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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: firstname.lastname@example.org 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
. . . . . . . . . . . . . . .3. Michael O. . 160 3. Ian M.5. 134 3. . . . . . . . . . . . . . . . . van Breda . . Kelleher. . . . .3. . . . . . . .6. . . . . . Kyrtopoulos . . . . . . de Kok. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anticarcinogenic effects of glucosinolate breakdown products John D. . . . . . . . . . . 170 Annex ECNIS participants . . . . . . Hayes. . . . . . . . . . . . . . . . . . . . . . . . . . 179 . . . . . . . . . . . . . . . . . . . . . . . . . . Eggleston . Probiotics and prebiotics — potential anticarcinogenic food components Joseph Rafter . . . . . . . . . . . . . . . . . . . Soterios A. . . . . . .4. . . . 140 3. . . . . Simone G. . . . . . . . . . . . . Anticarcinogenic compounds of olive oil and related biomarkers Theodore G. . . . . . . . . . . . . Sotiroudis. . Combined action of different dietary compounds preventive of cancer Theo M.
particular progress has been made here recently. especially those of relevance for the partners of the NoE ECNIS and its contacts. that of reviewing the mechanisms of action of some of the most promising anticarcinogenic compounds. Markers differ considerably in their characteristics. which to a large extent reflects the kinetics of metabolism and transport. These compounds are found in foods of many different types. and bioactive compounds. Current research . which is a very complex field indeed in view of the large number of cancer diseases. 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. pathogenetic mechanisms. vitamins and selenium on one hand. some of them mainly reflecting recent dietary intake and others indicating more the individual’s long-term nutritional status. on the other. only certain selected topics. food components and food preparation methods involved. The present review focuses on two major groups of food components. Biomarkers are a key tool for assessing nutritional status and dietary exposure to specific substances. although plantbased foods appear to be the richest source. Nutritional biomarkers are essential for studies of the links between dietary intake and the risk of cancer. and pool sizes of the substance in question. 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. the growth conditions and other factors. Also. The degree to which these compounds are present in plant tissue can vary markedly and be dependent on the plant variety.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. The ECNIS project aims at addressing important problems in this field and identifying knowledge gaps that exist. Many compounds contained in the diet have been proposed as having anticarcinogenic properties. are considered in detail. 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. Vitamins and selenium Although the measurement of vitamins in body fluids is a classical area for nutritional biomarker research. Particular attention is directed at identifying dietary factors that modulate environmental and lifestyle factors related to cancer risk. as outlined below. The microbial flora in the gut also play an important role in their action and composition through bioconversion and the release of various bioactive dietary components.
The antioxidant vitamins have been studied very much in relation to risk of cancer. The latter has the advantage of possessing lower detection limits. No studies are currently available on the relationship between lipid or protein oxidation markers and chronic disease. are controversial. but they are not useful as yet for large screenings. Regarding intervention studies using antioxidant supplementation. there are six investigations that have reported beneficial effects . Since DNA mutations are a crucial step in carcinogenesis and elevated levels of oxidative DNA lesions have been noted in many tumours. polyphenols. and the problems in defining the appropriate threshold for vitamin D sufficiency. Regarding vitamin E. The determination of vitamin D status is still a challenging task. There is limited evidence that plasma ascorbate is a functional antioxidant. The handling and storage of plasma for vitamin C determination has a strong impact on its accuracy. it appears to have only limited capacity as a direct antioxidant in vivo. Although it is likely that severe oxidative stress is a consequence rather than a cause of the development of many types of cancer. such damage is strongly implicated in the aetiology of cancer. epidemio-logic variations in vitamin D status and its determinants. at present it is impossible to assess the quantitative involvement of oxidative stress in the origin of cancer. Biomarkers of vitamins A. HPLC methods are those which are most sensitive. probably higher accuracy as well as the possibility it to determine ascorbate and dehydroascorbate simultaneously. including cancer. and that the intake of high doses of vitamin C can be used to decrease oxidative stress. strong research efforts in this area should be encouraged. as determined by isoprostanes and by inflammatory markers. which is water-soluble. Serum retinol is still the most reliable indicator for evaluating vitamin A deficiency. 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. Simpler tests using fluorescence or immunological techniques have a high level of accuracy and precision. The effects that have been postulated of high levels of vitamin E supplementation on exercise-induced lipid oxidation. 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. 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. C and E are an important part of the diet-cancer field. as assessed by currently available markers of lipid or protein oxidation in humans. 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. but there is a need of developing simpler methodologies. using oxidative DNA damage as a biomarker. The measurement of vitamin D metabolites presents a number of problems including the occurrence of vitamin D in different forms.Dietary vitamins. the variations in measurement between different assays and different laboratories. In contrast. and since metabolites of vitamin D have important biological effects. but colorimetric assays have a much higher throughput. Concerning the functions of vitamin A. Vitamin E intervention has not been shown to decrease the oxidation of plasma proteins. can readily be determined in plasma by automated colorimetric assays or by HPLC. both HPLC and GC-MS methodology have high precision and accuracy for its detection in plasma.
