In conclusion. Zerwekh JE. et al. Metabolism and excretion of 3H-1. Hollis BW. Noble JM. Musk AA. Several problems are important to bear in mind including the different forms of vitamin D (vitamin D2. vitamin D metabolites have various important biological effects. Binkley N. 7. and problems in defining the appropriate threshold for vitamin D sufficiency. J Clin Endocrinol Metab 2004. 2. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. 9. Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects. J Clin Endocrinol Metab 2004.89:3152–7. Clemens TL. Lemann J. Editorial: The determination of circulating 25-hydroxyvitamin D: no easy task. Gray RW. 11. 6. 8. Ann Clin Biochem 2003. Taranto M. Lindsay R.33:554–7. including the risk of cancers at various sites. Goldswain PR. Smith GA. Glendenning P. 10.41:272–81. the determination of vitamin D status is still a challenging task. the measurement variation introduced by different assays and different laboratories. Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D. Which circulating level of 25-hydroxyvitamin D is appropriate? J Steroid Biochem Mol Biol 2004. Competitive binding assay for vitamin D and 25-OH vitamin D.
J Clin Endocrinol Metab 1998. Galan P. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. Leidig-Bruckner G. Fenske JS. apparently healthy urban European population. Jones J. Body JJ. Chapuy MC. 18. Seasonal variation of biochemical indexes of bone turnover: results of a population-based study.89–90:621–2. Miller AB. 25-dihydroxyvitamin D in postmenopausal women.135:332–7.89–90:467–71. 19.59:57–63. 21. Am J Clin Nutr 2000. Davison KS. J Nutr 2005. Carter GD. 14. with particular reference to European countries. 22. Makin HL. Alsaker E. Prevalence of vitamin D inadequacy among postmenopausal North American women receiving osteoporosis therapy. Heaney RP. Armas LA. Lund E. Hanley DA. Brustad M. Carter GD. Norway: the Oslo Immigrant Health Study. Vitamin D status of middle-aged women at 65–71 degrees N in relation to dietary intake and exposure to ultraviolet radiation. Hercberg S. Vitamin D status: effects on parathyroid hormone and 1. MacFarlane GD. et al. Geographical differences in vitamin D status. 15. Maamer M. Kissling C. Need AG. Khan A. Brunvand L. Engelsen O. Haug E. 17. Jones J. Hypovitaminosis D in a normal. 13. et al. Sascha Abbas 70 12. Grauer A.60:658–9. Arnaud S. Horowitz M.89:5387–91.7:327–35. Beard MK. Prevalence of vitamin D insufficiency in an adult normal population. Scheidt-Nave C. Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation. Berry J. Andersen R. Nordin BC. Ovesen L. et al. Siris ES.Jakob Linseisen. Clin Chem 2004. Jakobsen J. Osteoporos Int 1997. Sackrison JL.62:813–21. Morris HA. Jones G.50:2195–7. Meyer K. Woitge HW. J Steroid Biochem Mol Biol 2004. Binkley N. Gunter E. et al. Vitamin D2 is much less effective than vitamin D3 in humans. Holvik K. Prevalence and predictors of vitamin D deficiency in five immigrant groups living in Oslo. 23.7:439–43. Carter R. Proc Nutr Soc 2003. 20. Public Health Nutr 2004. Katzer JT. Carter CR. Hollis BW. Vitamin D insufficiency in North America. Obstet Gynecol Surv 2005. 16. Metab 2004.83:68–75. Aksnes L.71:1577–81. . Ersfeld DL. Preziosi P. Eur J Clin Nutr 2005. J Clin Endocrinol. Holick MF. Meyer HE. J Steroid Biochem Mol Biol 2004.
Different forms of selenium With few exceptions. their functions and mechanisms of action. and methods employed in assessing an individual’s selenium nutritional status — both generally. nearly all of the selenium in animals. plants and microorganisms is bound within proteins. Selenium and cancer — selenoproteins as biomarkers in relation to selenium supplementation Björn Åkesson and Katharina Bruzelius Biomedical Nutrition. together with epidemiological findings on relations between the selenium status in the body and risk of cancer are reviewed. and the use of intervention trials to study the cancerpreventive effects of selenium supplementation. several low-molecular-weight selenium compounds have been shown to be present in different foods. the mechanisms of action involved. containing the amino acid selenocysteine (Sec. and Department of Clinical Nutrition.). it is incorporated non-specifically into proteins as an analogue of methionine . Other proteins can also bind selenium as a ligand . These include the forms of selenium present in the body and in the diet. The replacement of methionine by selenomethionine appears to be random and to be dependent on the relative concentrations of these amino acids . so-called selenoproteins.). When ingested by humans or other organisms.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 71 2. . Another selenium-containing amino acid is selenomethionine. but little is known of the role it has here. Lund. In the diet. and in epidemiological studies of the risk of cancer.9. Sweden Introduction The relationship between selenium and cancer involves many different factors. Selenite and selenate are inorganic forms of selenium used as dietary supplements. several protein-bound forms having been identified. some of these compounds being uncharacterized [4–6]. A variety of issues connected with this are reviewed in two of the chapters. Lund University. Pure and Applied Biochemistry. The present chapter concerns the various forms of selenium and their metabolism. as well as in connection with long-term trials for investigating the disease-preventive potential of selenium supplementation. selenoproteins are largely found in animal foods. which is synthesized by plants and by yeast.4. It is uncertain whether they occur naturally in foods to any major extent. In addition to the forms of selenium mentioned. In the chapter thereafter (Gromadziƒska et al. Lund University Hospital. the occurrence of different selenoproteins used as biomarkers of selenium status. A major part of the selenium in mammals is specifically incorporated into proteins of defined biological function. analogous to cysteine in which sulphur is replaced by selenium) (Table 2.
Selenomethionine and selenocysteine can also be converted to hydrogen selenide. 2.7. Several compounds can be converted into methylselenol (from ). Selenomethionine. selenium is removed as dimethylselenide via exha- Fig. The major selenium metabolite excreted in urine is selenosugar. which is turn is the precursor of selenocysteine in selenoproteins and various excreted forms of selenium. Metabolic pathways of selenium. At toxic doses.7.9. selenocysteine and selenite can be converted into the key metabolite hydrogen selenide (H2Se). dimethylselenide or trimethylselenonium ions for excretion.). Selenate and selenite are reduced by glutathione to hydrogen selenide. .Björn Åkesson. a much lesser amount being excreted as trimethylselenonium ions . 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). Katharina Bruzelius 72 Table 2. 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.
10.) [1.9]. mutations in gene associated with muscular diseases Unknown An antioxidant and selenium transport protein Reduces methionine-R-sulfoxides A membrane protein.10. such as Se-methylselenocysteine and selenomethionine. mainly in the gastrointestinal tract Reduction of phospholipid hydroperoxides Peroxide reduction. involved in the elimination of misfolded proteins from the ER Unknown Unknown. involved in many biological pathways Thioredoxin/glutathione reductase found mainly in testes . yet the functions of many of the proteins involved are still unknown (Table 2. found in embryos and in the olfactory epithelium Unknown Unknown Unkown. involved in many biological pathways Mitocondrial thioredoxin reductase. Several of these compounds can be converted to methylseleninic acid or methylselenol which have anticarcinogenic effects in vitro . The distribution and concentrations of selenoproteins Table 2. a membrane protein Involved in protein folding in the ER Unknown. are the selenium compounds most effective in cancer prevention . Selenoproteins A total of 25 selenoprotein genes were discovered in the human genome several years ago by sequence analysis. Several studies have suggested that methylated Se derivatives. only found in the testes Can reduce peroxides using glutathione as an electron donor Catalyses the formation of selenophosphate Cytoplasmic thioredoxin reductase.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 73 lation. 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.
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). Biosynthesis of selenoproteins Translation of the codon UGA to selenocysteine (Sec) and its insertion into proteins is a complicated process (Figure 2. which has two . selenoproteins also differing markedly in the nucleotide sequence of their SECIS elements. one internal loop. The SECIS consensus sequence consists of two helices. the Sec-specific elongation factor (EFsec). and the SECIS core. but the mechanisms involved have not yet been elucidated (Figure 2. The current conception is that the mRNA and the SBP2-EFsec loops back towards the translation complex [13. The cytosolic or cellular form of glutathione peroxidase (cGSHPx) was the first selenoprotein identified [10. In the first step.).). which is a short sequence of non Watson-Crick paired nucleotides that appear to exist in all selenoprotein mRNA:s. and the thioredoxin reductases. In addition to the SECIS element. one apical loop.11]. these organisms tending to have homologue proteins containing Cys instead of Sec . All known eukaryotic selenoproteins have one SECIS-element. except for SeP. 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 . This structure is located in the 3’-untranslated region of the mRNA in eukaryotes and immediately after the UGA codon in prokaryotes [14. Several other glutathione peroxidases containing selenocysteine have been found since then. Except for the SECIS element there are no features in the DNA-sequence that selenoprotein genes are known to have in common. . It is found in almost all tissues and is believed to play a part in the body’s antioxidant defence. This takes place when a special stem-loop structure called the selenocysteine insertion sequence (SECIS) is present in the mRNA. and the recently discovered L30 protein. but the catalytic ability of these latter proteins is much lower.15]. Many of the known selenoproteins catalyse redox reactions in which selenium is at the active site.17]. catalysing the reduction of oxidised thioredoxin and other substates [1. serine is bound to tRNASec and then transformed to selenocysteine .8. Katharina Bruzelius 74 in different tissues are also not well known.8.12].Björn Åkesson. that regulate thyroid hormones. The other two major groups of known selenoprotein enzymes are the iodothyronine deiodinases. There are no selenoproteins known to be present in yeast or higher plants. Several of the selenoproteins have an homologue protein containing Cys instead of Sec. The next major step is the interpretation of UGA as a signal to insert Sec instead of stopping the translation.
19]. there being a selenoprotein hierarchy. The stability of selenoprotein mRNAs is affected by the amount of selenium present in the cell. The role of the SECIS for mRNA stability was studied by combining the coding regions of giGSHPx. but it is not yet known how this regulation operates [18. Hypothesised Sec translation complex in eukaryotes. 2.8. but not with that from cGSHPx. at least there are factors in both the coding and the non-coding mRNA regions that influence its stability . Accordingly an overview of the individual selenoproteins is provided. thus contributing to the body’s defence against free radicals. After association with the ribosome the SBP2 and the L30 switch places. . 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. There is also a tissue selenium hierarchy that controls the retention of selenium in the tissues and organs under selenium-deficient conditions. This hierarchy is particularly noticeable in the glutathione peroxidase family. Individual selenoproteins The glutathione peroxidase family The glutathione peroxidases generally catalyse the reduction of peroxides using mainly glutathione as the electron donor. This indicates that in the cGSHPx mRNA.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 75 Fig. the brain and the testes retain selenium whereas the selenium concentrations in the liver and the kidneys decrease markedly . phGSHPx and cGSHPx with the 3’UTRs (3’-untranslated regions) of each mRNA and then to study the system during selenium deficiency. and hydrolysis of GTP . The links between selenium and cancer probably involve different selenoproteins. Five selenium-dependent glutathione peroxidases are known to date . It was shown that giGSHPx and phGSHPx containing each other’s 3’UTRs would remain stable. A special feature is that transcripts of some selenoproteins are much more stable than those of others. In rats and mice fed a seleniumdeficient diet. cGSHPx could not be stabilised by replacing its 3’UTR with those from more stable glutathione peroxidase mRNAs. where the order of stability is as follows: giGSHPx ≥ phGSHPx > cGSHPx = eGSHPx.
catalyses the reduction of phospholipid hydroperoxides and is expressed in a wide range of tissues. nonmitochondrial and sperm-nuclei-specific forms produced from the same gene  and has an important role in sperm functioning . and its functional significance is unclear.Björn Åkesson.24]. the level of cGSHPx in most tissues decreases considerably. In model systems with an over-expression of cGSHPx. skeletal muscle. 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. there also being a number of negative effects such as increased obesity and insulin resistance . phGSHPx. amniotic fluid. giGSHPx knock-out models show no particular changes in phenotype to occur compared with the wild type. Disruption of the phGSHPx gene is lethal embryonically. In mouse and rat. This is the only selenoprotein that has been identified in milk thus far. Extracellular glutathione peroxidase. milk. which has led to speculations that this enzyme is a form of selenium storage to some extent . 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. This glutathione peroxidase isoenzyme is also found in extracellular fluids such as blood plasma. is mainly found in the gastrointestinal tract and also in the human liver and in mammary cells and tissue according to certain studies [23. lung lavage and the aqueous humour [21. each containing one selenium atom. It differs from the three glutathione peroxidases just mentioned by being a monomer and having a different substrate specificity. eGSHPx.27–29]. . which consists of four subunits. without any obvious damages to the host organism. but knock-out of both cGSHPx and giGSHPx leads to colitis in mice . Although the glutathione concentration in the plasma is very low eGSHPx can also use other electron donors such as the thioredoxin system . thyroid. which has thus far only been found in olfactory epithelium and in embryos. Gastrointestinal glutathione peroxidase (giGSHPx). cGSHPx knock-out mice have been used to explore the effects of complete loss of cGSHPx activity. pancreatic. This enzyme has been implicated in inflammation and molecular signalling. which is found in most tissues.21]. catalyses the reduction of hydrogen peroxide and organic peroxides. the selenocysteine in this enzyme is replaced by cysteine . It consists of four identical subunits. protective adapatations against paraquat and other challenges were shown to take place. phGSHPx occurs in mitochondrial. Putative functions of this enzyme are those of protecting against ingested lipid hydroperoxides and reducing susceptibility to colon cancer . Katharina Bruzelius 76 Cellular (or cytosolic) glutathione peroxidase (cGSHPx). Under conditions of severe selenium deficiency. The most recently discovered member of the glutathione peroxidase family is glutathione peroxidase 6. Phospholipid hydroperoxide glutathione peroxidase. unlike disruption of cGSHPx or giGSHPx. Other tissues such as liver. is a tetrameric protein produced mainly in the kidney and then excreted into the extracellular environment .
The iodothyronine deiodinase family The iodothyronine deiodinase family consists of three enzymes. . Additional selenoproteins Selenophosphate synthetase 2 (SPS2) catalyses the formation of selenophosphate. and can stimulate the survival of nerve cells in culture . depending on the animal species. There are indications that it also acts as an antioxidant in the extracellular space. which catalyse the removal of different iodine groups from the thyroid hormones. TrxR 1 and 2 are ubiquitous being located in the cytosol and the mitochondria. SPS2 is a selenoprotein. which is the selenium donor for the formation of selenocysteine from serine bound in tRNASec. and expression of DI2 and 3 having been found in many tissues. such as vitamin C. including bovine mammary tissue [34–36]. Truncated isoforms of this protein have also been found. Selenophosphate synthetase 1. was designated “P” because of its being found in the blood plasma. DI1 decreases during selenium deficiency. DI2. Thioredoxin catalyses the reduction of protein disulfides and is involved in a number of vital processes.40]. such as DNA synthesis and the regulation of apoptosis. which is not a selenoprotein. DI1 is found in the liver. unless they are rescued by a high-selenium diet . DI3). is expressed in small amounts in many tissues but is primarily found in the testis . The third isoform. but a number of splice variants of TrxR 1 and 2 have also been reported. binding to heparin and related carbohydrates . thioredoxin/glutathione reductase (TGR). Selenoprotein P Selenoprotein P (SeP). Targeted disruption of the TrxR 1 or 2 genes in mouse models is embryonically lethal . The 15 kDa selenoprotein. It is localised in the endothelium. the second selenoprotein to be discovered.38]. The iodothyronine deiodinases have high priority in the selenium hierarchy. 2 and 3 (DI1. organs that normally are highly prioritised during selenium deficiency. their levels remaining virtually unaltered in the case of selenium deficiency. for example. Selenoprotein P is the major form of selenium in the plasma and is involved in selenium transport [37. but not in the thyroid. In some tissues. can also form complexes with mercury and cadmium . this property indicating the existence of a feedback step in the production of selenoproteins. iodothyronine deiodinase 1. SeP knock-out mice exhibit low levels of selenium in the brain and testes. respectively . It can contain from 1 to 17 selenocysteines. thus activating or deactivating them. kidneys and thyroid. These mice die after weaning of the young. but is produced primarily in the liver and is secreted then into the plasma. SeP is expressed in most tissues. but in mammals they can also reduce other substrates. It can reduce peroxynitrite and phospholipid hydroperoxides [39. There are three main isoforms of thioredoxin reductases.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 77 The thioredoxin reductase family The thioredoxin reductases (TrxR) catalyse the reduction mainly of thioredoxin. particularly DI2 and DI3. also catalyses the formation of selenophosphate .
SelN is the only selenoprotein in which a mutation of it has been shown to cause a disease . In contrast. the selenium levels in whole blood or the erythrocytes appear to primarily reflect the long-term intake of selenium . since above a certain selenium level they tend to reach saturation. Selenoproteins as biomarkers The use of selenoproteins as markers of selenium status has only been exploited thus far in a few large epidemiological studies. and it responds rapidly to changes in selenium status. Another important conception is that plasma selenoproteins may not be suitable biomarkers under conditions of high selenium status. Selenoprotein N (SelN) is a 70 kDa protein located in the ER. Selenoprotein R (SelR) reduces methionine-R-sulfoxides. also known as VIMP) is a membrane protein in the ER. Selenoprotein M (SelM) is structurally similar to the 15 kDa protein and is supposedly also involved in protein folding in ER . The responses to selenium intake obtained if other blood fractions are analysed can differ. 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. It has been identified in prokaryotes. since the turnover of erythrocyte selenium is slower. Selenium in the plasma or serum is the best known and most accessible index. eukaryotes and archaea . nails and urine has been employed. the measurement of selenoproteins can be expected to provide information on specific selenium functions. In addition. one that has been associated with the process of eliminating misfolded proteins by transferring them to the cytosol  and also with inflammation . the activity of GSHPx in plasma has been used by a variety of laboratories in selenium supplementation studies. as compared with plasma selenium. When data from . mutations in the gene have been associated with various muscular diseases. the determination of selenium in the hair. Selenoprotein S (SelS. is believed to be involved in protein folding . This is an important step in the regulation of biological processes and the management of oxidative stress in the cell. SelW occurs mainly in muscle and brain and has been shown to act as an antioxidant. In general. which also includes non-specifically bound selenium.Björn Åkesson. and may thus be suitable as an indicator of short-term changes. such as rigid spine muscular dystrophy. A number of other selenoproteins have been discovered in man but information regarding their function is very limited. For instance. utilising glutathione to reduce peroxides . The function of selenoprotein W (SelW) has not been elucidated fully but it has been implicated in white muscle disease . Katharina Bruzelius 78 located in the endoplasmatic reticulum (ER). usually responding rapidly to changes in selenium status or in the dietary intake of selenium . To date. Although its catalytic function is still unknown.