methylated selenocompounds having been found to have particularly strong chemoprotective effects. Some recent mechanistic findings on the flavonoids apigenin. Like other types of dietary chemopreventive agents. Flavonoids can also induce cell cycle arrest by modulating key components of cell cycle regulation. mitochondrial membrane depolarisation. Experimental studies have shown that selenium compounds affect cell growth. The suppressing activities include the inhibition of signalling pathways responsible for cell proliferation and survival.Executive summary 7 of it on oxidative DNA damage. the cell cycle. It is also important to determine whether there is an increased risk of certain forms of cancer after selenium supplementation. DNA repair and signal transduction. Most studies have involved healthy individuals. lung. mainly through intrinsic pathways involving members of the Bcl family. Several epidemiologic case-control studies have indicated a protective role of selenium in helping prevent the development of cancer. and the induction of apoptosis. 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. In these studies. These are usually divided up into blocking and suppressing activities. The optimal type and dose of selenium supplements needs to also be defined. release of cytochrome c and the activation of caspases. Several large human intervention studies have indicated that selenium supplementation can prevent the development of several different forms of cancer (prostate. cyclin-dependent kinases and inhibitors. whereas there are 13 studies that have reported a null effect. colon). 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. Both organic and inorganic forms of selenium have been evaluated. There is also a need of clarifying the mechanistic role of selenium in the etiology of cancer. The blocking activities include antioxidant effects and the modulation of drugmetabolising enzymes and multidrug resistant genes. use has been made of several biomarkers such as the concentration of total selenium in different body fluids as well as different selenoproteins. epigallo- . including cyclins. primarily glutathione peroxidase. The relationship of low selenium status and selenium supplementation to cancer disease is another important segment of focus in the diet-cancer field. Further studies are underway. 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. 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. It is important to explore the possibility of using other selenoproteins as biomarkers too. flavonoids exhibit a wide range of potentially beneficial activities in terms of cancer prevention.
its becoming increasingly clear that it is their breakdown products that can influence the initiation and progression of carcinogenesis. nitriles. as well as squalene and β-sitosterol. polyphenols. These are areas in need of further investigation. The bioavailability of different compounds varies markedly and for many compounds information on this point is scarce. Hydrolytic and clean-up steps are usually needed. is undesirable. 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. which reduces the formation of isothiocyanates from glucosinolates. Olive oil is one of the foods at which special interest has been directed in the diet-cancer field because of its containing a special mixture of agents that affect the initiation. quercetin. which also applies to analyses of these compounds in urine. resveratrol. it is difficult to relate responses of cells in culture to particular concentrations of phytochemicals to the situation in vivo. genistein. cyano-epithioalkanes and oxazolidine-2-thiones from glucosinolates. xanthohumol and the novel flavonol tricin have been summarised. and the sensitivity of detection may be a limiting factor although the increasing use of detection methods based on mass spectrometry will solve some of these problems including the identification of analytes. They also appear to influence apoptotic responses to chemotherapeutic agents. the measurement of polyphenols in body fluids is a difficult task. such as isoflavones and lignans. secoiridoids and lignans).Dietary vitamins. It is unclear whether the formation of thiocyanates. squalene and β-sitosterol) are reviewed. lignans. Without such information. but their utilisation in this way is hampered by several factors. which occurs at the expense of their forming isothiocyanates. It has also been pointed out that their efficacy is dependent on their bioavailability. In addition. These compounds have a number of different mechanisms of action. the availability of immunoassays is of great value. the chalone. relatively little is known of the pharmacokinetic properties of glucosinolate breakdown products in humans. The anticancerogenic properties of the glucosinolates have received a great deal of attention. It is also unclear whether the activity of the epithiospecifier protein (ESP). From an analytical point of view. but further study regarding this will be needed. the use of polyphenols as biomarkers of dietary intake has attracted considerable interest. For some compounds. Mammalian cells display marked dose responsiveness to phytochemicals. 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. hydroxytyrosol. Also. An especially interesting matter here is that intake of olive oil can increase the bioavailability of anticarcinogenic compounds. at low doses of phytochemicals cytoprotective adaptive responses . certain phenolic compounds (tyrosol. selenium and probiotics: biomarkers of exposure and mechanisms of anticarcinogenic action 8 catechin gallate. To clarify the matter. These include tocopherol and carotenoid antioxidants. The more recently developed metabolomic techniques may offer new possibilities in the future. 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. promotion and progression of multistage carcinogenesis.
evidence from human studies still being limited. This can well contribute to cancer prevention. The strongest evidence for the anticancer effects these can have comes from animal studies. considerable information of detailed character concerning the mechanisms governing the effects of bioactive food compounds and of nutrients on the cellular processes that relate to carcinogenesis has accumulated. Much hope is attached in this connection to the powerful techniques available today within the field of nutritional genomics. combinations of green tea flavonoids are more active than single compounds of this sort. whereas at higher doses cell cycle arrest and apoptosis occur. The synergistic effects of dietary phytochemicals need to be investigated further. As has been indicated here. Identification of the mechanisms that control such outcomes would be very useful. . It is presently unclear how these different types of responses are coordinated by the cell and how matters of whether adaptation.Executive summary 9 being activated. promising and as deserving of closer scrutiny. growth arrest or apoptosis is to occur are determined. Different strains of the bacteria may possibly target different mechanisms. 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. it appears that many combinations of complementary modes of action may be involved. Several possible mechanisms may explain how lactic acid bacteria can protect against tumour development in the colon. 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. Many healthful diet effects are attributed to probiotic bacteria. Although our understanding of the molecular mechanisms behind the observed combinational effects is still limited. The development of ideas regarding this has also been stimulated by findings that dietary supplements of only a single compound may have negative effects. In some systems. More work needs to be done to identify the specific strains and strain characteristics responsible for particular antitumour effects and the mediating mechanisms involved. 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. and green tea flavonoids can also show increased activity when present together with other phytochemicals. It is a highly challenging task to integrate this knowledge in such a way that useful hypotheses can be formulated that ultimately can be tested in human dietary intervention trials. An understanding of the mechanisms involved is a necessary basis for the development and validation of new biomarkers of nutritional status. Even with the above reservations and the limited number of human studies available in mind. one can regard the use of lactic cultures for human cancer suppression as interesting. It is important here to have insight into recent developments in analytical technology and thorough knowledge of biomarker kinetics. 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.
g. markers of nutritional status Markers of differing kinetics (response times) Markers of nutrients vs. the pathogenetic mechanisms involved. Pure and Applied Biochemistry.1. A number of textbooks and reviews are available within the field of nutritional biomarkers [see e. Lund University Hospital. Introductory overview and future prospects Björn Åkesson Biomedical Nutrition. Lund. 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. that role that various food components and food preparation methods play in connection with this. Introduction 1. A further aim is to examine and explore the mechanisms responsible for the action for different promising anticarcinogenic compounds. 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 brief overview of food components that appear to have an anticarcinogenic effect is provided in Appendix 1. The ECNIS project aims at addressing important problems in this field and identifying various knowledge gaps.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 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.2.1. This is a very complex field in view of the large number of different types of cancer.1. Table 1. . their physiological basis and the use of biomarkers in studies of different types. Some major issues regarding nutritional biomarkers Markers of dietary intake vs. 1–6] dealing with the basic theories and methodology. Sweden Dietary assessment methodology and the development and validation of biomarkers of nutritional status are key scientific fields within nutritional science. Various properties of biomarkers are summarised in Table 1. Lund University and Department of Clinical Nutrition.