20 (1. 1.69—5.66—1.07 (0.43) — — eGSHPx (mg/l) — 352 (306.34. In the following various results from their use in the authors’ laboratory are summarized (Table 2.95 (2.16) 0.38 (0.31) 2.47) 1.07) 1. 397)a 302 (259. 4.30—188.8.131.52. 0. Supplementation by selenate resulted in glutathione peroxidase activity plateauing at a plasma selenium concentration of 1. mU/mg protein.25) Plasma selenium (µmol/l) 1. glutathione peroxidase and protein-bound selenomethionine are the major selenium fractions contained in plasma .33.u.44.51 (6.41 (1. 1. Trial I baseline Finland.14 (1.78 (1.46—1.85) 1. Median values (and range) are given.47 (1. 0.52) 0. 0.83 (0.41.51. 346)a 6.13 (4.98.83) 1.52 (0.83) 0. c Data from subgroups having (top to bottom) low.39.03 (0. 2.86 (0. Selenoprotein P accounts for at least 40% of the plasma selenium . 1.69 (0. 1.11. 1.12) 0.44) 1.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 79 different cross-sectional studies was combined eGSHPx was found to approach a plateau at a plasma selenium concentration of approximately 1 mmol/l .92.64) 0.73) Values are means (95% CI). 1.75—2. Study European countries Prior to LDL-apheresis After LDL-apheresis Finland.14) 0. .00 (0. 0.91 (0. Trial II baseline Cancer cases Controls Elderly subjects (Malmö Food Study) Patients on HPN Latvians with differing fish intakec n 414 13 13 50 45 302 406 SeP (a.11. Immunoassays for measurement of this protein have been developed [57–59]. Plasma concentrations of SeP. Table 2. respectively.2 mmol/l . medium and high fish intake.43.91 (1.32) 3.41 (0.38 (2.86) 0. 1.88) 1.08. Selenoprotein P.24) 1.74)b — — — 126—205 38 21 16 31 1.11.18 (0. b GSHPx activity.). U/l.54—1. 1.22) 0. 1. a GSHPx activity.28.20—4.77 (1. 1.27) 1. selenium and eGSHPx in different studies (from ref.15) 1.23 (1.21) 1. 0.31—4.10 (1.38 (1.21.60. ).55 (0.69 (0.) 1. 0.56) 4.63—1.66) 1.83.65) 3.73 (0.16.56.51) 0.
eGSHPx and plasma selenium as markers of selenium status. Poland revealed an inverse relationship between the blood lead level and the level of selenoprotein P and of extracellular GSHPx . Concerning the association of selenium status and exposure to toxic metals. 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. with some indication of a plateau.Björn Åkesson. Katharina Bruzelius 80 Selenoprotein P as a biomarker Selenoprotein P levels were measured in healthy adults from 17 European Regions .9. plateauing within two weeks  (Figure 2. For women. Since the direction of causality was uncertain. the selenoprotein P values increased after selenium supplementation. and also between fish intake versus serum and urinary selenium were found.). Considerable variation between different regions was found. total selenium and selenoprotein P than control subjects did . In the Malmö Food Study  the relationship between the intake of different foods and selenium status was investigated. . studies of lead-exposed children in Katowice. in which the subjects were low in selenium status. A summary of the responses shown by the different selenium indices in the first of these two studies is provided in Figure 2. There was a close correlation between selenoprotein P and plasma selenium values. In the second study. but that at normal selenium status selenoprotein P usually correlated more highly with plasma selenium than eGSHPx did. 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. performed after the introduction of selenium-enriched fertilizers in Finland. no significant increase in selenoprotein P levels was observed after selenium supplementation and no differences were found between groups given different forms of selenium . The levels of selenoprotein P and eGSHPx were found to vary markedly for subjects from different regions and with different diseases.9. 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. the selenium status of the subjects thus being higher. Home parenteral nutrition patients with a variety of gastrointestinal diseases showed much lower levels of extracellular GSHPx. In a recent study of Chinese subjects of low selenium status given different doses of selenite and selenomethionine. significant relationships between milk intake versus selenoprotein P and urinary selenium. 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 . In a study of Latvian fisherman . however. Considerable attention has been directed at two studies conducted in Finland regarding the use of different forms of selenium [67. The scientists there compared the responses to oral supplementation of 200 mg selenium per day in healthy subjects on two occasions. In the first study. Regarding the relationships between SeP.68].
In a number of case-control studies. Generally speaking. . in turn found selenium supplementation to reduce total incidence and mortality of cancer in an American study group (see below).69]). 2.9. The strongest associations between premorbid plasma selenium levels and risk of cancer were observed for cancer of the respiratory and digestive tracts. The calculated degree of protection was found to differ between cancer sites [81.5–2 ppm) levels of selenium to the diet has a carcinostatic effect in animals treated with carcinogenic chemicals [71. 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. Biomarkers of selenium status and cancer Experimental studies have shown that the addition of high (0. 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. Interest in the preventive role of selenium supplementation in humans was stimulated markedly by results from two intervention studies.). β-carotene and α-tocopherol reduced the total cancer mortality and stomach cancer rate in a Chinese population. Blot and coworkers  found that a mixture of selenium.72]. Clark and associates . This suggested that selenoprotein P is a better indicator of selenium nutritional status than glutathione peroxidase is . These matters are reviewed in greater detail in the chapter that follows (Gromadziƒska et al. Fig. 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.87]. lower prediagnostic plasma selenium was found for cases of cancer than for controls. whereas in other studies no significant differences in this respect were found between cases and controls [82–86]. particularly among men [75–81].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.
05. in the lowest tertile as compared with the cases in the highest tertile. normal contour: p ≥ 0. 2. When cases were divided into subgroups according to cancer site. the ORs (adjusted for smoking) in tertiles of the SeP level were calculated for the respiratory.85. These were 6. respectively (p for trend = 0. whole blood selenium [89.2. the case-control differences of plasma selenium and SeP are compared for major cancer sites in several different study populations. 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 . the SeP levels for cancer of the respiratory tract were found to be significantly lower than in matched healthy control subjects.3. . 2. Fig.0. 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.4. Smokers were found to have significantly lower levels of selenoprotein P than nonsmokers . 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. For increasing quintiles. 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. In Figure 2.9. Smoking and biomarkers of selenium status Several factors other than selenium intake may affect biomarkers of selenium status.01). In addition.6 respectively.10. The factors contributing to the lower selenium status in smokers are unclear. plasma selenium has been used as a marker of selenium status.2. The circles represent selenium and the squares SeP.05. lower values of plasma selenium [77. 3. and toenail selenium [90–92] were observed in smokers than in non-smokers. and 0.Björn Åkesson. Percentage differences in the prediagnostic selenium and SeP plasma levels between cancer cases and controls (set at 0) for cancer at different sites.0. 2. The individual references are cited in .10.0 and 1.89]. erythrocyte selenium . This is probably due to SeP constituting at least 40% of the total selenium in human plasma . 2.90]. the ORs (adjusted for smoking) were 5. In other studies. The association between the relative risk of getting cancer and the SeP concentration found was also estimated from quintiles of the SeP level.
whereas smoking alone did not serve as a true predictor of this effect.25-year period. Smoking may also increase oxidative stress since cigarette smoke is a rich source of reactive nitrogen species. As reported by Sies and coworkers . α-tocopherol and selenium given for a 5.74]. It has been reported that smokers eat less selenium than non-smokers do. several intervention studies having indicated beneficial effects [73. in particular mortality due to stomach cancer . whereas the concentration of cadmium in the blood is significantly higher in smokers . and that these correlations are higher (more significant) in smokers. a small but significant reduction in total cancer mortality was obtained (RR = 0. both being acute-phase reactants. although this association disappeared when lead was included in the model. selenomethionine and glutathione peroxidase can scavenge peroxynitrite. The natural presence of cadmium in tobacco smoke may also contribute to the lower selenium status found in smokers. which together with superoxide can produce peroxynitrite . This may also explain the slightly lower selenoprotein P concentration in smokers. In addition. This agrees with findings of Dreher and co-workers  indicating that the human selenoprotein P promoter is rendered less active by cytokine treatment. In another study. which could exacerbate the risk of cancer associated with smoking . Multiple regression analysis also indicated the blood cadmium to increase significantly with a decrease in the selenoprotein P level.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. which could probably in part explain their lower selenium level . 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 . 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. smoking was found to be significantly associated with an unhealthy pattern of nutrient intake. a result that could possibly be explained by the covariance of lead and selenium in the blood . which suggests a repression of selenoprotein P expression during an acute phase reaction. In the Linxian trial involving supplementation of β-carotene. Multiple linear regression analysis of the data also suggested a depressive effect of cadmium on the concentration of selenium in the blood. 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. It has also been shown that selenoprotein P plays a role in the defence against peroxynitrite . Patients with a history of skin carcinomas who . suggests that the selenoprotein P levels are reduced by inflammatory activity. 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. in a meta-analysis of 51 published nutritional surveys of the relationship between smoking status and nutrient intakes.91).
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observed the development of different forms of liver tumours. however.1% bis-(4-acetamine)-phenylselenyl hydroxide. nevertheless. in response to selenium supplementation. Researchers of the former Soviet Union  feeding Se (as selenite) to rats at a 4.  observed that liver tumours occurred in rats fed protein-deficient fodder and grains originating from Serich (5–10 ppm) regions. it is quite possible that the organic selenium compound employed there had carcinogenic properties not linked with the element per se. Tscherkers et al. History Historically.  observed the development of tumours in male rats fed a diet containing 4.  published the alarming finding that feeding rats 0. ¸ódê.3 ppm Se. which suggests that the neoplastic changes that occurred could be attributed to Se. induced thyroid adenoma. Shapiro . 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. in addition to the occurrence of different selenoproteins. suggested that the pathological changes that occurred reflected regeneration of the liver rather than a carcinogenic process. Nelson et al. however. their use as biomarkers of selenium status. the methods used in assessing the nutritional status of selenium — in general.5. interest in selenium (Se) as a toxic trace element dates back to 1937 . Anticancerogenic activity of selenium — molecular mechanisms and epidemiological data Jolanta Gromadziƒska. In 1963. Poland Introduction There are many different aspects of the relationship between selenium and cancer. their functions and mechanisms of action. as well as epidemiological findings regarding the relationship between selenium level and risk of cancer are reviewed. equivalent to inclusion of 103–207 ppm Se in the diet for a 10-day period. Edyta Reszka. 11 were found to develop liver tumours.05–0. and concern for it as a carcinogenic agent dates to 1943 .3 ppm level. Seifter et al. Two of the chapters deal with these and related matters. and in epidemiological studies of risk of cancer — and the outcome of long-term trials concerned with the disease-preventive potential of selenium supplementation. Nofer Institute of Occupational Medicine. It should be noted. and intervention trials to investigate the effect of selenium supplementation on the incidence of cancer are summarized. In the present chapter the mechanisms behind the role that selenium can play in cancer prevention.Vitamins and selenium: Anticancerogenic activity of selenium 91 2. including the forms of selenium present in the diet and in the body. . whereas such tumours were found in less than 1% of the animals in the control group. Of 53 animals that survived for 18 months when thus fed. Wojciech Wàsowicz Department of Toxicology and Carcinogenesis.
) managed to inhibit the development of placental tumours in mice by use of Se compounds. In the control group. (cf. when Wassermann et al. tumours not only developed later.Jolanta Gromadziƒska. The animals were divided into several groups. a control group receiving a basal diet that contained 0. particularly when it was provided at both the initiation and the promotion stage of chemical carcinogenesis.5 ppm) reduced the incidence of cancer even more. In the groups given extra Se in their drinking water. Se supplied during only one of these two phases had a much weaker effect . respectively.12-dimethylbenzantracene (DMBA).0 ppm) they first developed at 17 months of age. An interesting study of the anticarcinogenic role of selenium was carried out by Schrauzer et al. 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 . These results indicated that supplying Se in larger amounts protects animals from developing cancer or delays . to 3%. in addition to this.1 to 1.1 to 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).4-dimethylaminobenzene (3’-MeDAB). Another early observation of such an effect was that of Clayton and Bauman . whereas in the group receiving the largest amount of Se in its drinking water (1. The maize oil content in the one case was high (25%) and in the other case low (5%). Supplying Se was found to decrease the number of tumours induced (from 97 to 46%). the first tumours developed when the animals were 4 months of age.  showed that adding 0. Wojciech Wàsowicz 92 The anticancerogenic effect of selenium in animals was first reported in 1911. but they also decreased in number with an increase in the amounts of selenium provided.15 ppm Se and the other groups receiving. Ip  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. 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]. Ip and Sinha  investigated the effect of various concentrations of Se on the development of breast cancer induced in rats by 7. who reported that in rats a diet enriched with 5 ppm Se decreased the incidence of liver tumours brought on by 3’-methyl-1. In both groups the incidence of cancer was found to decrease with increasing level of Se in the diet. in rats to which either of two different forms of Se were administered . Providing selenium at a still higher level (2. Two groups of animals received a diet containing maize oil. Harr et al.0 ppm Se in their drinking water. 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 . a strain characterized by the spontaneous development of breast adenoma in 80% or more of the cases. using female mice of the C3H strain. a supply of 0. . Edyta Reszka. At the same time. 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%.
proliferation and differentiation of the cells can be affected. however. and 2. Some reports suggest that the action of selenium toward cells that have been transformed earlier and toward normal cells differ . since reactive oxygen species can promote apoptosis in vitro . in which the metabolism. The anticarcinogenic effects of Se depend upon the chemical form of the Se-compound involved and the nature and dosage of the carcinogen. viruses or transplanted tumours .).30] (Tables 2. it has been postulated that the chemopreventive effect is related to the toxicity of selenium and the oxidative stress it induces. Thus a number of mechanisms for the anticarcinogenic effects of selenium and of many selenocompounds have been proposed. It has also been shown.23]. that selenium compounds can induce cell death through a mechanism distinct from oxidant toxicity [22. Cells adequately supplied with Se have been shown to be less sensitive to effects of both endogenous and exogenous carcinogens . affecting the cell cycle. depends on the cellular GSH/GSSG ratio.Vitamins and selenium: Anticancerogenic activity of selenium 93 the carcinogenic processes. It is also possible that selenium in the form of glutathione peroxidases. 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. 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. 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). including their providing protection against oxidative damage. 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 [24.13. 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 . At the same time. In addition. the activity of xenobiotic-metabolizing enzymes in vivo has been reported to increase when selenium compounds are given. Experiments in which no effect of Se was observed were rather rare. its dose and the agent that induces the development of cancer . and inhibiting angiogenesis [29. altering the metabolism of carcinogens. The activity of many cellular targets.25]. The effects can occur at a systemic. The anticarcinogenic effect of Se has been found to depend on the chemical form in which the element is administered. one should bear in mind that the results of animal studies cannot be directly extrapolated to humans. In several studies. cellular or nuclear level. and perhaps selenoprotein P and other selenoproteins as well. About 200 experiments have been performed aimed at assessing the effects of Se given to laboratory animals in doses higher than those usually employed in standard diets used to counteract the development of cancer induced by chemicals.12. enhancing immune responses. Regarding the mechanisms involved. Glutathione . can prevent mutations by serving as free radical scavengers [26–28]. in the majority of cases the reduction being quite significant .
necrosis and cell survival processes . Selenodiglutathione p-XSC. Se and the selenoproteins play a regulatory role in the following processes. DNA synthesis (in vitro) Cell cycle Apoptosis BSC and ist glutathione conjugates p-XSC. Edyta Reszka. This suggests that selenoproteins participate in the regulation of intracellular signal transduction . — Affecting apoptosis. — Controlling changes in the cell redox potential through inducing activation of the transcription factors and initiating de novo gene expression. intercellular communication. Wojciech Wàsowicz 94 peroxidases and thioredoxin reductases are involved in ROS scavenging. Yet non-protein selenium metabolites may also be important in the regulation of intracellular communication and metabolism. 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. — ROS-activated modification of the thiol and hydroxyl groups in the Cys and Tyr. ) Form of selenium Selenite Selenite Selenodiglutathione Selenite. 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. BSC ACF COX-2 PKC. The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis (ref. p-XSC. and changes in cell growth. — Regulating the expression of membrane and nuclear receptors responsible for cell maintenance.Jolanta Gromadziƒska. RNA. for example: — ROS-activation of protein kinases in the cytoplasm and nucleus. Table 2.12. BSC. PKA . Se-methylselenocysteine.
it cannot come about through activity of the selenoproteins. Table 2. cell detachment Yes ++++ ++++ S/G2-M ++++ Necrosis Late Fibronectin is an extracellular component that plays an important role in intercellular communication. selenic acid Ebselen P-XSC. Selenite activates the protein kinases involved in the pathways of cellular response to stimulation by inflammatory agents. COX-2 — cyclooxygenase.12. PKC — protein kinase A and C. PKA. JNK — Jun-N-kinase.Vitamins and selenium: Anticancerogenic activity of selenium 95 Table 2. In vitro effects of selenite and methylated selenocompounds  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. E — enhancement. The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis (ref. p-XSC — 1. ACF — aberrant crypt foci. selenite Se-methylselenocysteine Se-methyselenocysteine. Form of selenium Selenite p-XSC Selenite. ) — cont.4-phenylenebis(methylene)selenocyanate. W — weak. TK — thymidine kinase. whereas these processes can be inhibited by selenate . NE — no effect. BSC p-XSC. BSC — benzyl selenocyanate. Inorganic Se compounds such as selenite reduce the number of fibronectin receptors at the cell surface. 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. BSC. Oxidation of the thiol groups .13. AP-1 — activator protein 1. NF-κB — nuclear factor κB. Since this is an immediate action. selenium dioxide.
fos. Edyta Reszka. Se inhibits the expression of one of the activating zinc finger proteins that regulates the activation of the c-myc oncogene . NF-κB is also an important factor in regulating activation of the genes involved in the control of cell necrosis and apoptosis. The phosphorylation of mitogen-activated protein kinases (MAPK) represents one of these phases. 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 . Thioredoxin (Trx) plays a key role in this process. This process in turn results in the activation of AP-1 and of such transductional proteins as ras/rac.36]. the cell cycle. Reactive oxygen species (ROS) activates the phosphorylation of one of the NF-κB subunits. 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 . GSH-Px overexpression inhibits the translocation of another NF-κB subunit and reduces the phosphorylation of I-κB [40–42]. Oxidative stress induced by chemical or physical factors translocates Trx to the nucleus. In human hepatoma cells. the immune response. to the promotor regions. and in cellular proliferation or differentiation . degenerative and neoplastic diseases. such as cytokines. 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. GSH-Px and SeP are considered to have a particular role in MAPK inactivation . In human colon cancer cells. differentiation and senescence. for example. It regulates the effector genes in the promoter regions and can thus activate or repress gene expression. MAPK inactivation inhibits the cellular proliferation occurring in the course of the carcinogenic process. The breakdown of hydroperoxides by GSH-Px and the resulting decrease in the cellular hydroperoxide level inhibit the Se-activated kinase p38 . The reduced thiol group in Cys is essential for maintaining the activity of numerous transcription factors . intensifying the binding of NF-κB and AP-1 to the DNA. as well as the genes involved in apoptosis activation. GSH-Px in particular can act as a suppressor of protein kinases. such as those of proliferation. The redox potential is an important factor in regulating the nuclear factor κB (NF-κB) and the activation of AP-1 . NF-κB is a crucial target in the pathogenesis of various chronic inflammatory. and repair processes . selenite inactivates the c-myc oncogene and activates the c-fos genes that lead to the growth of normal cells . Through acting as a protein disulfide reductase. The strength of the binding of NF-κB to DNA is influenced by Se. growth suppression. TrxR thus affects the redox regulation of a variety of enzymes and receptors. directly. which can lead to their becoming activated. as well as such transcriptional and nuclear factors as NF-κB . I-κB. Wojciech Wàsowicz 96 in the transductional proteins results in their being modified structurally. myc and c-Jun N kinase [35. This process is made possible by intracellular reduction in the disulfide bridges (-S-S-). NF-κB activation involves changes in protein conformation and in the binding of numerous genes. The upor downregulation of the effector genes can modulate many different cell pathways.Jolanta Gromadziƒska. The process the translocation of NF-κB and its binding to DNA is multiphasic and highly complex. mainly in regions in which DNA binding to nuclear factors occurs .
a key enzyme in the deoxyribose formation needed for DNA synthesis. In cells with a high Se level. such as protein kinase A. ref-1. p53. ASK1) directly.. not only the selenocysteine incorporated into the protein structure. .54]. [50. Some low molecular weight Se compounds have been shown to influence the regulation of gene expression. Ca+2-dependent and -independent kinase C (PKC). but also its metabolites. AP-1.54.55]. selenodiglutathione. high concentrations of selenides tend to also be generated. and the glucocorticoid receptors [34. 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 . The various chemical forms of Se have been shown to differ widely in their anticancer properties. selenomethionine and Se-methyloselenocysteine can be transformed into selenides. they react with oxygen to produce superoxide and hydrogen peroxide . The anticancerogenic activities of various Se compounds are summarized in Table 2. Selenite has been shown to be more effective than selenomethionine in the inhibition of cancer cell growth during chemically induced carcinogenesis [31. When selenides are generated in sizeable amounts. 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 . It was also observed that methylated Se derivatives can induce apoptosis . the concentration of Se in the tissues also being found to be higher after the administration of selenomethionine than after selenite supplementation. cellular growth. modulation of tumour angiogenesis. Effect of different selenium compounds in cancer prevention The chemoprevention of cancer by selenium can involve the action of different selenometabolites.51]. the bioactivation of carcinogens. TrxR also regulates gene expression by affecting the cellular redox status and activating numerous DNA-binding transcriptional factors: NF-κB. but it is likely that one of Se metabolites is essential to this process. 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 .g. Se is also known to arrest several kinase pathways important in signal transduction. At the same time.12. reduction in the oxidative DNA damage that occurs.49]. 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 . Selenite. affect cellular metabolism. Trx inhibits certain kinases of the MAPK family (e. From this it can be concluded that a high concentration of Se is not crucial for the inhibition of carcinogenesis. . etc. Trx limits DNA synthesis.Vitamins and selenium: Anticancerogenic activity of selenium 97 and AP1 . It has been found that both sodium selenite and selenomethionine (SeMet) suppress tumour growth in many animal models involving a dose-dependent response [20. diacylglycerol kinase and thymidyl kinase .
Experimental studies have shown that the organic Se compounds induce apoptosis without DNA damage. Over 90% of the circulating Se is incorporated into the structure of the selenoproteins. any surplus of it is excreted from the body. modulates p53 activity. 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. Selenium compounds can also affect activation of the p53 gene since the main dietary selenium compound. in connection with studies of Se metabolism they conducted. Regarding the selenocompounds. in the previous chapter). In normal cells. 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. p53 regulates the activity of genes involved in DNA repair processes. Two other Se compounds. Fiala et al. only about 5% of it being found in other metabolites . sodium selenite and methylseleninic acid. however. p53-dependent DNA repair being activated within the concentration range of 10–20 µM. This modulation of the p53 molecule may be a specific mechanism of p53 activation.60]. DNA strand breaks after selenite treatment being an example.  showed that the selenium compounds as selenite. Wojciech Wàsowicz 98 When Se is supplied in high concentrations. which is not a DNA-damaging agent.4-phenylenebis-(methylene)selenocyanate and benzyl selenocyanate inhibit the activity of DNA cytosine methyltransferase. It is twice as active as selenomethionine in suppressing mammary carcinogenesis in rodents . . 1. In human cancer cells. responsible for single and double DNA strand breaks.7. even at rather high concentrations (10–50 µM) . p53 is often mutated and its functional activity suppressed. They suggested that this pathway may be the major mechanism involved in the chemoprevention that selenium compounds provide after the initiation of carcinogenesis. and of selenomethionine.Jolanta Gromadziƒska. an enzyme involved in DNA repair processes. SeMet oxidizes the thiols of the p53 molecules at 275 and 277 cysteine residues.13. which have been shown in in vitro experiments to affect apoptosis or to arrest the cell cycle (see Figure 2. Edyta Reszka. It appears then that the anticarcinogenic action of Se is due to the combined effect of selenium and selenoproteins . The metabolic pathways of Se-methylselenocysteine and selenobetaine involve the release of methylselenol or methylseleninic acid derivatives. Seo et al. Selenomethionine has been shown to act together with the p53 tumour suppressor protein in affecting the redox status. it is the actions of sodium selenite (Se IV). Ip and Ganther [59. were found to cause phosphorylation of the serines and threonines contained in the p53 molecule. The results of in vitro experiments on cells treated with selenite and methylated selenocompounds are summarized in Table 2.  demonstrated that the Se concentration is a determinant of basal p53 activity. that have been investigated most frequently . Methylseleninic acid in a range of concentration of 0. suggested that methylated Se derivatives are the most effective selenium compounds for cancer prevention . selenomethionine.5–1 µM was found to promote DNA repair processes by increasing the expression of two proteins associated with the p53 . Methylselenocysteine is one of the most important selenocompounds for chemopreventive activity .
68]. selenomethionine protects the cells from DNA damage through the induction of DNA repair. In vitro studies of a mammary cell line exposed to DMBA showed 1. this preventing DNA methylation from occurring. and 2-nitrophenylselenocyanate) were found to cause a dose-dependent decrease in the activity of formamidopyrimidine-DNA glycosylase (Fpg). In investigating the ability of inorganic Se compounds to induce apoptosis.  described the incorporation of Se into the factors involved in DNA repair regulation. as well as damaging the integrity of the genes of the DNA repair enzymes [67. which has an essential role in recognizing DNA lesions in the nucleotides and in the excision of damaged nucleotides in mammalian cells . The postsynthetic methylation of DNA is catalyzed by the family of S-adenosylmethionine-dependent DNA methyltransferases . 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. ebselen. 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. Seo et al. Selenium can also affect DNA methylation.66]. an important epigenetic mechanism that exerts control over gene expression . and to be involved in the pathway connected with the p53-dependent activation of DNA repair processes . Selenium can also act as an inductor of the enzymes participating in phase II detoxification  and it modulates expression of the enzymes involved in phase I detoxification. One possibility is that a small fraction of the SeMet is metabolized to a low-molecular-weight compound . which is a dominant mechanism of the cell death these compounds bring about .65]. They also affect the integrity of the XPA protein (xeroderma pigmentosum group A protein).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 . This decreased GSH concentration in the cells induced apoptosis. one of the enzymes involved in DNA repair processes. Blessing et al. 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. such as benzo(a)pyrene and arsenic . . Limitations in the DNA repair capacity appear to be a key determinant of predisposition to cancer [49. Reducible selenium compounds (phenylseleninic acid. The methylation of Se compounds to Se(CH3)2 and Se(CH3) results in a decrease in methyl donation. It has been found that Se derivatives can activate the p53 gene. These selenocompounds affect the DNA repair processes through oxidation of the zinc finger structures and the release of zinc from DNA. but that the induction of apoptosis is not a simple result of the regulation of p53 by selenium .Vitamins and selenium: Anticancerogenic activity of selenium 99 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. DNA methylation is one of the first steps in the carcinogenesis induced by certain chemicals.  showed that in normal human fibroblasts. selenocystine.
methylselenol). including nutrients. Case-control studies tend to reflect the short-term Se concentrations in the organism. Selenium regulates the immune response by stimulating natural killer cells and activating antigens of various types to destroy the tumour cells . the results of epidemiologic studies . Research over the last decade points to a significant protective role of Se in preventing the development of malignant neoplasms. such as in connection with antibody production and specific cell immunity . but no such relationship was found for lung cancer .74]. Cellular membranes of the T lymphocytes are particularly sensitive to Se deficiency. These metabolites that are synthetised in the cell can be involved in the cell’s metabolic pathways and destroy the integrity of tumour cells or induce apoptosis in these cells [51. stimulate the expression of cytokine receptors and the proliferation of lymphocytes [73. and impairment of the body’s ability to reject implants and to destroy neoplastic tumours [75. In a Chinese study. the inhibition of prostaglandin and immunoglobulin synthesis. The decrease in the number and the activity of cytotoxic T lymphocytes (CTL) is accompanied by the reduced excretion of lymphotoxins and the inhibition of both leukocyte and macrophage migration. NK cells and macrophages. a statistically significant increase in morbidity from oesophagus and stomach cancer was noted in a population with low levels of selenium. Se can stimulate the expression of IL-2 receptors found on activated T lymphocytes and on NK cells . such as suppression of the host immune response to bacterial and viral infections. Edyta Reszka. such as the Se level associated with the dietary pattern at the time of sampling in cases and controls. Low plasma selenium concentrations are thought to be associated with increased morbidity and mortality from cancer. however. 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.Jolanta Gromadziƒska.g. due to the large amounts of unsaturated fatty acids present in their structure.82]. Low Se concentrations in the tissues and in human body fluids also appear to be associated with numerous changes in the immune system. micronutrients and phytochemicals [81. The anticarcinogenic effect of selenium is also partly based on its ability to produce antitumour metabolites (e. To date.76]. 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. Wojciech Wàsowicz 100 Effects of selenium on immune functions Certain anticancer properties of the selenocompounds may be associated with their effects on cellular immunity. 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. reduction in the activity of T lymphocytes. Insufficient dietary intake of selenium results in many types of defects of the immune processes. An inverse association between selenium and risk of cancer has been reported both in case-control studies and in followup cohort studies. Selenium compounds can activate cytotoxic cells.80].
A review of epidemiologic studies of lung cancer and of the selenium concentration found in biological material. 95% CI: 0.86 (95% CI: 0. .09–0.14.80 (95% CI: 0. 95% CI: 0. representing a daily Se intake of 55 mg.41. but only in populations with a low mean Se level.57–0.Vitamins and selenium: Anticancerogenic activity of selenium 101 have been rather inconsistent.20.77–1. Lung cancer Studies to test the hypothesis that low Se concentrations contribute to the development of lung cancer have been conducted in many countries (Table 2. In a metaanalysis of 14 epidemiologic studies. 95% CI: 0.17–0. The protective effect of Se in connection with lung cancer could also be noted in studies examining Se concentration in the toenails.50. to 1. Of the studies considered in the meta-analysis.30–0.). those with high toenail Se levels were found to have a particularly high relative risk of lung cancer (RR: 4.94 and RR = 0.87] in which Se in both serum and toenail samples was analyzed. to 0.). In most of the reports. whereas others have obtained null results [84. In the control group of non-cancer patients the mean serum Se concentration was 100 ng/ml blood serum.58–1. whereas the value for groups with a low basic level of Se was RR = 0.60) after adjustments for smoking status were made . 95% CI: 0. dosedependent protective activity of Se was documented in Finnish and Dutch studies [86. The findings for three major forms of cancer are summarized below. the mean value obtained for groups showing a high basic concentration of Se was RR = 0.74 (95% CI: 0.00 (95% CI: 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: 0.30) 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.14. It is notable that of the women investigated within the Nurses Health Study.46 (95% CI: 0.16). 11 of which had a prospective design. the risk of lung cancer at high Se concentrations was found to be RR= 0.89]. indicated selenium to have a certain protective effect. Some authors have reported there to be an association between cancer risk and Se status. These finding confirm the assumption that the Se level in the toenails can be used as a marker of long-term Se concentration .85].45–1. when the study population was divided up according to Se level.22).81) revealed a statistically significant protective effect of high Se concentrations. undertaken by Zhou et al . the protective activity of Se could be clearly discerned when the reference group consisted of subjects showing the lowest level of this trace element.54–34.61–1. Interestingly.33.10) for the serum Se level.44) and for the Dutch population (RR = 0. The significant. The total RR values for lung cancer in patients showing high Se levels ranged from 0. only the findings for the two Finnish populations (RR = 0.24–0.97) (Table 2.
 NCC Serum 0. c data from 91 individuals.0 µg/l) 120 (119. controls (mean Se level) RR (95% CI) (high vs.  NCC Serum Garland et al.5) µg/l) USA 15—18 258 (0.8 (16. 2459 (0.  cohort Diet Zhou et al.  Male Female China The Netherlands CC Diet All China — 25 — 1.001 0.27–1.88) 0.30–0.49 NA Comstock et al.575 (0.36) 0.94) 1. 112.44)e 0.537 (0.7) µg/l)b 153 (61.60–2.41–2.30 (0.98 (0.70–2.61 (0.206) µg/g male.102 Table 2.2 (2.3 317 (0.33 (0.1 (19.81) 0.07 µg/day) 0.110 ppm) Study Se index Gender Population Years of follow up No.20 (0. Epidemiological studies of the selenium level in the body and risk of lung cancer (according to Zhuo et al.  NCCa Serum Goodman et al.0 (13.43) P for dose-response trend 0.3) µg/l)c Finland 8—12 153 (57.52 0.  CC Diet Jolanta Gromadziƒska.2) µg/day) 290 (39.3) µg/l F) Male All Male Female USA Male Finland 5—8 3.  NCC Serum Knekt et al.1 (2.41) f 0.20 (0.129) mg/kg) Japan 11—13 77 (113.6) µg/l) 356 (117.5 µg/l) Finland 16—19 77 (53.10 (16.80 (33.  NCC Serum Kabuto et al.65 (0. d data from 177 individuals. ) Author All All All M.60) 0. g data from 370 individuals.20 (0.134) mg/kg) China 4—5 108 (46.56 (0.006 Hu et al.08 0. . Wojciech Wàsowicz a Nested case-control.12) µg/day 227 (33.529 (0.76 (0.47–1.537 (0.547 (0.17 NA Knekt et al.0) µg/day) 870 (64. b data from 189 sets.9) µg/l)d 216 (45 µg/l) 47 (0.20–1. Edyta Reszka.54–34.0) µg/l USA 5—14 356 (119.  NCC Toenail Van den Brandt et al.50 (0.897 (0.02) 1.126) µg/g male. 0.7 (18. low Se) 0.0 (16. cases (mean Se level) No.14.5 47 (0.09–0.811 (0.66 (0.11 Kromhout et al.48 >0.308) µg/g) 250 (0.166) µg/g) 250 (0.01 0. e male subjects randomized in the earliest in the trial.0) µg/day) 0.  NCC Serum Ratnasinghe et al.41–1.080) µg/g female)g 0.19) 0.550 (0.46 0.108 ppm) 515 (0.20) 63 (NA) 290 (36.  NCC Toenail Hartman et al.5) µg/l)b 145 (57.41 (0.37–1.17–0.6 (15.15) 0.40) 4. f male subjects randomized in the 5th year.77–1.109) µg/g female) 227 (33.  cohort Toenail All The Netherlands 3.2 (24.
in the Physicians’ Health Study.25–0. 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. 95% CI: 0.103]. no protective effect of higher Se levels was found in either of the groups investigated .5.96) . A nested case-cohort study conducted on Japanese-American men (at a > 20-year follow-up) also confirmed the association between a high Se level in the plasma and a decreased risk of prostate cancer (OR = 0.72 (95% CI: 0.61–1. 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. was 0. indicating that the intake of selenium was able to reduce the risk of prostate cancer .84) in cohort studies and 0.49 (95% CI: 0.Vitamins and selenium: Anticancerogenic activity of selenium 103 Prostate cancer A majority of prospective and case-control studies show high levels of selenium intake to have a protective role in preventing the development of prostate cancer (Table 2.39) in case-control studies.). A lower Se concentration in the serum was found to be associated with a stronger tendency to be afflicted with a colorectal tumour . defined as the average of the 1st and 4th quintiles or the 1st and 3rd quartiles. 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 2.3–0. The lack of a protective effect of Se in connection with risk of prostate cancer risk was observed in the men participating in the Carotene and Retinol Efficacy Trial (CARET) . which involved a 6.61-0.16.9) . Several studies have shown that selenium can be of specific help in combatting prostate cancer . 95%CI: 0. A high prediagnostic Se level was found. Also. In the Netherlands Cohort Study.85). In a US case-control study of the relation between the risk of colonic malignant or benign tumour and the serum Se level. In two large case-control studies of male populations in Great Britain and Canada.15. although a protective effect of a high Se level (fifth quintile) was found (OR = 0. colorectal cancer patients with low Se concentrations were found to have a significantly lower survival time . especially men who had a PSA concentration equal to or higher than 4 ng/ml .74 (95% CI: 0. in which the Se concentration in the toenails served as a measure of long-term Se intake. 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 . to be associated (at a 13-year follow up) with a significantly reduced risk of prostate cancer. In addition. In the Health Professionals Follow-Up Study. high Se concentration in the toenails was associated with an appreciably lower risk of prostate cancer . The findings of this review showed that the pooled RR of prostate cancer for a particular Se intake. however.17–0. 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.38. the odds ratio (OR) for the development of cancer that was associated with a high level of Se intake was 0.3 years’ follow-up period.
73—2.24 (0.40) 1. .  NCC Helzlsouer et al.38 (0.018) µg/g) 300 (0.02 (0.  Cohort 1211 (0.10) 0.84) 0.  NCC Yoshizawa et al.13) 1.707 0.78 (0. controls (mean Se level) RR (95% CI) (high vs. cases (mean Se level) No. low Se) 1.02 Nomura et al.65—1. Epidemiological studies of the selenium level in the body and risk of prostate cancer Author Serum Serum Toenail Toenail USA 1—6 117 (0.611 µg/g) 0.5 (0.  Case-control Nails Jolanta Gromadziƒska.126) µg/g) 249 (131.4) µg/l) Finland 8—12 46 (59.008 0.4) µg/l)b 46 (58.  NCC Plasma 577 (0.6 µg/l) 0.15.69 0.622 µg/g) 13 586 (0.090) µg/g) NCC Van den Brandt et al.3 540 (0.018) µg/g) — 249 (128.  NCCa Goodman et al.99) 0.18—0.0 µg/l) The Netherlands 6.42—2.  (2000) Toenail USA (JapaneseAmericans) USA UK — 300 (0.00 (0.108 (0.05 0.12 Knekt et al.9) 0. Wojciech Wàsowicz a Nested case-control.104 Table 2.530 (0.3—0.3 (20.17—0.8 (19.3 (14. Edyta Reszka.60) 0.48—0.79 µg/g) USA 5 181 (0.85) P for dose-response trend 0.547 (0.8) µg/l)b Study Se indicator Population Years of follow up No.39 (0.6) µg/l) 456 (114.82 µg/g) 181 (0.106 (0.54—1.69 (0.77 µg/g) 233 (0. b data from 51 sets.6 (19.96 µg/g) USA 5—14 235 (114.16 0.581 Allen et al.  Case-control Serum Li et al.