plasma. The amount of a substance occurring in the erythrocytes. 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. Some methods. such as the analysis of cobalamine and folate in plasma. variations in metabolism or in excretory pathways. are in routine daily use in clinical laboratories. and they may not respond to overexposure of nutrients at all. trace elements are coupled to the levels of enzyme activities.Björn Åkesson 12 Dietary components as biomarkers Many biomarkers involve the measurement of a nutrient or some other dietary component in the blood. Biomarkers show temporal variation. different nutrient-dependent physiological tests can be employed . . Various diet-related compounds in the plasma also differ considerably in turnover times. 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 . Also. 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. 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. 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. One such example is olive oil discussed in this volume by Sotiroudis and Kyrtopoulos. Also proteins with a transport function as well as other functions are used as biomarkers. since the erythrocyte cells have a lifetime of about 120 days . the gut flora. The level of a biomarker often varies as well with age. diurnal variations. for example zinc and copper to superoxide dismutase activity and selenium to the activity of glutathione peroxidase and other selenoproteins. 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. urine or hair. 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. often reflects much more the long-term intake if it than the plasma level does. and inflammatory and other diseases can influence the levels of different biomarkers . gender and gene polymorphism (as outlined below). interactions between dietary or endogenous components or xenobiotics. such as the retinol-binding protein and ceruloplasmin. whereas others are performed only in a few specialised research laboratories. . In addition. the main function of some markers being that of serving as indicators of malnutrition.
For this reason.12]. 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. The assessment of dietary intake in epidemiological studies has been a major use of biochemical markers of nutritional status. and 4) eventually provide the basis for gene-based screening tests”. An overview of this matter is provided in . 2) help establish causality of food and nutrient associations in epidemiology studies. buccal mucosa. faeces. These matters are outside the scope of the present review. it should be borne in mind that there are very few (if any) “ideal” biomarkers that have been validated for studies of different types. 3) aid in distinguishing causal components of complex dietary mixtures. As summarised by Hunter . urine. however. 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]. Also. This topic is only taken up briefly in the present volume. 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. Some of these samplings require much skill and effort and can only be used in small studies. information on the interactions between dietary intake and gene polymorphisms “can serve to 1) define susceptible subpopulations. if the analytical methodology improves so that smaller amounts of sample suffice. . A number of statistical methods for the correction and evaluation of such data are available . 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. thus strengthening dietary associations. together with an assessment of dietary intake by a food frequency questionnaire or by some other methods. although epidemiological findings on the association between the selenium level and cancer are reviewed by Gromadziƒska et al. Folate has served as a prototype here for demonstrating how genetic make-up can influence the nutrient status of an individual. Their use may increase.Introductory overview and future prospects 13 Although several of the biomarkers mentioned here are widely used. as exemplified in the present volume by Linseisen and his colleagues in connection with vitamin D and polyphenols. or for the calibration of the dietary assessment methods . methods for measuring nutrients or nutrient-dependent variables in cells in the blood (mainly leucocytes). The two contributions mentioned also illustrate the difficulty in defining valid cut-off points. adipose tissue or biopies from other organs have been developed. Although the use of the biomarker concept for these purposes is very attractive. There are particularly good reasons for using the 25-hydroxy vitamin D as a biomarker in extended studies in the future. 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. Biomarkers in studies of that type have been used either alone.
It was also stated that “in four trials (three with unclear/inadequate methodology). apoptosis.17]. C and E on biomarkers of oxidative stress. de Kok and their colleagues in this volume. 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. although for other nutrients and their biomarkers there may be little or no response due to homeostatic regulation of their plasma levels. vitamin A and/or vitamin E . especially β-carotene. certain metabolites and various effects on DNA and its metabolism . The potential preventive effect of selenium should be studied in adequate randomised trials” . Other aspects of cancer-related biomarkers have been the subject of a previous review conducted within the ECNIS project . selenium and polyenoic fatty acids for example. selenium showed significant beneficial effect on the incidence of gastrointestinal cancer.Björn Åkesson 14 Biomarkers and response to dietary supplements An important use of biomarkers is to aid in the assessment of compliance in meal studies and dietary intervention studies. a meta-analysis showing there to be an increase in mortality in studies of antioxidant supplementation by β-carotene. a matter reviewed recently [16. Biomarkers of this category include tumours and the mortality in animal models. Studies in which nutrient antioxidants or combinations of those are employed are of special relevance for various topics taken up in the present volume. gut-lumen enzymes and carcinogen-metabolising enzymes. In the present volume Loft et al. 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.15]. usually responds to an increase in the supply of them. such enzymes as cyclo-oxygenase 2. . work that is as reviewed by Manson. review the evidence that individual antioxidants as well as for antioxidant-rich diets affecting oxidative DNA damage. cell proliferation and differentiation. The content or profile in the blood of β-carotene. To the surprise of the scientific community it was found that some antioxidants. the criteria to be employed for the evaluation of efficacy in the future being proposed [14. Biomarkers are also used in long-term human intervention studies in which there are clinical endpoints. as well as precancerous lesions. had negative effects. the existence of a renal threshold or other factors. A possible further use to which such biomarkers could be put would be to substantiate ‘anticancer ’ claims regarding various food components. α-tocopherol. Hayes. adenomatous polyps. 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.
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.
3. 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. 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 University. These protective compounds are found basically in all types of food. Plant tissues can also vary enormously in the store of such compounds. for example. This information was collected with use of a PubMed search conducted in 2005. emphasis on recent reviews. Lund. but plant-based foods appear to be their richest source. of refs 305256 3351 4173 869 181 146 The forty-seven reviews listed below were selected from among the 146 reviews arrived at last in this search. Table 1. and avoiding of similar articles by any given author.). Different plant families harbor different mixtures of compounds of this sort. 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.2.Per Mercke 18 Appendix 1.2. a list of putative anticarcinogenic food components has been generated (Table 1. The criteria used for the selection were their relevance as adjudged from their title. Overview of possible anticarcinogenic food components Per Mercke Biomedical Nutrition. depending on the plant variety and the growth conditions. Sweden There is a vast range of dietary compounds proclaimed to have anti-carcinogenic properties as investigated by the scientific community. .
Milner .and probiotics Butyrate Vitamins Vitamin A including retinol and retinoic acids α. . . Kelloff et al. Ferguson . . Milner . Mayne . . . Ferguson . Wild et al. Branca et al. . . Turini and DuBois  Branca et al. methionine) . Konety and Getzenberg . Gill and Rowland .  Branca et al. Seifried et al. 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. Wild et al. Sanderson et al. Milner . Mason . Milner . Mayne . Ferguson . Wild et al.  Branca et al. Manson . Rafter et al. Kelloff et al. Ferguson . riboflavin. . . Ferguson  Starch and resistant starch Fibre/non-starch polysaccaride Vegetables Branca et al. Sharma and Farmer . Milner . Sharma and Farmer . Vlastos et al. Rafter et al. . Mayne . Manson et al. Gill and Rowland . . . . Bowen et al. Greenwald .  Pre. Vlastos et al. Kelloff et al. Ferguson . Vlastos et al. Crews et al. Vlastos et al.  Branca et al. Sharma and Farmer . Crews et al. Kelloff et al.  Branca et al. choline. . Seifried et al.and β-Carotene Lycopene Lutein Zeaxanthin β-Cryptoxanthin Carotenoids Orange vegetables Tomatoes Green vegetables Branca et al. Turini and DuBois  Branca et al. . Sanderson et al. Rafter et al. . Rafter . Greenwald . . Maruvada and Srivastava . pyridoxine. Loft and Paulsen . . Sanderson et al. Seifried et al. . Ferguson . Loft and Paulsen . . .  Branca et al. . Sanderson et al. Ferguson . . Gill and Rowland . . . Loft and Paulsen . . Ferguson . Rafter et al. . Greenwald . . . . Wagner et al. . . Vitamin E Vitamin D Vitamin C B vitamins (Folic acid cobalamine. . Turini and DuBois .3.Overview of possible anticarcinogenic food components 19 Table 1. Greenwald . Kelloff et al. Wild et al. Maruvada and Srivastava . . . Vlastos et al. . . Granado et al. . Manson et al. Manson et al.