91 (0.123) µg/g) The Netherlands 3.43—1.00) 0.77 (0. 105 .  NCC Serum Nelson et al.7) µg/l) 276 (130. controls (mean Se level) RR (95% CI) (high vs.  a Nested case-control.092) µg/g) Rectal 76 (0.106)) Rectal 36 (0.547 (0.3) µg/l)c Finland 8—12 29 (63.5 (19.  NCCa Serum Knekt et al.5—5.3 Colon 112 1246 (0.8) µg/l) Finland 8—12 48 (64.326 Garland et al.7 (0.45) 0.0 (18.863 (0.5 89 (0.9) µg/l)b 1.273 0.724 0.10 (0.9) All All USA — 25 (138 µg/l) 138 (134 µg/l) USA 1—4 276 (131.2) µg/l)b 0.41—2.575 (0.  NCC Serum Wallace et al.  Van den Brandt Cohort Toenail et al. low Se) P for dose-response trend Knekt et al.92) 0.44—1.76 (0.593 (0.  Case Serum -control Female Male (0.30) 1.7) µg/l)c 48 (65.22) 0.88—4.578 (0.3 (18.643 0. b data from 32 sets.091)) The Netherlands 3.58) 0.108)) 1.3 (17.Table 2.82 (0.16.0 (18. c data from 59 sets.50 NA Study Se indicator Gender Population Years of follow up No.  NCC Toenail Van den Brandt Cohort Toenail Vitamins and selenium: Anticancerogenic activity of selenium et al.146) µg/g) 47 (0.75) 0.04 (0.535 (0.41—1. cases (mean Se level) No.58 (0.186) µg/g) 2.733 (0.12 0.18—5.560 (0.42—2.843 (0.411) µg/g) Female (0.3 Colon 121 1209 USA 3.01 (0.131 0. Epidemiological studies of the selenium level in the body and risk of and colorectal cancer Author Male Female 1.3 (15.65) 29 (64.59—4.
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. Summary Results of epidemiologic and laboratory studies have indicated selenium to have a protective role in counteracting or preventing the development of cancer. and the Polyp Prevention Study. would need further verification as well. No inverse association of Se level and cancer risk was found for lung cancer in females. 95% CI: 0. no significant association being found between risk of cancer and toenail Se status. there are some reports of a significant inverse relationship between blood Se levels and the prevalence of adenomatous polyps [117.118].19–0.Jolanta Gromadziƒska. the Polyp Prevention Trial. a pooled analysis of data from three clinical trials of colorectal adenoma was conducted: the Wheat Bran Fiber Trial. 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. to be associated with an elevated risk of lung and prostate cancer. Edyta Reszka. although this result was only statistically significant in the case of the Polyp Prevention Study (OR: 0.34–0. a significant inverse association was obtained between toenail Se level and risk of colon cancer (OR = 0. a variety of confounding factors such as geographical location. To gain further insight into the anti-carcinogenic potential of selenium. A low level of selenium concentration in the body was shown to be associated with a higher risk of lung. gender. a number of epidemiologic studies show a low Se level. The majority of studies on relations between selenium level and occurrence of colorectal cancer have yielded null results for both males and females.112]. After adjustment for age. To sum up. possibly due in part to the rather low proportion of women in the study population . Colorectal cancer risk was also analyzed in a 41-month follow-up of the Nurses’ Health Study . There is a need of clarifying the role of Se . especially in males. smoking status and study site. The potential role of dietary Se in the early prevention of colorectal neoplasms would need to be confirmed. selenoprotein P (SeP) concentration or glutathione peroxidase activity in the plasma . environmental exposure. A similar result was obtained in a 3. breast cancer was not found to be influenced by the selenium level [114–116]. age. as found in the Polyp Prevention Study . genetic susceptibility.95) . 95%CI: 0. However. and the preventive role of Se regarding cancer of this type. gender.42. In contrast to prostate cancer. However. The selenium level was measured in blood specimens of 1763 trial participants.93) . However. 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. and the like need to be taken into account. however.57. Wojciech Wàsowicz 106 and a lower cumulative cancer-related survival rate than such patients with higher Se concentrations did . prostate or colorectal cancer. In a Canadian case-control study.3-year follow-up study of a Dutch cohort in which toenail Se level was used as an indicator .
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Amongst their health-promoting properties are antioxidant. both by blocking initiation and by suppressing the later stages involving promotion. These polyphenolic compounds are classified. Examples include quercetin (a flavonol in vegetables. Several recent reviews have summarised the potential chemopreventive mechanisms for a number of flavonoids [1–4]. epigallocatechin gallate (EGCG). chocolate and soy. Those flavonoids which have been studied in most detail exhibit many properties which could be protective against heart disease. angiogenesis. UK Introduction Many thousands of different flavonoids are found in plant species. Anticarcinogenic effects of flavonoids Margaret M. are summarised here. An early study by Duthie et al  reported that quercetin protected human lymphocytes from hydrogen peroxide-induced DNA damage. apples and onions). resveratrol. pelargonidin. Blocking mechanisms Possible ways in which initiation of carcinogenesis can be blocked include prevention of reactive oxygen species attack on DNA. volunteers consumed quercetin-rich blueberry/apple juice for 4 weeks. xanthohumol and the novel flavonol. xanthohumol (a chalone in hops and beer) and genistein (an isoflavone in soy). narginin. daidzein). anti-allergic. anthocyanins (cyanidin. . Such chemopreventive agents can be effective at different stages of the carcinogenic process. according to structure. ageing and cancer. apigenin). Many flavonoids possess antioxidant or free radical scavenging potential. isoflavones (genistein. progression. tea.1. Leicester. anti-inflammatory. tricin. with major dietary sources including fruit. altered metabolism of procarcinogens in favour of conjugation and excretion of reactive metabolites. epicatechin-3-gallate) and chalones (xanthohumol). inhibition of carcinogen uptake into cells and enhanced DNA repair. Bioactive components in foods 3. catechins (epicatechin. and anticancer activities. Some recent data for the well-studied flavonoids apigenin. Also in this study. which varies depending on the hydroxylation status of the benzene rings. quercetin. University of Leicester.3. invasion and metastasis. which led to a significant increase in antioxidant capacity of plasma. petunidin). the chalone. flavanones (myricetin. hesperetin). as flavonols (quercetin. Manson Cancer Biomarkers and Prevention Group. flavones (luteolin. vegetables. Total daily intake can range from 50–800 mg. Similar findings were reported by Wilms et al . antiviral. kaempferol). who also found that quercetin protected human lymphocyte DNA from bulky adduct formation following treatment with benzo[a]pyrene. genistein.
However. In another recent study . genistein and EGCG (reviewed in ) have a number of effects in common with those detailed here for apigenin and quercetin. the flavonols. . The latter led to a 34% reduction of PGE2 levels in small intestinal mucosa and blood. or even better. AP-1. cdc2. and pAkt. p53. but not apoptosis . a novel flavonol in rice bran.1. A range of tumour suppressing activities is shown for apigenin (Table 3. Such interactions influence the expression of drug metabolising enzymes such as cytochromes P450 . namely inhibition of signalling through the EGFR family. while the isoflavones.and down-regulate key molecules.Margaret M. Suppressing mechanisms Mechanisms which result in suppression.2. cyclin D1. In a subsequent study by the same group . induction of quinone reductase activity).). with inhibition of COX-1 and COX-2 activity. Such interactions might influence bioavailability of anti-cancer drugs in vivo and could be considered for combination therapies . and PI3K. Xanthohumol possesses several useful properties to block carcinogenesis including modulation of enzymes involved in carcinogen metabolism and detoxification (inhibition of Cyp1A. depending on cell type and experimental conditions. p27. flavonoids can both up. induction of cell cycle arrest involving a decrease in cyclin D1 and phosphorylation of Rb. was shown to inhibit the growth of breast tumour cells. During later stages of carcinogenesis additional useful mechanisms include inhibition of angiogenesis. including JNK. . Xanthohumol was also found to inhibit COX-1 and COX-2 activities. genistein and daidzein. including hydroxyl and peroxyl radicals. and to be antiestrogenic . NF-κB. A significant number of flavonoids. without affecting expression.) and quercetin (Table 3. Quercetin and silymarin were found to inhibit MRP1/4/5-mediated drug transport from intact erythrocytes with high affinity. Tricin. alone and in combination. causing G2/M arrest. in a manner which suggested that they interact at the substrate-binding sites. Manson 116 Flavonoids can interact with the aryl hydrocarbon receptor (AhR) as agonists or antagonists. have been shown to induce G2/M arrest in SW480 and CaCo2 human colon carcinoma cells . tricin decreased the number of intestinal adenomas in Apcmin mice by 33%. quercetin and kaempferol. and induction of apoptosis involving release of cytochrome c from mitochondria. invasion and metastasis. elimination of tumour cells. The inhibitory effect of other flavonoids on COX-2 expression and activity has been reviewed by O’Leary et al. depending on structure and cell context. along with inhibition of superoxide anion radical formation and nitric oxide production . They have also been shown to influence the multi-drug resistance phenotype acquired by many tumour cells. include growth inhibition by induction of cell cycle arrest or apoptosis. Resveratrol. p21. activation of caspases 3 and 9 and downregulation of Bcl family members. reduced P-glycoprotein expression and function in multi-drug resistant human cervical carcinoma KB-IV cells. modulated intracellular drug levels by inhibiting function. accompanied by upregulation of p21 and p27. scavenging of ROS.
↑APAF-1. ↑cyt c release. ↑cleavage of caspase 3  Apoptosis (p53-dependent) ↓Her2. NOS-2. ↓VEGF and in nude mice OVCAR-3 and A2780/CP70 ovarian cancer cells ↓Akt. ↓Akt. ↓ colony formation  PC-3 prostate cancer cells ↓p50. altered Bax:Bcl2 ratio. ↓cyclin D1/D3 & cdk4. ↓p70S6K1. p27.  . LAN-5. ↑IκBα Apoptosis Effect Reference  PWR-1E.1.7. ↓p70S6K1. p16.Bioactive components in foods: Anticarcinogenic effects of flavonoids 117 Table 3.8.9 and cIAP-2 ↓fatty acid synthase activity Apoptosis   Growth inhibition and apoptosis ↑p53. G1 arrest. ↓NF-κB-DNA-binding. ↑IκBα . apoptosis HER2-overexpressing breast tumor cells ↓PI3K & Akt activity. ↑cleavage of caspase 3. ↓IκBα degradation and phosphorylation. ↓Bcl2. PC-3. ↓NF-κB p50 & p65  Growth inhibition. ↓TNFα activation of NF-κB ↓Bcl2. LNCaP. VEGF Apoptosis  DU145 prostate cancer cells ↓cyclin D1/2 & E. cyclin D1. ↓p65. MMP9. Chemopreventive suppressing effects of apigenin Tissue/cell type Jurkat T cells Mechanism ↓chymotrypsin-like activity of 20S and 26S proteosomes. ↓PI3K-HER2 docking. ↓TNF a-induced NF-κB activation Apoptosis. SK-N-BE neuroblastoma cells Breast cancer cells ↑ROS.↓HIF-1α. ↑p27 Apoptosis. ↑HER2 degradation Apoptosis  A549 lung cancer cells in vitro ↓Akt. ↑p53. ↓CDK2/4/6. ↓HDM2 Growth and angiogenesis inhibition Angiogenesis inhibition   HCT 116 colon carcinoma cells ↑ERK & p38 phosphorylation and activity ↑phosphorylation of Elk & ATF2  HCT 116. ↑cleavage of caspases 3. p18. ↑p21. COX-2. ↑Bax. ↓HER2/neu phosphorylation.↓HIF-1α. DU145 prostate tumour cells Breast and prostate cancer cells NUB-7. HT-29 colon cancer cells ↓CK2. ↑Bax. ↑p21. ↑DFF-45 cleavage. ↓IKKα actvity. ↓VEGF.
↑p21 ↓HSP70 ↓c-myc Growth inhibition.2. ↑survivin. ↓Pgp Apoptosis Effect Reference  Inhibiting chymotrypsin-like activity of 20S and 26S proteosomes. epithelial cells migrate to the apex of the crypt. ↑IκBα Apoptosis  Breast and prostate cancer cells Colonic aberrant crypt foci ↓fatty acid synthase activity Growth inhibition and apoptosis  ↑Bax. ↑Bax. Chemopreventive suppressing effects of quercetin Tissue/cell type HL60 human myeloid leukeamia cells Jurkat T cells Mechanism ↑Bax. a process involving the APC gene. ↑cleavage of caspase 9 Suppression by 4-fold. PC3 prostate tumour cells HT29. epicatechin. G2/M arrest Apoptosis    One recent report by Fenton and Hord  has suggested a novel chemopreventive mechanism for flavonoids. apoptosis ↑3-fold  MiaPaCa pancreatic tumour cells MCF7 breast tumour cells ↓phosphoFAK Decreased invasion  ↑PTEN. ↑c-jun Inhibition of androgen receptor activity  ↓ErbB2/3. ↓Bcl2. ↓Bcl2. SW480 colon cancer cells SW480 colon cancer cells A549. naringin and hesperidin induced a greater migratory response in APCMin/+ cells compared to those expressing wild type APC. resulting in under. ↓Akt Growth inhibition and apoptosis  LNCaP. ↑p27. such as the cell cycle inhibitor p16 and the retinoic . In normal colon. During the carcinogenic process. both hypermethylation of the promoter regions of tumour suppressor genes and hypomethylation of oncogenes can occur. Both EGCG  and genistein  have been shown to reactivate a number of key genes.Margaret M. These authors reported that apigenin. which is often mutated in colon cancer. ↑phosphoBcl2. ↑phospho cdc2. ↓phosphoAkt Growth inhibition and apoptosis  ↓β-catenin/Tcf transcriptional activity ↑cyclin B1. ↑p53. H1299 human lung carcinoma cells PC3 prostate cancer cells ↓Sp1 interaction with AR. Manson 118 Table 3. Such flavonoid-induced migration was dependent on matrix metalloproteinase activity.or over-expression.
and induction of apoptosis. Duthie SJ. Sarkar FH. Duthie GG. Mut Res Gen Toxicol Environ Mutagenesis 1997. Mut Res 2004. Inhibition of survival signalling by dietary polyphenols and indole-3-carbinol. mainly through intrinsic pathways involving Bcl family members. suggesting the potential use of ‘flavonoid cocktails’ to reverse multi-drug resistance in treatment of this cancer .Bioactive components in foods: Anticarcinogenic effects of flavonoids 119 acid receptor (RARβ). References 1. 4.9:11–8. like other types of dietary chemopreventive agents. 3. For example Mertens-Talcott et al. Phosphorylation of JNK1/2 and p38 was increased by the combination. exhibit a wide range of potentially useful activities for cancer prevention. Collins AR. synergistic or antagonistic effects of combinations of more than one chemopreventive agent.555:53–64. . Manson MM. Conclusions Flavonoids. Ellagic acid potentiated the inducing effect of quercetin on levels of p21 and phosphorylation of p53 at serine 15. showed that quercetin and ellagic acid acted synergistically in inducing apoptosis. mitochondrial membrane depolarisation. modulation of drug metabolising enzymes and multidrug resistant genes. . Manson MM. Combinations of flavonoids were found to have an inhibitory effect on the breast cancer resistance protein (ABCG2). including cyclins. Neither the generation of ROS. Cancer Prevention — the potential for diet to modulate molecular signalling. Li YW. 5. Trends Mol Med 2003. Surh Y-J. which. 2. involved direct interaction with the enzyme. in several different cancer cell types. They can also induce cell cycle arrest by modulating key components of cell cycle regulation.393:223–31. Combined effects There is now accumulating evidence for the additive. nor quercetin stability were affected by ellagic acid.41:1842–53. Dodson VL Quercetin and myricetin protect against hydrogen peroxide-induced DNA damage (strand breaks and oxidised pyrimidines) in human lymphocytes. Suppressing activities include inhibition of signalling pathways responsible for cell proliferation and survival. while quercetin alone only induced p38 phosphorylation. Cell signaling pathways altered by natural chemopreventive agents. Their blocking activities include antioxidant effects. cyclin dependent kinases and inhibitors. in the case of EGCG. cytochrome c release and activation of caspases. Eur J Cancer 2005.3:768–80. using MOLT-4 human leukaemia cells. Cancer chemoprevention with dietary phytochemicals. Nature Rev Cancer 2003. The mechanism proposed was through inhibition of DNA methyltransferase.
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Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells. The 70 kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer. Apigenin induces apoptosis through proteosomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway. Park S.25:2017–25. 27. Human Mol Genetics 2005. Am J Physiol 2003. Flavonoid quercetin. Chao JI. Volate SR. Reed E. silymarin. Wargovich MJ. Kim ES. a potent inhibitor against beta-catenin/Tcf signaling in SW480 colon cancer cells. Carcinogenesis 2005. Cao ZX. Jiang BH. 31. Parhar K.285:G919–28. Jiang BH. Sulikova M. Way TD. curcumin.19:342–53.26:1450–6. Shukla S. Zheng JZ. J Nutr 2003. Fang J. 32. Hahm ER. Lin JK. 24. 30. Zhou Q. thelial growth factor and angiogenesis in human lung cancer cells. Pelling JC.Bioactive components in foods: Anticarcinogenic effects of flavonoids 121 21. Eng C. but not apigenin or luteolin. Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells. Salh B.52:273–9. Quercetin decreases the expression of ErbB2 and ERbB3 proteins in HT-29 human colon cancer cells. Stevenson MA.68:635–43. Anticancer Res 2005. ginseng and rutin). Zazrivcova K. Sinden MR. Mol Pharmacol 2005. Liu HF. Hu XW Shi XL.6-dichloro-ribifuranosylbenzimidazoleand apigenin-induced sensitization of colon cancer cells to TNF-alpha-mediated apoptosis. . FASEB J 2005. Flavonoids promote cell migration in nontumorigenic colon epithelial cells differing in APC genotype: implications of matrix metalloproteinase activity. Jung KC. Kao MC. 28. Huang YT. Davenport DM. 34. 25.328:227–34. Lui LZ. 35. Zhao MJ. Kang NE.26:793–801. 39:114–24. Yuan HQ. Gupta S. Chang JY. Bang MH. Calderwood SK. 26. Xue Y.48:182–8. Farah M. Van Dross R. Yang CH.14:1457–63. J Biol Chem 2004. Lee PPH. Biochem Biophys Res Comm 2005. Sedlak J.20:835–49. Young CYF. 33. Modulation of aberrant crypt foci and apoptosis by dietary herbal supplements (quercetin. Hord NG. Bodo J. Muga SJ. Neoplasma 2005.16:155–62. Kim WK. Fang J.279:4479–89. Kim HK. Park CH. Moussavi M. Kuo PC.133:3800S–4S. Eivemark S. Mol Carcinogenesis 2004. induced apoptosis in human myeloid leukemia cells and their resistant variants. Cho HJ et al. Phytoestrogen exposure elevates PTEN levels. 22. 36. Knudson A. Lee MT. 29. Int J Hyperthermia 2004. Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K and HDM2/p53. Apigenin inhibits expression of vascular endo. Targeting of focal adhesion kinase by flavonoids and small interfering RNAs reduces tumor cell migration ability. Jones EL. Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells. The chemopreventive bioflavonoid apigenin modulates signal transduction pathways in keratinocyte and colon carcinoma cell lines. 5. J Biol Chem 2004. Gong AY. Duraj J. Lee LT. Waite KA. Quercetin. Lin YS. Carcinogenesis 2005. 23. Nutr Cancer 2004.279:55875–85. J Nutr Biochem 2005. Fenton JI.