Manson . broccoli. 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. Ferguson . . Branca et al. Sharma and Farmer  Branca et al. . horse-radish. . Zhang  Ferguson . . Turini and DuBois  Maruvada and Srivastava . sorghum. soybeans) Branca et al. kale. Manson  Iron Iodine Isothiocyanates Benzyl isothiocyanate Cruciferous vegetables Phenethyl isothiocyanate Sulphoraphane Oltipraz (synthetic) Cruciferous vegetables Sulphones Dithiolthiones Glucosinolates Indole-3-carbinol 3. Mayne . Manson . squash. . Seifried et al. broad beans. garlic.  Ferguson . chocolate Epicatechin Epigallocatechin (EGC) Epigallocatechin gallate Caffeic acid Ferulic acid Ellagic acid Genistein Cereals. Ferguson . Sharma and Farmer  Flavonoid polyphenolics Catechins Catechin Tea.3’-Diindolylmethane Indole-3-acetonitrile Diallyl sulphide Allylmethyl trisulphide Tangeretin Apigenin Nobiletin Rutin Quercetin Kaempferol Taxifolin Cruciferous vegetables Bianchini and Vainio . Kelloff et al. Greenwald . Kelloff et al. Ferguson . Sanderson et al. Manson .  Allium compounds Onions. . Ferguson . Manson et al. . . . Duthie and Crozier . potatoes. . Ferguson . Sanderson et al. . Manson . Kelloff et al. Manson et al. Seifried et al. . scallions. . Wild et al. Wild et al. An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected — cont. Rafter et al. . . Saleem et al. . . Gill and Rowland . . . . . Rafter et al. Branca et al. . Manson . . Seifried et al. Manson et al. Kelloff et al. . pulses (millet. Seifried et al. Manson  Crews et al. Gill and Rowland . Ferguson . Kelloff et al. . Greenwald .3. Milner . Rafter et al. . Wiseman  . Milner . tomatoes. Crews et al. Rafter et al.Per Mercke 20 Table 1. Kelloff et al. endive Citrus fruits Branca et al.  Bianchini and Vainio . Milner . Sanderson et al. berries. . Sharma and Farmer . Manson  Phenolic acids Isoflavones Adlercreutz . onion Radish. Manson et al. Atkinson and Bingham . . Sanderson et al. chives Citrus fruits. . . Maruvada and Srivastava .
Gescher A. J Nutr 2003. 2. Greenwald P. sis: potential role of lipid peroxidation. Nair J. Steward WP. 6. Manson . Biomarkers in disease and health.25 Suppl:29–36. et al. Bartsch et al.133 Suppl 1:3820S–6S. 7. et al. Farmer PB. Powers HJ. Hematol Oncol Clin North Am 2002. cacao Manson  Reference Monoterpenes Citrus fruits (peel) Ferguson . Scientific basis of biomarkers and benefits of functional foods for reduction of disease risk: cancer. Primary prevention: phytoprevention and chemoprevention of colorectal cancer. Kelloff et al. raspberries. Cereals. PASSCLAIM — dietrelated cancer. Bartsch H. black-berries. Innovative agents in cancer prevention. walnuts. Martel P. strawberries.88 Suppl 1:S73–87.  Other non-flavonoid phenolics Lignans Olive oil Adlercreutz . Biol Chem 2002. . Rafter J. . Br J Nutr 2002. Downes CS.Overview of possible anticarcinogenic food components 21 Table 1. Branca F. dietary fat and antioxidants.383:915–21. DuBois RN.43 Suppl 2:II47–84. 9. olive oil References (selected from the PubMed search described above) 1. Br J Nutr 2001.16:811–40. Verhagen H. pecans. Milner JA. Br J Nutr 2002. 3. Class of compound Methylxanthines Example of bioactive substance Caffeine Theophylline Theobromine Limonene Perillyl alcohol Geraniol Menthol Carvone Hydroxytyrosol Curcumin Resveratrol Food source Tea. Mutat Res 1999. Rafter et al. Turini ME. Recent Results Cancer Res 2005. Rowland IR.166:257–75.91:315–23. Prospects for cancer prevention. 11. Ferguson LR. Pannemans D. 8.86 Suppl 1:S55–S92. J Cell Biochem 1996. Turmeric Branca et al. . Manson MM. Wiseman  Grapes. 4. Cancer risk factors for selecting cohorts for large-scale chemoprevention trials. Rafter JJ. Diet and cancer: assessing the risk. Sanderson P. Incorporating basic nutrition science into health interventions for cancer prevention.88 Suppl 2:S219–24. 10. Owen RW Exocyclic DNA adducts as oxidative stress markers in colon carcinogene. Govers M. Pool-Zobel B.3. McGlynn AP. Mathers JC. Johnson IT. Gill CI. Emerging dietrelated surrogate end points for colorectal cancer: UK Food Standards Agency diet and colonic health workshop report. Rechkemmer G. 5. Br J Nutr 2004. An overview of putative anticarcinogenic food components as assembled from 33 out of the 47 selected reviews that were selected — cont. Pool-Zobel B. coffee.428:329–38. . Eur J Nutr 2004. Hanley AB. cola.