Manson 122 37. Talcott ST. Wang Y. Chen D. Bomser JA. Reversal of hypermethylation and reactivation of p16 ink4a. Christman JK.21:1263–73. et al.63:7563–70. Percival SS.11:7033–41. Fang MZ. Yang CS. Clin Cancer Res 2005. Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Ai N. 40. Mertens-Talcott SU. 38.Margaret M.135:609–14. Romero C. Sun Y. Zhang SZ. Cancer Res 2003. Combined effects of multiple flavonoids on breast cancer resistance protein (ABCG2)-mediated transport. Fang MZ. Hou Z. Lu H. 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. 39. Yang XN. . Sun Y. Pharmaceutical Res 2004. RARβ and MGMT genes by genistein and other isoflavones from soy. Morris ME. J Nutr 2005.
followed . Germany Introduction The primary aim of analytical cancer epidemiology is to detect associations between exposure variables and disease endpoints. Nutritional epidemiology deals with dietary factors that are difficult to assess. whereas others are limited to only a few kinds of food (such as apigenin found in parsley and celery).2. such as various secondary plant products. 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. reaching plasma concentrations of 0–4 µmol/l at an intake of 50 mg aglycone equivalents . the sugar moiety of the compound in question and its further metabolism by the gut microflora. lignans. and components for which bioavailability is low. phenolic acids consist of two main subgroups. German Cancer Research Centre. these including the flavonols. the hydroxybenzoic and the hydroxycinnamic acids. including beverages. The basic flavonoid structure and various examples of them are given in Figure 3. however. for example. The use of biomarkers for such compounds should overcome some of the methodological problems in nutritional epidemiology just referred to. flavones. from imprecision.1. Even more problematic. however. 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. isoflavones. Dietary measurements tend to suffer. is the fact that the polyphenols markedly differ from one another in their bioavailability and intestinal metabolism. measurement error being independent of that contained in the corresponding questionnaire data. Some of the polyphenols are found in a rather wide variety of foods (kaempferol contained in many vegetables. Isoflavones and gallic acid are polyphenols that are absorbed to the highest extent. Flavonoids are classified in several different sub-groups.3 to 43% (based on urinary excretion) of the dose administered. Current evidence from bioavailability studies suggests that the bioavailability varies from about 0. Heidelberg. anthocyanins and proanthocyanins. Estimates of dietary intake estimations are hampered by incomplete or missing data in food composition tables. flavanones. Bioavailability is determined by different factors. needs to be demonstrated in advance of their use in epidemiological and etiological studies . for example). Biomarkers of dietary polyphenol intake for studying diet-cancer relationships Jakob Linseisen and Sabine Rohrmann Division of Clinical Epidemiology. flavanols.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 123 3. Polyphenols Polyphenols are provided by plant-derived foods. The analytical data obtained are objective and more precise. particularly concerning dietary components provided by only certain kinds of foods.
Thus far. data on the phenolic acids are very limited. 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. 3. The polyphenol concentrations contained in biological specimens are estimated in most studies after hydrolysis of the glycosides involved. ideally 24-hour samples. and glucuronidation. The basic structure of flavonoids and the chemical structure of selected polyphenols. The steady-state levels of the plasma polyphenols should only be achievable through regular intake of the foods in question. Sabine Rohrmann 124 by the catechins. is a promising approach. flavanones and quercetin glucosides. Flavonoids undergo extensive first-pass phase II metabolism in the intestinal epithelial cells and the liver. Fig. the galloylated tea catechins and the anthocyanins are much less available. concentrations of them reaching baseline levels within 24 hours . due mainly due to the higher concentrations of polyphenols — at least after extraction and enrichment — as compared with plasma samples. Measurement of the compounds found in urine samples.1. The proanthocyanidins. although the kinetics involved differ considerable. sulfation.Jakob Linseisen. . For many of the polyphenols. the aglycones being quantified. their half-life time in the plasma is short.
or urine samples. although a part of them is excreted unmodified. LC-MS) to characterise clusters of polyphenolic compounds that can potentially be used as biomarkers. Some techniques such as liquid chromatography-mass spectrometry (LC-MS). However. a single mass-selective detector often fails to fulfil the requirements for sensitivity. the flavonoids are usually glycosylated and the phenolic acids are ester-bound. such as plasma. the clean-up procedures for this type of samples may be more complicated than required for many food systems. For the flavonoids. Phenolic acids undergo further metabolism and degradation. frequent use is made of enzymic hydrolysis by means of sulfatase and glucuronidase unless a study aims at determining the exact metabolites.3. Thus far. serum. Separation and detection systems The two major separation techniques for the quantification of polyphenolics are HPLC and GC.). . Hydrolysis In foods. the respective aglycones and acids being subsequently detected and quantified. the fluorescence emitted is recorded by means of a plate reader. new developments include the creation of HPLC-ESI-MS-MS systems and similarly coupled devices. sulfated and glucuronidated compounds.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. Identification of compounds by means of mass fragmentation is used as a gold standard. combined with different detection systems (Table 3. solid phase extraction (SPE)-columns provide the most convenient solution for removing from a sample any matrix compounds that would disturb the analysis. flavonoids mainly undergo modification by means of phase II enzymes leading to methylated. Clean-up procedures For human specimens. and the phenolic acids are genarally quantified by means of GC after derivatisation. Accordingly. Thus. HPLC has become the method of choice. For the purpose of quantification. with the option of time-resolved measurement. the scientific literature contains no report on use of the metabonomics techniques (NMR. although the use of LC-MS is becoming increasingly common. Although in principle these methods can be applied to human specimens.5]. Hydrolysis (acidic or enzymatic) is frequently used to simplify the analytical procedure. For the structural characterization of compounds. do require only minor sample-preparation steps. In biological specimens. The availability of antibodies for isoflavones and lignans has enabled the antibodybased assays with a high degree of sensitivity to be developed . mass spectrometric techniques need to be used.
4 . Phenolic acids Phenolic acids Flavonoids.46 (0.1) 5.04 1.88 (0. GC — gas chromatography.6 4.7) 7.Jakob Linseisen.26–4.1 6.8) 8. Phenolic acids Flavonoids.5 42.0 5.3 0.1 (0.26 42.0) 6. LC — liquid chromatography.2 Cmax (µmol/l) Mean(SEM) 1.0 7. Most of the studies concerned only one or some few compounds within a given subclass of polyphenols.7–9.8 (0.52) 1.84 (0.14 0.6) 5.4.9 (1. even after the administration of polyphenol preparations or polyphenol-rich food corresponding to 50 mg aglycone equivalents.3 (3.3.8) Range 3.4–62.6 6.8–29. (according to ).5 15.6) 4.9 2.3 5.87 8.21) Range 0. ) Tmax (h) Mean (SEM) Range Daidzin Daidzein Genistin Genistein Glycitin Hesperidin 6.4–5. Sabine Rohrmann 126 Table 3.76–3. Bioavailability studies and other short-term interventional studies A report was published vey recently summarizing all scientific studies (n = 97) that has been conducted thus far on the bioavailability of polyphenols . Table 3.21–0.57 (0.5 (0.5 (0. lignans GC LC Immunoassays Mass spectrometry Mass spectrometry Time-resolved fluorescence HPLC — high-performance liquid chromatography.00) 1.3 (0.38) 0.00 0.50–2. Differences between the various compounds and of classes of polyphenol are striking nevertheless. Kinetic data from the experiments in question are summarized in Table 3.7 7.36–3.50 Urinary excretion2 (% of intake) Mean(SEM) Range Elimination half-life (h) Mean (SEM) 5. Relatively low plasma concentrations were obtained in each case.0 3.4–8.92 (0.0) 19.8–7.0 27.3 3.6) 4.5 (0.0) 21.0) 3–24. Phenolic acids Flavonoids.8 4.4–9.9 (12.0–5.8) 8.2 1. Phenolic acids Isoflavones.1 (0. Phenolic acids Flavonoids.6 (4.7–10.3) 8. 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.0–6.56 (1.0–55.46–4. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al.25) 1.4.3 (0.27) 2.6 (1.0–9.
0 0.0 (0.4) Range 1.5–2.1–30.0–0.26) 0.0 0.004–5.40) 0.7–2.03 All the data were converted so as to correspond to a supply of 50 mg aglycone equivalent.40 (0.46 (0.6) 3.17) 2.0 1.8 2.38 0.0 4.0 1.4 (0.7) 1.9 (2.5–2.3 0.6 (0.07–1.1) 2.1–4.1–55. 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 .80 0.2) 10.2) 3.57) 0.1 Elimination half-life (h) Mean (SEM) 2.45–1. The authors examined evidence relating to the effects of these polyphenols had on biomarkers used to test various pathophysiologically relevant hypotheses in vivo.1) 11.5) Range 1.5 (1.6 (17.09–0.2–15.10 0. Usually represent 24-h urine samples. ) — cont.1) 1.2) 1.35 10.10 (0.70 Urinary excretion2 (% of intake) Mean(SEM) 8.7) 11.0 1.02) 0. Rutin (Epi)catechin EGC EGCG Gallic acid Chlorogenic acid Caffeic acid Ferulic acid Anthocyanins Proanthocyanidins 1 2 Cmax (µmol/l) Mean(SEM) 0.26 Range 0. AUC — area under the plasma concentration-time curve.51–3.8–28.3–1.8 (3.5 0.4) 2.001–0.1 (3.5 (0.1–1.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 127 Table 3.01) 0.1 2.20 0.03) 4.3 (0. Tmax (h) Mean (SEM) Range Naringin Quercetingluc. EGC — epigallocatechin.3–2.2 0.52 0.9 (8.0 19.57–4.45) 0.1–61.1 1.09) 1.5–2.3) 18.70 37.1) 1.5 0.09–1.4–39.06) 0.03 0.3–9.1 (0.5 (0.6–5.5 0.6) 2.12 (0.0 2.06 (0.03 (0.00 (0. Tmax — time to reach Cmax. Observational studies: cross-sectional studies and studies related to cancer Various studies have dealt with the suitability of using fasting plasma or urinary concentrations of polyphenols (mainly flavonols.1) 1.96 (0.33) 1.03–0. The magnitude of the variation in the plasma polyphenol concentrations found between free-living subjects following their usual diet habits was .5 (0.3) 6.1 0.1 1.3 (0.4 0.9–28. EGCG — epigallocatechin gallate.7 (0.008–0.5 (0.6 0.30–2.7–2.50 0.5 (5.2) 0.8 (0.13–1.5–5.2 1.4 (0.0) 36.7–4.6–3. although one study in particular failed to support this conclusion . 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.50 (0.3 0.4.1 (0.02 (0.3) 0.7 27.20 (0. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al.4 (0.7 5.31–6.3 (0.9 4.3) 1.03) 2.5 17.0 4.6 0. flavanones or isoflavones) as biomarkers of polyphenol intake [9–16].2) 1.7 (1.7 0.4) 2.
1 (2.7 (78.5. participating in a cross-sectional study (according to Bolarinwa and Linseisen .6 (275.0a 78.0a 0.4 159.1 (41.5) 1304.Jakob Linseisen.1 1357. Table 3.9) 140.0a 0. 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. The correlation coefficients between estimates of the dietary intake of polyphenols of the day before blood sampling took place and fasting plasma concentrations polyphenols were as high as 0.3 644.9 7103.4 104468.1 53.8) 108.6 (254.5 114.6) 477. close to of steady-state plasma concentrations of them can only be achieved if the substance in question is consumed regularly.1) 520. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet.9) 0.2 0.7 (15.8 (77.0a 48.7 561.6 413.0a 0.7) 277.6) 6990.6 339.4 2904. the intra-individual variation also being high .9 372.7 2290.0 6103.7 (48.4 59.1 (94. 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.0a 58.8 26.3 (67.0a 0.).6) 2160.0a 0.75.0 (3077.8 82.0a 0. a precondition most likely to be fulfilled by compounds such as kaempferol that are widely distributed in plant foods.7 1348. 91.0 478. and if the bioavailability of the compound is not particularly low. and that the correlations are much lower when intake calculations are based on data obtained either three or seven days prior to blood sampling.9 2641. (nmol/l) Max.3) 652.6 2542.9) 267.2) 260.1 6849.5 Mean (SD) Min.8 . Due to the short half-life time of most polyphenols.0a 0.4) 107.5) 908.8 0.0a 0.9 563.0a 19.4) 10. Sabine Rohrmann 128 found to be rather high (Table 3.8 158.9 4528.8 1121.3 (153.2) 1031.0a 0.3 388.8 (15.2 (17.0 742.3 1253.3 (42.5.0 0.1 (203.6 460.
little use has been made of biomarker measurements. One study reported correlation coefficients of between 0.0 108. several studies on associations with the risk of cardiovascular diseases and with cancer at different sites.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 129 Table 3.8 5. although this has not been investigated tested extensively.9) 31. . To give an example of the polyphenol concentrations found in 24-h urine samples of subjects on a habitual diet. Nielsen and coworkers  reported average (SD) concentrations of quercetin.7) 545.3) 0.8 (80.1 (40.8 Below the detection limit.8) 157. (nmol/l) Max.6). In the following. 122. 701(659).0a 0. just as was indicated already for the plasma concentrations. some of which appear very promising. 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. In large-scale epidemiologic (etiological) studies of disease-related effects of dietary polyphenol levels. participating in a cross-sectional study (according to Bolarinwa and Linseisen  — cont. 1638(1316) µg/24 h.7 388.2 639.9) 126. The urinary polyphenol excretion rates show a high degree of variability. 50(32).0a 0. Their work provided the basis for the estimation of dietary flavonol and flavone intake.6 36.8 52.24 and 0.3 (1.0a 0. respectively. the correlation coefficients for selected flavonols and flavanones being between 0. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet.1 (24. are taken up. Compound Naringenin Luteolin Genistein Hesperetin Kaempferol Apigenin Isorhamnetin a Median 79.1 27.74 for plasma isoflavone concentrations.0a 0. except as regards the intake of phytoestrogens (isoflavones and lignans).3 14. naringenin.28 and 0.4) 9.7 1356. and total flavonoids as being 25(23).0a 0.38 . 76(110). Only few studies concerning the use of biomarkers of polyphenol intake were found to be available (Table 3.0a 533.5 (21. Plasma and urinary polyphenol concentrations are not expected to reflect long-term or habitual dietary intake.5. the dietary intake estimates being based on FFQ data . Hertog and colleagues were the first to analyze commonly consumed foods in terms of their flavonol and flavone content by means of HPLC.0a 0.2 Mean (SD) Min. kaempferol.2 (4.1) 37. phloretin.1 2555.0 (7.7 151.5 204.
6.  Cohort: nested CCS 97/187 114/219 Serum. genistein. 1999  CCS 60/60 Urine. Sabine Rohrmann 130 Table 3. NS — not significant. equol.  Zheng et al. total phenols Breast NS Sun et al. Table 3. citrus flavonoids Cancer site Breast Result NS Zheng et al. enterolactone Urine.7. 2002  Type CCS N. enterolactone Result Significant inverse association NS Significant inverse association Significant inverse association Significant inverse association Significant inverse association NS 248/492 Plasma. isoflavones Urine. isoflavones. enterolactone Urine. enterolactone Serum. 2002  Cohort 232/772 Urine. Author Ingram et al. Urine. lignans Higher risk when isoflavone estimates are higher CCS — case-control study. cases/ controls 144/144 60/60 18/20 250/250 220/237 194/208 88/268 Specimen.Jakob Linseisen. Epidemiological studies of the risk of breast cancer risk in which biomarkers of dietary isoflavone and mammalian lignans intake were employed. NS — not significant.7.  Dai et al.) may be the ready availability of appropriate immunoassays suitable for measurements on large numbers of samples. isoflavones. 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. tea polyphenols Gastric and esophageal Significant inverse association CCS — case-control study. enterolactone for highest and lowest categories Significant positive association Grace et al. cases/ controls 250/250 Specimen.  den Tonkelaar et al.  Murkies et al. . One of the major reasons for the frequent use of biomarkers of phytoestrogen intake in epidemiological studies (see Table 3. Phytoestrogen Urine.  Piller et al.  Type CCS CCS CCS CCS CCS CCS Cohort: nested CCS Cohort: nested CCS N.  Pietinen et al. Flavonoid Urine.  Hulten et al. isoflavones Urine. lignans Plasma. enabling the attainment of the requirement of sufficient statistical power.
Reproducibility. such as analytical time and costs). To the best of our knowledge. having coefficients of variation close to or above 10% and recovery rates frequently at < 90%. Accounts of analytical procedures that permit a wide variety of polyphenols to be determined in a single run were published recently [16. LC-MS. the immuno-assays being located at the other end of the scale. For all these methods. no systematic findings on repeated within-subject samples or on losses during sample storage are available. except for some few small studies . in which peak identity cannot be confirmed with sufficient certainty. and reports on the quality of the method in question are usually obtained clearly above the detection limit. in which the sample volumes available are usually very small. where the characteristic fragments may be absent. or use of fluorescence detectors. validity and reliability For each well-developed method. This differs from other methods. MS-based systems also have their problems in connection with concentrations close to the detection limit. the concentrations were found to be high enough to enable the characteristic mass fragments to be identified. satisfactory figures concerning the analytical precision and accuracy are available. Antibody-based assays are also subject to failures in specificity due to cross-reactions with other matrix compounds. recovery 90–105%). Detection limits very close to 1 nmol/l of polyphenols have been found for techniques involving HPLC-ECD. Specificity and sensitivity In applying mass-selective detection methods at least to standard mixtures or biological specimens spiked with the compound in question. MS-based systems also have their problems when the concentrations involved are very low. The sensitivity achievable with HPLC-MS. and TR-FIA (immunoassay). HPLC-MS-MS. The higher values here are those for the MS-based techniques (coefficient of variation < 5–7%. the analysis of polyphenol content is restricted in most cases to only a few compounds or classes of compounds. ECD (electron capture detection). . GC-MS and HPLC with use of fluorometric detection is slightly lower.20]. confirmation of their results by use of MS-based techniques is necessary. As already mentioned.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. Sensitivity is a major issue with regard to analytical methods for determining polyphenols in biological specimens. however. such as HPLC-UV/VIS. working at the detection limit of a method is always a challenge. In epidemiological studies in particular. the degree of sensitivity a method provides can be decisive for whether it can be used or not (along with other factors. However.
91:447–57.86:415–21. Rantala M. Scalbert A.54:143–9. Bolarinwa A.823:143–51. van Staveren WA. 142. 8. Eur J Clin Nutr 2002. Br J Nutr 2001. Key TJ. Riboli E. 4. Donovan JL. Am J Clin Nutr 2005. Am J Clin Nutr 2005. J Chromatogr B Analyt Technol Biomed Life Sci 2005. Sabine Rohrmann 132 References 1. Merken HM. Crozier A. Measurement of food flavonoids by high-performance liquid chromatography: A review. Alfthan G. Hollman PC.81 Suppl 1:243S–55S. Prediction of dietary flavonol consumption from fasting plasma concentration or urinary excretion. Flavonoids in human urine as biomarkers for intake of fruits and vegetables. 2. Polyphenols: food sources and bioavailability. Free Radic Res 2004. 14.41:203–9. Am J Clin Nutr 2004. 3. Sinha R.65:339–48. Williamson G. Beecher GR. IARC Scientific Publications No. Hampl R.56:891–8. Eur J Nutr 2002. Jimenez L. Pharmacokinetics and metabolism of dietary flavonoids in humans. Linseisen J. Review of 97 bioavailability studies. 9. Verkasalo PK. Noroozi M. Am J Clin Nutr 1998. 11. Eur J Clin Nutr 2000. 10. In: Toniolo P et al. 12. 6. Kesaniemi YA. 15. Wang GJ. Ritchie MR. Burns J. Lyon: IARC. Manach C. Review of 93 intervention studies. Linseisen J. Radtke J. Morton MS. Stumpf K. Plasma concentrations of the flavonoids hesperetin. Bioavailability and bioefficacy of polyphenols in humans. Silaste ML. editors. Biochemical markers of dietary intake. Williamson G. Appleby PN.11:459–66. Lean ME. 13. Meyboom S. et al. Deighton N. J Agric Food Chem 2000. Morand C. Manach C.Jakob Linseisen. Kelly IE. Robbins RJ.81 Suppl 1:230S–42S.68:60–5. Phenolic acids in foods: an overview of analytical methodology.79:727–47. 5. Buysman MN. . Nielsen SE. et al.48:577–99. Cummings JH. II. J Agric Food Chem 2003. Plasma and urinary phyto-oestrogens as biomarkers of intake: validation by duplicate diet analysis. Cancer Epidemiol Biomarkers Prev 2002. 7. Scalbert A.38:771–85. Application of Biomarkers in Cancer Epidemiology. Blake A. Zock PL. Fasting plasma concentrations of selected flavonoids as markers of their ordinary dietary intake. 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). and diets high or low in fruit and vegetables. Freese R. Wolfram G. 1997. Kaaks R. Erlund I. Lapcik O. Mutanen M. de Vries JH. Adlercreutz H. Manach C. Remesy C. Allen NE. Time-resolved fluoroimmunoassay of plasma daidzein and genistein. Manach C. naringenin and quercetin in human subjects following their habitual diets. 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. Morand C.. Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake. Remesy C. 16. Al-Maharik N. Davey G. I.51:2866–87. Aro A. Steroids 2000. Kleemola P. Br J Nutr 2004. Bioavailability and bioefficacy of polyphenols in humans. Uehara M.
22:241–3. Kataja V. Wen WQ. Biofactors 2004. Carcinogenesis 2002. Plasma enterolactone and genistein and the risk of premenopausal breast cancer. et al. . Lee MJ. Sun CL. Briganti EM. et al. Eur J Nutr 2002.11:815–21.7:289–96. Cancer Epidemiol Biomarkers Prev 2004.10:223–8. Lancet 1997. 19. Yuan JM. Veer PV.13:698–708. Zheng W. et al. Chang-Claude J. et al. Hebert JR.8:35–40. China. Shu XO. Ito H.15:225–32. Den Tonkelaar I. Adlercreutz H. 25.41:168–76. Urinary excretion of phytoestrogens and risk of breast cancer among Chinese women in Shanghai. Manach C. Dai Q. 22. Kolybaba M. Winkvist A. Grace PB. Johansson R. Custer LJ. Murkies A. et al. Phytoestrogens and breast cancer in postmenopausal women: a case control study. Hallmans G. Yang CS. Healy DL. 18. Low YL. Lenner P. Urinary tea polyphenols in relation to gastric and esophageal cancers: a prospective study of men in Shanghai. Stumpf K. Remesy C. et al. Burger HG. 27. Adlercreutz H.10:339–44. Phytoestrogen concentrations in serum and spot urine as biomarkers for dietary phytoestrogen intake and their relation to breast cancer risk in European prospective investigation of cancer and nutritionnorfolk. Thijssen JH. Urinary phytoestrogens and postmenopausal breast cancer risk. Case-control study of phyto-oestrogens and breast cancer. 20. Cancer Epidemiol Biomarkers Prev 2001. Wahlqvist ML. Keinan-Boker L. Taylor JI.350:990–4. Jin F. Linseisen J. Mannisto S. Cancer Epidemiol Biomarkers Prev 2002. Franke AA. Mennen L. Jin F. Arts CJ. Urinary excretion of isoflavonoids and the risk of breast cancer.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 133 17. 23. Cancer Epidemiol Biomarkers Prev 2001. Dalais FS. Adlercreutz H.23:1497–503. Botting NP. Lopez D. Uusitupa M. Mulligan AA. Gao YT. Shu XO. Dai Q. Hulten K. 26. Serum enterolactone and risk of breast cancer: a case-control study in eastern Finland. Sanders K. et al. 21. Piller R. Gonthiera MP. Cancer Epidemiol Biomarkers Prev 1999. Menopause 2000. Eur J Cancer Prev 2006. Ross RK. Ingram D. Luben RN. An incident casereferent study on plasma enterolactone and breast cancer risk. Custer LJ. High-throughput profiling of dietary polyphenols and their metabolites by HPLC-ESI-MS-MS in human urine. Morand C. 24. Pietinen P.
Soterios A. which represent major antioxidants in olive oil. considered as putative nutritional biomarkers in relation to cancer the incidence of cancer. a dose-dependent decrease in the urinary excretion of the F2-isoprostane 8-iso-PGF2α. are dose-dependently absorbed in humans after the ingestion of realistic doses of virgin olive oil. the excretion of both HValc and HVA also being significantly correlated with the dose of administered hydroxytyrosol. a number of simple and bound phenolics (tyrosol.2.. Sotiroudis and Soterios A.).8. Anticarcinogenic compounds of olive oil and related biomarkers Theodore G. Kyrtopoulos 134 3. two secoiridoids (dialdehydes related to oleuropein and ligstroside but lacking the carboxymethyl group at C4). a statistically significant negative correlation has been found between homovanillyl alcohol (HValc. A brief overview is presented of recent findings concerning the bioavailability of certain minor but important olive oil minor components (polyphenols.2]. together with homovanillic acid — HVA) and isoprostane excretion. Epidemiological studies demonstrate rather conclusively that populations within Europe consuming this diet have a particularly low incidence of a number of common cancers [1. This indicates olive oil phenolics to maintain their antioxidant activities in vivo. a biomarker of in vivo lipid peroxidation processes. Greece Olive oil is an important ingredient of the Mediterranean diet. that have been thoroughly studied. tyrosol. 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.3. the triterpene hydrocarbon squalene and the phytosterol β-sitosterol [1–3]. Athens. These include tocopherol and carotenoid antioxidants. Kyrtopoulos Institute of Biological Research and Biotechnology. When olive oil samples containing increasing amounts of a phenolic extract of olive oil were administered to human volunteers. the aglycone of ligstroside. oleuropein. Figure 3. hydroxytyrosol. In addition. lignans. squalene and β-sitosterol). secoiridoids and lignans). The National Hellenic Research Foundation. Sotiroudis. was observed. A plethora of minor constituents in olive oil have been identified as being effective agents mitigating against the initiation. 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 . Phenolic compounds HPLC chromatography of the methanol extract of virgin olive oil reveals seven major polyphenol peaks corresponding to hydroxytyrosol. Thus. HValc in urine reflects the in vivo .Theodore G. and a peak containing the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol [1–3] (Table 3. Oleuropein and its metabolites tyrosol and hydroxytyrosol. a major metabolite of hydroxytyrosol. promotion and progression of multistage carcinogenesis.
Concentrations of the major phenolic compounds found in virgin olive oil Compounds Tyrosol Hydroxytyrosol Oleuropein aglycone Total secoiridoids Lignans References  and . An HPLC method for the simultaneous determination of oleuropein and of its metabolites hydroxytyrosol and tyrosol in human plasma has been developed [13. Structures of certain phenolic compounds detected in olive oil. 3.75  14. It was observed in this respect.83–4. Hydroxytyrosol (A). 38–65  Fig. After the ingestion of olive oil of low phenolic content plasma glutathione peroxidase activity was found to decrease postprandially. Recent findings suggest that olive oil may also affect the bioavailability of other food bioactive components with a chemopreventive potential.45 .2.Bioactive components in foods: Anticarcinogenic compounds of olive oil and related biomarkers 135 concentration of hydroxytyrosol [5–11]. 1.8. oleuropein aglycone (B). a biomarker of the intake of tomato-rich food and hypothesized to be responsible for reducing the risk of various . that the concentration in human plasma of lycopene.65–4.42 . mg/kg 27. (+)-pinoresinol (C). but this was not observed after the intake of olive oils of moderate to high phenolic content .72  41.14].53 .71  103–205  27. 2. Table 3.
It has been observed that postprandial squalene metabolism is age dependent . The consumption of tomato products prepared together with olive oil. particularly in premenopausal women . Nevertheless. proapoptotic and anti-oxidant mechanisms established for them [17. as compared to the consumption of tomatoes cooked without olive oil . Lignans are plant compounds metabolized in the gut to produce the phytoestrogens enterolactone and enterodiol. their reaching a steady state. This triterpene hydrocarbon is found mainly in nonedible shark liver oil. Recent findings suggest that enterolactone is more rapidly metabolized in human colon epithelial cells and/or excreted by them than enterodiol is. If virgin olive oil were the sole source of dietary fat. suggesting that dietary lignans may be important in the etiology of breast cancer. leading to a reduced farnesyl pyrophosphate availability for prenylation of the ras oncogene . while virgin olive oil is a major source of phytosqualene. its content ranging from 800 to 12. Experiments in vitro and animal models suggest squalene to play a tumour-inhibiting role. restricted almost entirely to estrogen-receptor alpha negative breast cancer has been found. a tendency for a lower risk of breast cancer to be associated with higher plasma concentrations of enterolactone. 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. increased dramatically after the consumption of tomatoes cooked in olive oil. Sotiroudis.000 mg/kg. that the phase II metabolism of enterolactone and enterodiol already may take place during their uptake in the colon. Soterios A. Although animal studies have enhanced our understanding of the possible . but not with sunflower oil. 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 ). and that the epithelial cells in the colon may be responsible for this metabolism . Nevertheless. which expands the plasma pool of this metabolically active hydrocarbon . very little is known concerning the postprandial metabolism of squalene.Theodore G. the squalene intake would be more than 200 mg/d . was found to improve the antioxidant activity of plasma . 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. Kyrtopoulos 136 cancers. which is most probably based on its strong inhibitory action on the catalytic activity of beta-hydroxy-beta-methylglutaryl-CoA reductase. and that the content of squalene in the whole plasma and in the lipoprotein fractions (where its ratio to cholesterol is highest in the VLDL and the intermediate density lipoproteins ) varies directly with the triglyceride content and is increased in hypertriglyceridemia. Phytoestrogens have an anticarcinogenic potential through the anti-estrogenic. whereas the findings of epidemiological studies are controversial . Plasma enterolignan concentrations can thus be considered to be good biomarkers of dietary lignan exposure and be used to evaluate the effects of lignans .18]. anti-angiogenic. 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 . 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 . Together with squalene, they represent markers of cholesterol synthesis and absorption and are transported together with cholesterol in serum lipoproteins . β-Sitosterol, one of the most common phytosterols and the main olive oil sterol , 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 . 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 , 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.
gluconapin. Subsequently. formed by the plant from any one of eight amino acids.3. the olefinic glucosinolates sinigrin. The types of neoplastic disease in man that these vegetables appear to protect against include colorectal cancer . Claverton Down. Hayes1. used in their biosynthesis. which is in turn conjugated with glucuronic acid to form a desulfoglucosinolate before finally being sulfated to yield the glucosinolate . Glucosinolates are substituted β-thioglucoside N-hydroxysulfates. phenylalanine and methionine. This endeavour is simplified to some extent by the fact that relatively few glucosinolates are present in the human diet. Ninewells Hospital and Medical School. Glucoraphanin has been reported to be abundant in broccoli . isomerisation and decarboxylation reactions .) [9. Each cruciferous vegetable contains a mixture of glucosinolates that varies according to the strain of the plant [8. tyrosine and tryptophan .2]. glucobrassicanapin and progoitrin.20]. Michael O.John D. methionine. the synthesis of glucosinolates proceeds through the conversion of elongated amino acids to their oxime derivatives. Kelleher. though it can be influenced by environmental factors [16. Much of the diversity amongst glucosinolates arises from the addition of different sized alkyl groups to the side chain of those amino acids. United Kingdom Glucosinolates and their association with cancer chemoprevention Epidemiological studies have revealed that regular consumption of cruciferous vegetables. Cruciferous vegetables uniquely contain glucosinolates at approximately 20 µmol/g dry mass of vegetable [8. alanine. Feeding experiments in animals have also suggested broccoli can protect against liver cancer . namely. cauliflower. though certain broccoli . Dundee DD1 9SY Department of Pharmacy and Pharmacology. and it is thought that these phytochemicals are primarily responsible for the putative cancer chemoprevention conferred by eating diets that contain significant quantities of these vegetables [10. Furthermore.9.4. Michael O. Ian M. The task of establishing a link between the ingestion of particular glucosinolates and their possible health benefits is not straightforward. Kelleher1 and Ian M. leucine. the oxime is metabolised to a thiohydroximate. lung cancer . As shown in Figure 3.17]. catalysed by members of the cytochrome P450 (CYP) 79 family . Over 115 naturally occurring glucosinolates have been identified. Eggleston 140 3. Brussels sprouts. The glucosinolate content is primarily under genetic control.11]. this variable elongation of amino acid side chains entails repetitive additions of methyl groups through a series of transamination. phenylalanine. greater health benefit may be obtained from raw as opposed to cooked vegetables . condensation. and possibly prostate cancer . University of Bath. and the aromatic glucosinolate gluconasturtiin (Table 3. The most common of these are the methylsulfinylalkyl glucosinolates glucoiberin and glucoraphanin. kale. swede and turnip.. valine. principally valine. Bath BA2 7AY. Anticarcinogenic effects of glucosinolate breakdown products John D. University of Dundee. isoleucine. Hayes. cabbage. Eggleston2 1 2 Biomedical Research Centre. is associated with a reduced incidence of cancer [1.12–15]. such as broccoli.9].
The R group is derived from the original amino acid and is highly variable.3. Brussels sprouts and cauliflower [9. The aromatic glucosinolate gluconasturtiin is present in watercress. cauliflower and kale . Substantial amounts of progoitrin are present in many cruciferous vegetables .Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 141 strains also contain substantial amounts of glucoiberin . 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. Sinigrin has been reported to be the predominant glucosinolate in Brussels sprouts. gluconapin is also found in high levels in Brussels sprouts .22]. . The indolyl glucosinolate glucobrassicin is present in Savoy cabbage. cabbage. and whilst not abundant it can elicit distinct pharmacological effects.9. 3. Table 3. Synthesis of glucosinolates.
). suggesting that glucosinolates can be hydrolysed in the gastrointestinal tract during digestion of food [24. Kelleher. Michael O.147). Ian M. thiocyanates.3.4A. myrosinase is released from the “myrosin” cells and is able to hydrolyse glucosinolates within the damaged plant. resulting in the formation of an epithioalkane. Hydrolysis of these phytochemicals is catalysed by myrosinase (β-thioglucoside glucohydrolase. Hydrolysis of glucosinolates.4B. At high or neutral pH the formation of isothiocyanates is favoured while at low pH the formation of nitriles is favoured.2. myrosinase activity may be present in human colonic microflora. Eggleston 142 Production of isothiocyanates. or during chewing whilst eating. 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 3.4A. 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 . Upon wounding of the vegetable. but rather it appears to be due to certain of their breakdown products. Fig.25]. nitriles. 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. In addition. EC 3. as well as the reaction temperature.John D. 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. Hayes. during freeze-thawing. during food preparation. and 3. 3. . The outcome of the reaction with myrosinase depends on the nature of the aglycone. for example during harvesting.
olefinic and aromatic glucosinolates undergo a Lossen rearrangement. Following damage to the plant tissue the glucosinolate sinigrin is hydrolysed by myrosinase resulting in the formation of four distinct compounds. Elemental sulfur is also formed in certain circumstances. glucoraphanin. The thiohydroximate-O-sulfates formed from methylsulfinylalkyl. to form their respective isothiocyanates (ITCs). gluconapin. On the right-hand side of the figure. gluconapin and glucobrassicanapin) may. at pH 4 and in the presence of Fe2+ ions. Hydrolysis of sinigrin. Thus.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 143 ESP — epithiospecifier protein.g. such as allyl-ITC. Certain thiocyanates that are formed during a Lossen rearrangement. thiocyanates or nitriles [10. the thiohydroximate-O-sulfates formed by myrosinase from glucosinolates with a side chain containing a double bond (e. In this case. in the presence of an epithiospecifier protein (ESP) and ferrous ions. hydrolysis of glucosinolates with aliphatic or aromatic side chains gives rise primarily to isothiocyanates (ITCs). glucobrassicanapin and sinigrin yield 3-methylsulfinylpropyl-ITC. 4-methylsulfinylbutyl-ITC (sulforaphane). can convert spontaneously to form allyl isothiocyanate . 3. Fig. give rise to a cyano-epithioalkane . 3-butenyl-ITC. 4-pentenyl-ITC and 2-propenyl-ITC (allyl-ITC).23]. sinigrin. At neutral pH. respectively. an arrow shows that allyl thiocyanate. At low pH. formed from sinigrin. are unstable and can undergo a relatively slow spontaneous conversion to their respective isothiocyanate . with the elimination of sulfate. .4B. The glucosinolates glucoiberin. 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 .
Production of indoles from glucosinolates. those aglycones from glucosinolates that contain β-hydroxylated sidechains form oxazolidine-2-thiones.5.5. Ian M. 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. Likewise.31]. and is diminished by heating . Kelleher. Hayes. coli [35. Eggleston 144 myrosinase and ESP convert sinigrin to 1-cyano-2.34].5-epithiopentane . Upon hydrolysis by myrosinase. ESP and Fe2+ ions to an epithionitrile  and in the case of epi-progoitrin ((2S)-hydroxy-3-butenyl glucosinolate). it can be hydrolysed by myrosinase to crambene (1-cyano-2-hydroxy-3-butene) [33. If the aglycone generated by myrosinase is from a glucosinolate with a side chain lacking a double bond. This reaction may involve ESP. The best studied of these is glucobrassicin.3-epithiopropane . Production of indoles from glucosinolates The indolyl glucosinolates glucobrassicin and neoglucobrassicin are synthesised by the plant from tryptophan. At neutral pH. Two cDNAs for ESP have recently been cloned from Arabidopsis and broccoli and the purified proteins characterized following their heterologous expression in E. as a consequence of spontaneous cyclization. glucoconringiin and gluconapoleiferin .4-epithiobutane [30. but rather gives rise to indole-3-carbinol and a thiocyanate ion (Figure 3. 3. a (2R)-hydroxy-3-butenyl glucosinolate. Gluconapin can similarly be converted by the combined actions of myrosinase and ESP to 1-cyano-3. Examples of these include progoitrin. is also converted in the presence of myrosinase. Progoitrin. this reaction probably proceeds Fig. glucobrassicanapin can be hydrolysed to 1-cyano-4. .John D. A few glucosinolates produce thiocyanates though the mechanism involved is unclear .36]. Michael O.). the sulfur atom may be lost and a nitrile formed [37–39]. hydrolysis of glucobrassicin by myrosinase does not generate an ITC.