Stacewicz-Sapuntzakis M. Br J Nutr 2003. 15. A critical assessment of some biomarker approaches linked with dietary intake. A critical evaluation of the application of biomarkers in epidemiological studies on diet and health. Kelloff GJ. . Cancer Res 2003. Zhang Y. Clin Cancer Res 2004. Granado F. Konety BR. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action.9:11–8. 19. 28. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research.4:1–4. Isothiocyanates in cancer prevention. Saleem M. J Mol Med 1996. Anderson DE. McDonald SS.48:169–88. Mammographic breast density as a biomarker of effects of isoflavones on the female breast. 17. Olmedilla B. Trends Mol Med 2003. Mayne ST. Greenwald P. Sharma RA. J Steroid Biochem Mol Biol 2002. Vlastos AT.134 Suppl:1640S–5S. Duthie G. Tea beverage in chemoprevention of prostate cancer: a mini-review. Andersen R. Nutr Cancer 2003. Crowell JA. Biomarkers for cancer diagnosis: implications for nutritional research. Vainio H. Duncan C. Mukhtar H. Mason JB.227:886–93. 26.130 Suppl:467S–71S. Gamma-tocopherol — an underestimated vitamin? Ann Nutr Metab 2004. Schottenfeld D.133 Suppl 3:933S–40S. 29. Bingham SA. Lubet RA. 20.46:261–73. Follen M. Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis. Getzenberg RH.Per Mercke 22 12. Manson MM. Boone CW. Biomarkers and their use in cervical cancer chemoprevention. Loft S. Blanco I. Drug Metab Rev 2004. 21. Adhami VM. Braesco V. Exp Biol Med (Maywood) 2002. Urol Clin North Am 2002. J Nutr 2003. et al. Sharifi R. O'Brien NM. 22. Cancer prevention — the potential for diet to modulate molecular signalling.86 Suppl 1:S5–35. Br J Nutr 2001. Elmadfa I.90:487–502.555:173–90. Breast Cancer Res. Milner JA. 14. 32.63:4295–8.74:297–312. Alink G. 25. Curr Opin Clin Nutr Metab Care 2000. 16.29:95–106. Mutat Res 2004. Wagner KH. Poulsen HE. 13.133 Suppl 3:941S–7S. J Nutr 2003.83:113–8. 30. Steele VE. Bianchini F. 24. Nutritional and clinical relevance of lutein in human health. Andersson C. Cancer risk and oxidative DNA damage in man. Vitamin D and prostate cancer. Br J Nutr 2001. Wild CP.3:447–51. 27. Maiani G. 18.86 Suppl 1:S37–53. Siddiqui IA. Kamal-Eldin A. J Nutr 2000. Chen L.36:655–67. et al. et al. Holst B. Crews H. Wilson L. Bowen P. Atkinson C.10:4901–12. The antioxidant conundrum in cancer. Biological relevance of adduct detection to the chemoprevention of cancer. Adlercreutz H. Progress in cancer chemoprevention: development of diet-derived chemopreventive agents. 2002. Seifried HE. Biomarkers of nutrient exposure and status in one-carbon (methyl) metabolism. Phytoestrogens and breast cancer. Srivastava S. Malone WA. Farmer PB. Maruvada P.47:13–23. 23. J Nutr 2004. Crit Rev Oncol Hematol 2003. Woods JA. Ghosh L. Plant-derived phenolic antioxidants. Crozier A. 31.