Agents such as ITC that increase GST and NQO1 . nitriles.). 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. both in vivo and in vitro [45.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 145 through a Lossen rearrangement generating an unstable ITC intermediate . A major problem exists in interpreting experiments utilizing vegetable extracts because of uncertainty about attributing biological effects to specific phytochemicals. Induction of gene expression mediated by Nrf2 Isothiocyanates increase the expression of antioxidant and detoxication proteins.3∂-diindolylmethane. In the acidic environment of the stomach.44]. cyanoepithioalkanes. 3. both of which have potent pharmacological effects . there is a relative abundance of information about isothiocyanates and indoles. 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. hydrogen sulfide and elemental sulfur .46]. and oxazolidine-2-thiones.2-b] carbazole (ICZ) and ascorbigen (ASG). whereas activation of cell cycle arrest and apoptosis occurs at higher levels of phytochemical [43.6. There is a dose-dependency in these responses: generally. when compared with the data about thiocyanates. and stimulation of apoptosis. These include induction of antioxidant and detoxifying genes. Fig.6. indole-3-carbinol condenses to form various compounds including indolo[3. Structures of indoles produced from glucosinolates — 3. A challenge in evaluating the literature arises from the emphasis placed on certain glucosinolate breakdown products and the dearth of data relating to others. such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1).2-b]carbazole and 3. inhibition of CYP enzyme activity. indolo [3. hydrolysis of glucobrassicin yields indole-3-acetonitrile. Thus. cell cycle arrest. It can also combine with ascorbic acid to form ascorbigen  (Figure 3.3’-Diindolylmethane (DIM). At acidic pH. induction of cytoprotective genes and inhibition of CYP activity occurs at relatively low concentrations of phytochemical.
Sulforaphane. leading to its inhibition and inability to serve as a substrate adaptor . ITCs modify cysteine residues in the Cullin 3:Rbx1 E3 ubiquitin ligase substrate adaptor Keap1. Michael O. induce NQO1 in the mouse Hepa-1c1c7 hepatoma cell line [48. allyl-ITC. all genes that are induced by ITCs contain an ARE in their promoters and are regulated by Nrf2.John D. metallothionein. Eggleston 146 enzymes without increasing arylhydrocarbon hydroxylase activity. 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 . Importantly.7. 3) Benzyl-ITC. suggests AREs are present in the promoters of at least 100 genes [53–55]. multidrug resistance-associated protein. thioredoxin reductase. GST. ferritin.49] (Figure 3. 4) 3-methylsulfinylpropyl-ITC. catalysed by the phase I drug-metabolising enzyme cytochrome P450 (CYP) 1A1.7. GST and NQO1 in the gastrointestinal tract in an Nrf2-dependent fashion . as far as is known. Indeed. small intestine and liver of rodents [53. as does the rat GSTP1 gene (called GPEI) . Through this characteristic. Hayes. 5) 7-methylsulfinylheptyl-ITC. Fig. are sometimes called mono-functional inducers . Examination of nrf2–/– and wild-type mice. glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits. The mouse nqo1 gene contains a functional antioxidant response element (ARE) in its 5∂-upstream region . The battery of genes regulated by Nrf2 includes those encoding aldo-keto reductase (AKR). 7-methylsulfinylheptyl-ITC. Ian M. It is thought that ITCs possess the ability to induce ARE-driven gene expression because they are thiol-active . Many of these genes are induced by sulforaphane in vivo in an Nrf2-dependent fashion in the stomach. feeding broccoli seed to mice increased the levels of GCLC. microsomal epoxide hydrolase. carboxyl esterase. 6) 8-methylsulfinyloctyl-ITC. 3-methylsulfinylpropyl-ITC.54. 2) Phenethyl-ITC. 3.55–58]. NQO1. UDP-glucuronosyl transferase. 8-methylsulfinyloctyl-ITC. Structures of isothiocyanates which induce NQO1: 1) Allyl-ITC. Kelleher. Many of these compounds induce GST P1-1 in the rat liver RL-34 cells .). thioredoxin. heme oxygenase 1. benzyl-ITC and phenethyl-ITC.
rat UGT1A1. and thereby generate reactive oxygen species. and rat UGT1A6 contain an XRE. and this may also contribute to gene induction .69]. it is a good inducer of GST enzyme activity in mouse liver and small intestine . a fact that implies it works through Nrf2.2-b]carbazole and 3. Examination of the dose of indole required to double XRE-driven reporter gene expression shows that indolo[3. rat and human CYP1A1 are prototypic XRE-regulated genes. rat GSTA2. Consistent with this view.3∂-diindolylmethane. Amongst genes for drugmetabolising enzymes. However. The promoters of other genes including rat and human NQO1.67].2-b]carbazole is a much more potent inducing agent than 3. Although indole-3-acetonitrile has not been studied in the Nrf2 knockout mouse.64]. indole-3-carbinol or ascorbigen [43. and there is abundant evidence from enzyme assays. Indole-3-carbinol is a modest inducer of Nrf2-dependent ARE-driven gene expression in the liver and small intestine of mice [52. rat ALDH-3. as does the mouse BAX gene.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 147 required for the ubiquitination of Nrf2 under homeostatic conditions [60–62]. see ).3∂-diindolylmethane . but unless they are metabolised by CYPs. suggesting it is a mono-functional inducer . 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. By comparison with sulforaphane. The identity of this metabolite is not known. but not CYP1A1. Treatment of cells with benzyl-ITC causes a rapid increase in the level of reactive oxygen species. The indole-containing glucobrassicin breakdown products can activate gene expression by several mechanisms. Surprisingly. and it is therefore anticipated that activation of AhR by glucobrassicin-derived indoles will influence significantly the metabolism of xenobiotics (for a review. exposure of RL-34 cells or HepG2 cells to sulforaphane causes stabilisation and rapid accumulation of Nrf2 [63.2-b]carbazole and 3. .3∂-diindolylmethane can increase the stability of Nrf2 protein is not known. 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. 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. This cytochrome has O-deethylase activity towards ethoxyresorufin. Interestingly. this seems unlikely . allyl-ITC does not increase Nrf2 stability  and there may therefore be other factors involved in enzyme induction by these phytochemicals. this may not necessarily lead to an increase in Nrf2 protein levels. western and northern blotting that CYP1A1 is inducible by indolo[3.2-b]carbazole and 3. It has been found that administration of cranbene to Fischer 344 rats causes an elevation in hepatic GST and NQO1 enzyme activities. cranbene was found to be an approximately equally potent inducer in the rat . indicating that AhR ligands could lead to an increased production of Nrf2 mRNA. mouse.3∂-diindolylmethane. the mouse nrf2 gene promoter also contains an XRE .
2-b]carbazole before exposure to benzo[a]pyrene provides a small measure of protection against DNA damage as measured by the comet assay. This function is likely to alter gene expression substantially. the basic structural unit of chromatin. sulfotransferase and UGT1 . Hayes.84]. 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. Michael O. Furthermore. 3. This viewpoint is probably an oversimplification and does not take into account the multiple changes in gene expression that indoles affect. In the case of the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. such as growth arrest and DNA damage (GADD) GADD34. NF-IL6 and the GADD proteins are regulated through XREs and whether the process is mediated by AhR. It is not however clear whether the genes for ATF3. GADD45 and GADD153 [75. Tumorigenesis caused by the carcinogens 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and N-nitrosobis-(2-oxopropyl)amine can be prevented by phenethyl-ITC through a process that involves inhibition of activation of pro-carcinogens by CYP isoenzymes [78. such as CYP1B1 and CYP19 . GST T1-1. DNA is tightly coiled around an octamer of histone proteins in a structure known as a nucleosome.2-b]carbazole and sulforaphane before exposure to benzo[a]pyrene was found to confer substantial protection against genotoxicity. Also. prior treatment of the LS-174 cells with both indolo[3.81]. Eggleston 148 In addition to induction of CYP1A1 by indoles. Inhibition of CYP can also be achieved by sulforaphane [80. Each of the histone proteins contains an evolutionary conserved amino tail protruding from . other cytochromes are inducible. c-Jun and NF-IL6 as well as genes involved in cell growth. and this protection was greater than was achieved by either phytochemical alone . recognition of this possibility has lead to considerable recent interest in the ability of HDAC inhibitors to act as both chemopreventive and chemotherapeutic agents. 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. the ITCs with highest lipophilicity and low reactivity of their NCS group had the greatest ability to inhibit lung tumorigenesis . p21 is induced by indole-3-carbinole .3∂-diindolylmethane can induce expression of the transcription factors ATF3. Inhibition of carcinogen activation by glucosinolate breakdown products Certain isothiocyanates can block the activation of several carcinogens to their ultimate carcinogenic forms.76]. c-Jun. Within the cell. Most importantly.79]. Indeed. as are the drug metabolising enzymes AKR. Kelleher.John D. Bonnesen et al  have reported that treatment of human colon LS-174 cells with indolo[3. Inhibition of histone deacetylase by isothiocyanates The isothiocyanate sulforaphane has been shown to inhibit histone deacetylase (HDAC) [83. Ian M.
For example. along with other dietary HDAC inhibitors such as diallyl disulfide .85]. Thus the ability of sulforaphane.92]. by deacetylation of histones associated with the p53 gene. to affect chemoprevention via its ability to alter chromatin structure is of acute importance to the welfare of the cell. The addition and removal of the acetyl groups is carried out by HAT and HDAC. The tail is also subject to many post-translational modifications including acetylation. this was associated with a higher level of apoptosis and lower proliferation in animals on the ITC containing diet . Equally. In addition. allowing transcription factors access to the regulatory regions of genes. In pre-cancerous and cancerous cells. sulforaphane has been found to cause a G2/M phase delay with an increase in apoptotic cell fraction in a timeand dose. Conversely. Relocation of Cdc25C was found to be controlled by its phosphorylation at Ser-216. 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. In human PC-3 prostate cancer cells. and was accompanied by its translocation from the nucleus to the cytoplasm . The loss of Cdc25C was reported to be due to proteasomal activity. However. resulting in a reduction in its transcriptional activity. mediated through activation of checkpoint kinase 2 (Chk2) . p53  and Bax [84.85]. removal of acetyl groups causes the tail to wrap tightly around the DNA thereby preventing interaction with the transcription machinery. . 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 majority of the studies into mechanisms by which this class of chemical inhibit cell growth have focussed on sulforaphane and phenethyl-ITC. apoptosis and differentiation . Sulforaphane has been shown to diminish HDAC activity with a concomitant rise in histone acetylation in prostate cancer cells . mammalian HDAC is capable of downregulating p53 function.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 149 the nucleosome.dependent fashion . The link between inhibition of HDAC activity and the resultant increase in transcription of tumour suppressor genes has been reported for p21 [84. Cell proliferation by ITCs may also be achieved by distrupting cytoskeletal structure and tubulin polymerisation [93. Inhibition of HDAC may prevent the removal of acetyl moieties from histones thus allowing transcription of the tumour suppressor genes. which can determine the accessibility of the DNA to transcription factors. tumour suppressor genes are associated with deacetylated histones resulting in the inactivation of these genes. human embryonic kidney cells  and human colorectal cancer cells .94]. 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. 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. respectively.
and this is thought to amplify the effects of Bak and Bax . Eggleston 150 Administration of ITCs to cells at growth suppressive concentrations results in the rapid generation of reactive oxygen species (ROS). ITCs down-regulate the anti-apoptotic proteins Mcl-1 and Bcl-xL . are increased by phenethyl-ITC and sulforaphane in PC-3 prostate cancer cells. in human leukaemia HL60 cells. though this may be cell specific. However.95]. ERK1/2 . in PC-3 cells sulforaphane can increase Fas protein levels and activate caspase-8 whilst simultaneously targeting mitochondria and activating caspase-9 . the mechanism by which JNK activates caspases remains unclear. Cell cycle arrest at G1 occurs as a consequence of indole-3-carbinol inhibiting both cyclin-dependent kinase (CDK) 2 and CDK6. For example. Besides increasing the levels of these pro-apoptotic proteins. It therefore appears that cyclin D-CDK6 activity can be inhibited by dual mechanisms. Kelleher. as does pre-treatment with N-acetylcysteine . Various ITCs have been shown to activate c-Jun N-terminal kinase (JNK) [102. Furthermore.97]. Furthermore. Michael O. Hayes. In the case of CDK2. There is evidence that ITCs can activate both the intrinsic and extrinsic caspase cascades. in HaCaT keratinocytes. benzylITC and phenethyl-ITC are more effective at activating caspase-9 than caspase-8 . treatment with 400 µM indole-3carbinol induces the CDK4/6 inhibitor p15INK4b mRNA and protein causing hypophosphorylation of Rb protein . Furthermore. By contrast.92. In the case of CDK6. addition of GSH subsequent to ITC treatment can block apoptosis [44. within 1 h of exposure. and this may lead to induction of Apaf-1 [91. In human bladder cancer UM-UC-3 cells. 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 .103].John D. 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 . 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 . expression of the gene is reduced because indole-3-carbinol attenuates recruitment of the Sp1 transcription factor to the CDK6 promoter . The levels of pro-apoptotic proteins Bak and Bax. Ian M. This may in part be due to 3. which neutralize the antiapoptotic effects of Bcl-2.3∂-diindolylmethane rather than indole-3-carbinol as significant quantities of the indole spontaneously condense to the dimer in culture conditions . which appears to be necessary for cell death [92. and this is mediated by extracellular signal-regulated kinases. Consistent with the view that production of ROS is necessary for apoptosis. though the effect is cell-specific .101]. overexpression of catalase suppresses ITC-initiated programmed cell death. indole-3-carbinol has been . the pro-apoptotic proteins Bok and Bim EL are also induced by ITCs. caspase-8 plays a major role in apoptosis stimulated by phenethyl-ITC . Treatment with ITCs leads to a loss of mitochondrial membrane potential and release of cytochrome c from mitochondria .
a TGF-β family member associated with pro-apoptotic activities .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 . at the expense of forming isothiocyanates. It is unclear whether formation of thiocyanates. and the larger complex also contains an additional 75 kDa cyclin E immunoreactive protein. cyanoepithioalkanes and oxazolidine-2-thiones. These changes result in prevention of the CDK2-mediated G1/S transition . the reduction in CDK2 activity is accompanied by redistribution of the kinase in the 200 kDa complex from the nucleus to the cytoplasm. Thus. Indole-3-carbinol. suggesting that indole-3-carbinol can influence the nucleocytoplasmic shuttling of the kinase . ESP should possibly . from an active form within a 90 kDa complex to a lower activity form within a 200 kDa complex . This may also mediate the anti-tumour effects of indoles. Concluding comments It is becoming clear that glucosinolate breakdown products can influence the initiation and progression of carcinogenesis.115]. Treatment of PC-3 prostate cancer cells with 60 µM indole-3-carbinol inhibits the EGF-induced autophosphorylation of PI3K and Akt . an increase in p53 protein levels. is undesirable. such as tamoxifen . nitriles. treatment with 300 µM indole-3-carbinol or 30 µM 3. induction of p21 . They also appear to influence apoptotic responses to chemotherapeutic agents. The reduction in CDK2 activity is attributed to a selective alteration in the size of the complex in which it is contained. Furthermore. cyano-epithioalkanes and oxazolidine-2-thiones from glucosinolates. If so. In HCT-116 human colon cells. Indoles can affect apoptosis in breast and prostate cancer cells. indole-3-carbinol can induce nonsteroidal antiinflammatory drug-activated gene-1 (NAG-1). 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. 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 . but not 3. It is unclear whether the activity of ESP. the Akt/PI3K cell survival pathway appears to be targeted by indole-3-carbinol. is undesirable from a cancer chemoprevention perspective. It is possible that down-regulation of this receptor represents an antiproliferative mechanism in prostate cells. In cells that contain wild-type p53.3∂-diindolylmethane. such as MCF-10A. which reduces the formation of isothiocyanates from glucosinolates. Also.3∂-diindolylmethane has been found to result in activation of the ATM signalling pathway. nitriles. Specifically. the 90 kDa and 200 kDa complexes include forms of cyclin E that differ in size. nuclear translocation of NF-κB is inhibited by 3.3∂-diindolylmethane through a reduction in phosphorylation of IκBα [114. there are few data about chemopreventative activities of thiocyanates.
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are far from certain. whole grains. The combinations of phytochemicals that natural foods contain can reduce the risk of cancer by affecting different overlapping and complementary mechanisms. in contrast. van Breda 160 3. Evaluating the effects of phytochemicals that act synergistically Carcinogenesis is an extremely complex multistep process in which numerous molecular mechanisms play an important role. Table 3. summarizes various mechanisms by which phytochemicals can modulate the risk of cancer [1. and there are even reports of increased occurrence of lung cancer in smokers receiving dietary β-carotene supplements [5.5. which are believed to have cancer-preventive effects. 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. . In this chapter. 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. de Kok. Cancer-preventive dietary compounds may interfere at a variety of different levels with these processes. In the research conducted. 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.2]. vegetables. The Netherlands Introduction The relatively consistent epidemiological finding that the consumption of whole foods of different types such as fruits. Although relatively high doses of single bioactive agents may show potent anticarcinogenic effects. Simone G.Theo M. 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. however. Some studies show no reduced incidence of cancer as a result of taking supplements of vitamin C  or β-carotene . de Kok and Simone G. Combined action of different dietary compounds preventive of cancer Theo M. therefore.6]. evaluation of the effects of synergistically acting phytochemicals is needed. and their potential health-promoting effects evaluated extensively. in contrast.10. In addition to characterizing the chemopreventive effects of individual compounds. 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. Isolated and purified compounds. and the like. 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. One of the key problems of research in this field. van Breda Department of Health Risk Analysis and Toxicology. Maastricht. both in vitro and in vivo. numerous bioactive compounds have been isolated and identified. The intended positive effects of an increased intake of β-carotene or vitamin C as dietary supplements. University of Maastricht. evidence that bioactive compounds act synergistically is reviewed.
another green tea polyphenol.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 161 Table 3. Epicatechin was found to promote the occurrence .11. 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. 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. . Suganuma et al.  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. one without a galloyl moiety.10.
EGC and ECG or gallic acid and α-tocopherol Reduced oxidative stress in micelles and human LDL . C and ß-carotene Reduction in α-tocopheryl radicals. reduced co-oxidation of vitamins E. 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. [10. Simone G.  Green tea infusions and grape Reduced tumour cell growth or grape skin extracts Polyphenols. were also enhanced in a dose-dependent way by EC. reduced release of TNF-α Increased production of caspases-3.  Zhou et al. vitamin E.  Liu et al. Inhibition of tNOX.  Zhou et al. Extracellular production of oxygen species Antagonism of TCDD-induced transcription of human CYP1A1 via interaction with the Ah-receptor Suganuma et al. These effects. reduced levels of estrogen receptor-α protein and of serum IGF-I.  hydroperoxides and malondialdehydes.  EGCG and EC Inhibition of cell growth and induction of apoptosis in gastric carcinoma cells Horie et al. induction of apoptosis. 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.  EGCG.  EGCG. enhanced apoptosis. Zhou et al. -8 and -9. induced by a tea polyphenol having a galloyl moiety.11] Polyphenols and other phytochemicals Green/black tea and soy (SPC)* Green/black tea and soy (SPC) Inhibition of prostate tumours. sulindac or tamoxifen Synergistic effect Mechanisms involved Reference Inhibition of growth of human lung cancer cells Enhanced cellular uptake of EGCG. Table 3. 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. trap-ping of lipid peroxyl radicals and regene-ration of vitamin E Zhou et al. de Kok. tumour weight and metastasis Inhibition of breast tumour cell growth Reduction in serum levels of testosteron and DHT Inhibition of tumour angiogenesis. EGC and ECG Modulation of CYP1A1 expression in human hepatocytes Williams et al.11. A and ß-carotene Reduced oxidative stress Morré and Morré  Reduced formation of lipid Gorelik et al. EC. EC.Theo M.
and down-regulation of genes involved in carcinogenesis.  Van Breda et al. Selection of studies of the synergistic effects of mixtures of polyphenols and of combinations of these with other types of phytochemicals — cont. and epicatechin gallate (ECG) — and it was not found to be improved further by the addition of other compounds . 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. 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.  Jørgensen et al. carcinogens or cytotoxic substances . . being due instead to the effects of the complex mixture involved. the modulation of their expression and activity by phytochemicals is a potential mechanism by which the risk of cancer can be reduced. Synergistic effects of green tea catechins. both on the inhibition of cell growth and the induction of apoptosis. epigallocatechin (EGC). EGCG. The optimal degree of synergy was achieved by a combination of the four major tea catechins — EC. 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) . indicating them to be involved in catechin-induced apoptosis. Enzyme induction usually enhances detoxification. but under some circumstances substrates may be activated to become mutagens. Some of the cytochrome P450 genes are expressed constitutively. After this combined treatment. catalase was found to block the synergistic effects of EC and EGCG.24] Different types of vegetables (cauliflower. Since the cytochrome P450 (CYP) enzymes are responsible for the metabolism of many environmental carcinogens.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 163 Table 3. whereas others are inducible by xenobiotic compounds or by phytochemicals. 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. suggesting that the reactive oxidative species and production of hydrogen peroxide play a part in the synergistic mechanisms here . Various gastric cell lines were shown to differ in their susceptibility to EGCG treatment.11. Williams et al. carrots. but it had a significant synergistic effect on the induction of apoptosis when combined with other catechins. peas and unions) Up. 8 and 9 became elevated.  demonstrated that complex green tea extracts exert mixed agonist/antagonist activity on the Ah-receptors. interpreted mainly in preventive terms No indication of specific synergetic mechanisms * SPC — Soy Phytochemical Concentrate. Interestingly. the activity levels of caspases 3. whereas EGCG acts as a strict AhR antagonist. were also found in gastric carcinoma cells . EC alone had virtually no effect on carcinomic cell growth or induction of apoptosis.
the latter a biologically more active metabolite of testosterone and a prerequisite for the development of benign prostatic hyperplasia and prostate cancer . The modulation of these two different mechanisms may be an explanation of the synergistic effects of the combined phytochemicals . These results suggest that more effective cancer prevention and cancer therapy could be achieved by use of combinations of different phytochemicals. Cells in which NOX activity is blocked.). 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. In further investigations it was shown that bioactive compounds in tea (particularly EGCG ) and soy (the soy isoflavone genistein as well as SPC) inhibit growth of prostate cancer and tumour metastasis in vivo . vitamin C can enhance the activity of polyphenols through a synergistic antioxidant effect.Theo M. In line with this. Recently. reduced expression of estrogen-receptor (ER) alpha and reduced serum levels of the insulin-like growth factor (IGF)-I. NOX proteins are located at the cell surface and are responsible for the increase in cell size that occurs following cell division . The strongest synergistic activity was found for ethanol extracts of grape skins.11. In an immunedeficient mouse model in which MCF-7 human breast cancer cells were implanted. Morré and Morré  showed there to be an exceptionally strong (10-fold) synergy between grape polyphenols and tea catechins in the inhibition of tNOX. which is found predominantly in the seeds.13]. van Breda 164 Synergistic effects of polyphenols and other phytochemicals In two different studies. are unable to enlarge. Simone G. Polyphenols and dietary antioxidant vitamins can also have synergistic inhibitory effects on lipid peroxidation and on the co-oxidation of dietary antioxidants. In the gastric fluid. 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 . They cease to divide and eventually undergo apoptosis. This was accompanied by inhibition of tumour angiogenesis. 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. all of these being factors in breast cancer development. increasing at the same time the amounts of vitamin antioxidants reaching the blood system. The authors indicated that the resulting synergistic increase . whereas no activity was detected for extracts of grape seeds. SPC in combination with green tea showed a synergistic inhibition of tumour cell growth. A phytochemical soy concentrate (SPC) and green and black tea infusions were employed (Table 3. indicating that the effects were not caused by resveratrol. Zhou et al. de Kok. 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. the authors suggested that the antioxidant network in the stomach can decrease the level of hydroperoxides and other cytotoxic compounds. such as by phytochemicals. though to a lesser extent) .
This. indicating that combinations of different foods containing different complex mixtures of phytochemicals can also have an antagonistic effect on gene expression. the individual vegetables were able to modulate genes which were not significantly modulated by the mixture. These green tea polyphenols were also found to trap the initiating radicals (ROO•) as well as the propagating lipid peroxyl radicals (LOO•). 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.24]. ECG and gallic acid) can effectively reduce α-tocopheroxy radicals so as to allow α-tocopherol to be regenerated. plays a major role in enhancing the antioxidant efficiency of vitamin E. 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. . When the contributions of the separate phenolic compounds were calculated. and assuming their concentrations to be those typical for red wines. By studying the kinetics of the reaction of α-tocopherolyl radicals with green tea polyphenols by means of stopped-flow electron paramagnetic resonance. In two recent animal studies on the effects of vegetable consumption on the modulation of gene expression.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. Zhou et al. In one study. the synergistic effects of taking whole foods or complex mixtures of compounds have been reported. 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 .22]. it was found that most of the genes that were differentially expressed before and after vegetables were consumed. EGC. the changes in gene expression could be interpreted as a cancer preventive effect [23. 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.  demonstrated clearly that several green tea polyphenols (EC. 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. the Trolox equivalent antioxidant capacity (TEAC) value and phenolic composition were determined for a large number of pinotage wines . Taking account of the different mixtures found of the 12 phenolic compounds that were present. On the other hand. 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 . EGCG. 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. 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.
synergistic effects between these and the other wine constituents may have contributed to the TEAC values.  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. its also reducing side effects. 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. without a loss in treatment potency. Other synergistic effects In addition to investigating the synergistic effects of various phytochemicals. 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 .  demonstrated.28]. de Kok. it appears . however. A review of the effects of various combinations of chemotherapeutic and chemopreventive approaches in dealing with cancer has appeared recently . In that study. Various combinations of drugs have been proposed for further clinical development based on their synergistic activity as shown in vitro or in animal studies. 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 . 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. Administering multiple agents can increase the efficacy and potency of the chemopreventive measures taken and reduce toxic side-effects. This suggests that the regeneration of phenolic compounds from phenoxy radicals is a mechanism that can contribute to the synergistic effects observed. in addition to the synergistic effects between the different phenolic compounds. 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. the regeneration of quercetin from its phenoxyl radical by the presence of (+)-catechin. This implies that. Simone G. Also. van Breda 166 indicated 16 to 23% of the antioxidant activity to be synergistic. 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. Jørgensen et al. particularly those of sulindac. Although our understanding of the molecular mechanism behind the observed combinatorial effects of these chemopreventive agents is still limited. EGCG and sulindac were also found to synergistically enhance apoptosis.30] are examples of this.Theo M. the effects reported by Suganuma et al. Use of retinoids in combination with such SERMs (selective estrogen receptor modulators) as tamoxifen or raloxifene [29.
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relieving vaginitis to name but a few. Evidence from a wide range of sources supports the view that the colonic microflora is involved in the etiology of CRC. acidophilus to eleven volunteers on a fried meat diet known to increase faecal mutagenicity. animal models. Probiotics usually refer to highly selected lactic acid bacteria.or prebiotics alone. anticancer effects. This has led to intense interest in factors. Karolinska Institute. Lactobacillus spp. with defined gut survival properties and associated biological activities and which can be ingested in fermented milk products or as a supplement.6. The list of healthful effects attributed to probiotic bacteria is extensive  and includes: alleviation of lactose intolerance symptoms. that have the potential to improve host health’)  and synbiotics (combinations of pro.Joseph Rafter 170 3. 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. although there are some on breast  and bladder cancer . alleviating constipation. Huddinge. Probiotics and prebiotics — potential anticarcinogenic food components Joseph Rafter Department of Medical Nutrition. The evidence from animal studies provides strongest support for anticancer effects and data from human studies (epidemiology and experimental) are limited.2]. 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. e.and prebiotics on cancer is derived from in vitro studies.and prebiotics). 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. 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. 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. Evidence for protective effects of pro. resulted in a lower faecal mutagenic activity after 3 days . The vast majority of studies on the anticancer effects deal with colorectal cancer (CRC). and Streptococcus spp. Overall. Bifidobacterium spp. serum cholesterol reduction. Sweden Introduction An example of a functional food..g. that can modulate gut microflora and its metabolism. such as probiotics. epidemiology and human intervention studies. Diet makes an important contribution to CRC risk  implying that risks of CRC are potentially reducible. Administration of L.
Hayatsu and Hayatsu  also demonstrated a marked suppressing effect of orally administered L. There will also be. at-risk groups and patients. the opportunity to exploit genomics and proteomics in investigations of effects of pro/prebiotics on gene expression and post-transcription .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) . In two population-based case-control studies of colon cancer. it will become possible to perform studies in healthy volunteers. data from human intervention studies are of paramount importance in providing evidence that probiotics. two companion American prospective studies. the 1980–1988 follow-up of the Nurses’ Health Study and the 1986–1990 Health Professionals follow-up study. acidophilus administration.11]. there are few epidemiological studies addressing the association between fermented dairy products and colorectal cancer. did not provide evidence that intake of dairy products is associated with a decreased risk of colon cancer . In most cases.and prebiotics. and other dairy products [10. in the near future. although a weak non-significant inverse association with colon cancer was observed . an inverse relationship for yoghurt consumption with risk of large colon adenomas in men and women was reported . In a cohort study in the Netherlands. particularly in urine.15]. it would appear that the case control studies indicate protective effects while the prospective studies do not. High levels of mutagenicity also appeared in urine on days 2 and 3 of the fried meat and ordinary fermented milk dietary regimen. the most profitable approach would be to use combinations of pro. despite the high fat intake. colon cancer incidence was lower than in other countries because of the high consumption of milk. It will be important to define dose and time relationships and it would appear at present. On the other hand. During L. In another case control study. yoghurt. In conclusion. Once such markers are available. an increase in the number of faecal lactobacilli corresponded to a lower mutagen excretion. the urinary mutagenic activity on days 2 and 3 was significantly lower compared to the ordinary fermented milk period. 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 . In summary. As yet. an inverse association was observed for yoghurt  and cultured milk consumption . adjusted for potential confounding variables. 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. 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. prebiotics or fermented milk consumption are causally related to reduction in cancer risk. An epidemiological study performed in Finland demonstrated that. Thus. casei on the urinary mutagenicity arising from ingestion of fried ground beef in the human. 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. from animal studies. this is an area of high priority for future studies.
http://www. all of the ”state of the art” colon cancer risk biomarkers. Oral administration of lactic acid bacteria has been shown to effectively reduce DNA damage. acidophilus cells. longum were antigenotoxic toward N’-nitro-N-nitrosoguanidine (MNNG). 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. It involves a twelve-week randomised.  showed that a specific strain of L. 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.2-dimethylhydrazine (DMH)-induced genotoxicity. casei subsp. Goldin et al. and involving 8 research centres in Europe. Although B. in gastric and colonic mucosa in rats. L. that Lactobacillus acidophilus. including colonic mucosal markers. while heat-treated L. Bifidobacterium breve and B. Certain strains of lactic acid bacteria have also been found to prevent putative preneoplastic lesions or tumours induced by carcinogens.  reported. longum or inulin was associated with a decrease in AOM-induced colonic small ACF in rats and combined administration .syncan.24]. using the comet assay. which clearly demonstrate a protective effect of dietary supplements of lactic acid bacteria against colon tumour development. Pool-Zobel et al. Consumption of B. Of relevance here is a clinical trial which is presently ongoing i. ACF are putative preneoplastic lesions from which adenomas and carcinomas may develop. placebo controlled trial of a food supplement containing Lactobacillus GG. confusus. acidophilus also inhibited the formation of ACF. It will also be particularly important to use data on mechanisms of action to develop hypothesis based intervention studies in humans. L. Metabolically active L. faecal water markers and immunological markers. there have been many studies.22] or DMH [23. These bacteria were also protective toward 1. prevented MNNG-induced DNA damage. In this study. to examine the effect of a synbiotic preparation on colon cancer risk biomarkers in humans (SYNCAN project. Bifidobacterium Bb-12 and Raftilose Synergyl in adenoma patients. are being measured. using these models. End points used are the tumours themselves or early lesions. feeding of bifidobacteria suppressed the ACF formation induced by AOM [21.e. Overnight cultures of L. Streptococcus thermophilus. the peptidoglycan fraction and whole freeze-dried cells were antigenotoxic. as well as an acetone extract of the culture. such as aberrant crypt foci (ACF). gasseri. adolescentis culture and its supernatant did not show an inhibitory effect in this study . induced by chemical carcinogens. 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. acidophilus was not antigenotoxic. acidophilus. funded by EU.Joseph Rafter 172 events in colonic biopsies and to identify human groups responsive to pro/prebiotic intervention. Among different cell fractions from L. double blind. In recent years. induced by azoxymethane (AOM) .be).
longum administered in the diet to rats inhibited liver. acidophilus not only suppressed the incidence of DMH-induced colon carcinogenesis but also increased the latency period in rats. colon and mammary tumours. In addition. Clostridium butyricum TO-A and Bacillus mesentericus TO-A.  using whole peptidoglycan isolated from B. It has been . used in this study contained Streptococcus faecalis T-110. infantis strain ATCC15697. The probiotic mixture. breve reduced the number of ACF with four or more crypts/focus and crypt multiplicity which are reliable predictors of malignancy . Goldin and Gorbach  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 . Sekine et al. Indeed. we exploited human flora associated (HFA) mice to test the effects of a probiotic mixture on a parameter of relevance for colon carcinogenesis i. but the data presented suggested that they may act through a combination of mechanisms involving an increase in short-chain fatty acid (SCFA) production. which do not survive contact with gastric acid. induced by the food mutagen 2-amino-3-methyl-3H-imidazo(4. which are acid resistant in contrast to most bacteria. Recently. DNA adduct formation . ornithine decarboxylase activity and expression of the ras-p21 oncoprotein.33]. Biothree®. In addition.e. in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis in the AOM-induced colon carcinogenesis rat model . Dietary administration of a lyophilised culture of B. There is additional direct evidence for anti-tumour activities of lactic acid bacteria obtained in studies using pre-implanted tumour cells in animal models. the results from a previous report from our laboratory. the results indicated that the prebiotic decreased AOM-induced carcinogenesis. enriched with oligofructose. longum also significantly inhibited AOM-induced cell proliferation. while a possible protective effect of probiotics was observed.5-f)quinoline (IQ). Reddy and Rivenson  reported that lyophilised cultures of B. Ingestion of B. 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. More recently. and that the additional colonisation of B. lower proliferative activity and a variation in the expression of some enzymes involved in the pathogenesis of colon cancer. reported that a single subcutaneous injection significantly suppressed tumour growth. longum resulted in a significant suppression of colon tumour incidence and tumour multiplicity and also reduced tumour volume induced by AOM in rats . there was a report on the antitumourigenic activity of the prebiotic inulin. Feeding of fermented milk increased the survival rate of rats with chemically induced colon cancer . mindful of the fact that the composition and metabolic activities of the intestinal flora of experimental animals are significantly different from those of humans . 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. The authors concluded that.Bioactive components in foods: Probiotics and prebiotics — potential anticarcinogenic food components 173 significantly decreased the incidence of large ACF . five intralesional injections resulted in 70% tumour regression in the mice. The mechanisms by which they act are less clear.
It is effective for the improvement of symptoms caused by abnormal intestinal flora. However. diarrhoea and constipation. the use of lactic cultures for human cancer suppression is interesting. i. quantitative and/or qualitative alterations in the intestinal microflora incriminated in producing putative carcinogen(s) and promoters (e. Interestingly.40]. effects on physiology of the host. difficile and C. 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. AAC). The strongest evidence for anticancer effects of probiotics comes from animal studies and evidence from human studies (epidemiology and experimental) is still limited. binding and degrading potential carcinogens.Joseph Rafter 174 reported that S. even with the above reservations in mind and mindful of the limited number of human studies available. An important goal for the future should be carefully designed human clinical trials to corroborate the wealth of experimental studies. It has also been reported that B. alteration of physicochemical conditions in the colon. 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. Biothree® is used as a clinical therapy in Japan. Thus. Salmonella typhimurium. there are several possible mechanisms which might explain how lactic acid bacteria might protect against tumour development in the colon. It is possible that different strains target different mechanisms. bile acid-metabolizing bacteria). 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. Also. holds promise and certainly deserves more scrutiny. All of the mechanisms have various degrees of support. production of antitumourigenic or antimutagenic compounds. faecalis T-110 and C.e. However. given by gavage.3-b]indole (2-amino-alpha-carboline. enhancing the host's immune response. C. botulinum) was inhibited in mixed cultures of S. faecalis T-110 and C. mesentericus TO-A stimulated the growth of Bifidobacterium by producing 3. 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. Vibrio parahaemolyticus. as mentioned above. . butyricum TO-A showed strong symbiosis with each other and the growth of enteropathogens (enterotoxigenic Escherichia coli.3-dihydroxyazetidine [39.g. butyricum TO-A . Conclusion Many healthful effects are attributed to the probiotic bacteria and some of these effects have more scientific support than the anticancer effect. mainly originating from in vitro and animal experiments and some of them even have some support from human clinical studies. such mechanisms might include: alteration of the metabolic activities of intestinal microflora.
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Viale Settimo Severo 65. Paradisgatan 5C.antsz. 55099 Mainz. SE-22100 Lund. Topeliuksenkatu 41 a A. Pleinlaan 2. Strada Della Carita 10. steffen.org Deutsches Krebsforsforschungszentrum (DKFZ) Germany. 10133 Turin. 91-348 ¸ódê.be Lund University (ULUND) Sweden.se Fondazione ISI (ISI) Italy.Annex ECNIS participants Nofer Institute of Occupational Medicine (NIOM) Poland. dan. J.uk . 20135 Milan. Teresy 8. 00250 Helsinki.de Finnish Institute of Occupational Health (FIOH) Finland. Vassileos Constantinou Avenue 48. SW7 3RP London.akesson@tbiokem. Âw.loft@farmakol. bjorn. coordinator.segerback@biosci. firstname.lastname@example.org University of Leicester (ULEIC) United Kingdom.email@example.com Karolinska Institutet (KI) Sweden. Saarstrasse 21.hu Nicolaus Copernicus University. schoketb@okk. DK-1017 Copenhagen.it Johannes Gutenberg-Universität Mainz (JOGU) Germany. 1050 Brussels. LEI 7 RH Leicester. 11635 Athens.matullo@unito. taioli@policlinico. giuseppe. skyrt@eie.Hirvonen@ttl. H-1097 Budapest.ki. University Road. Neuenheimer Feld 280.pl Genetics Research Institute & Ospedale Policlinico (GRI) Italy. Jagielloƒska 13.se Institute of Cancer Research (ICR) United Kingdom. Old Brompton Road 123. Ari.fi Vrije Universiteit Brussel (VUB) Belgium.uk National Institute of Environmental Health (NIEH) Hungary.ku.lth. 69-120 Heidelberg.ac.de University of Copenhagen (UC) Denmark. mkirschv@vub. ryszardo@cm. firstname.lastname@example.org The National Hellenic Research Foundation (NHRF) Greece. david. SE-171 77 Stockholm. Norregade 10.Nair@dkfz. Collegium Medicum in Bydgoszcz Poland.ac. ecnis@ecnis. 85-067 Bydgoszcz. Gyáli út 2-6.
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