36. McCall MR.55:46–67. 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. Nutrition and cancer prevention: new insights into the role of phytochemicals. Biomarkers.11:181–8. Viner JL.31:289–300. Carroll PR.Overview of possible anticarcinogenic food components 23 33. Antioxidant vitamins: evidence from biomarkers in humans. 41. Kyrtopoulos SA.5:977–84. Bibl Nutr Dieta 2001. Adv Exp Med Biol 2001. . Tahir A. observational versus experimental human studies. Arts IC.492:255–62. Am J Clin Nutr 1997. Hawk ET. Int J Vitam Nutr Res 2003. Sarhill N. Georgiadis P.82:905–41. Halliwell B.9:1829–40. Christie R. low-dose carcinogenesis and dietary exposures. Establishing the significance and optimal intake of dietary antioxidants: the biomarker concept. Frei B. Am J Hosp Palliat Care 2003. Plant foods versus compounds in carcinogenesis. Molecular epidemiological approaches to the study of the genotoxic effects of urban air pollution. Wiseman H. Research strategies and the use of nutrient biomarkers in studies of diet and chronic disease. Nutr Rev 1999. Moyad MA. Epidemiology and prevention of colorectal cancer. Methylation and colorectal cancer.73:70–8. 34. 39. part I: time to redirect our attention? Urol Clin North Am 2004. J Pathol 2001. Patterson R. Wang CY. 38. Bell SM. 35. Public Health Nutr 2002. Who's afraid of n-6 polyunsaturated fatty acids? Methodological considerations for assessing whether they are harmful. 46.29:115–24. 37.57:104–13. 44. Moyad MA.428:91–8. Berry EM. Surg Clin North Am 2002. Rivlin RS.65 Suppl 4:1240S–5S. Mutat Res 1999. 40. Limburg PJ. Lifestyle/dietary supplement partial androgen suppression and/or estrogen manipulation. Prentice RL. 47. 43. Neuhouser M. Kampman E. Sugar E. The therapeutic potential of phytoestrogens. 45. Lifestyle recommendations to prevent prostate cancer.6:147–51. 42. Future directions. Ocke MC. Nutr Metab Cardiovasc Dis 2001. Jubb AM. A novel PSA reducer and preventive/treatment option for prostate cancer? Urol Clin North Am 2002.195:111–34.20:465–73. Quirke P. Hollman PC. Assessment of nutritional status and fluid deficits in advanced cancer. Kaaks RJ. Eur J Cancer Prev 1997. Vineis P. Mahmoud FA. Expert Opin Investig Drugs 2000.
a technique that was later superseded by various chromatographic and fluorescent techniques.1. . these techniques took over and today retinol can be determined in serum routinely by direct.2]. 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]. Denmark Since antioxidant vitamins can affect an organism’s capacity for defence against reactive oxygen species. except for markers of DNA damage.[3. Copenhagen. tocopherols and tocotrienols.4] or reversed-phase [5–7] liquid chromatography . has also been demonstrated . 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.1. 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. The direct-phase methods can also typically measure a large number of other lipid-soluble vitamin isomers in the same run. With the advent of high performance liquid chromatography (HPLC). xanthophylls. Vitamins and selenium 2. the retinol binding protein (RBP) [1. which is advantageous from a collection standpoint. the development of fast and simple methods for the determination of vitamin A status has long been given a high priority. which are dealt with elsewhere in this volume. biological markers of the dietary exposure to these vitamins is of importance. such as pro-vitamin A carotenoids. Dragsted University of Copenhagen. menadione and phylloquinones. C and E Lars O. This chapter is concerned with such markers. The possibility of using dried blood spots. although reversed-phase methods able to separate certain of the retinol isomers have been published .2. The reversed-phase techniques are faster and smaller sample volumes suffice but they are generally unable to discriminate between the various isomers of vitamin A to the same extent as the direct-phase methods can. 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. Due to worldwide concern for vitamin A deficiency (VAD). The structure of vitamin A and pro-vitamin A carotenoids is shown in Figure 2. Biomarkers of exposure to and effects of vitamins A.
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 .
If dehydroascorbate is physiologically present. The colorimetric and fluorometric methods are generally based on redox-reactions. possibly reflecting higher leakage of haem in this group. which can be photometrically measured by use of manual or automated equipment. and about 1. but a few of them use assay blanks produced by adding ascorbate oxidase to the sample. erythorbate. with ascorbate and dihydroascorbate leading to the formation of a chromophore or a fluorophore. 2. together constituting vitamin C. Fig. Most of these methods are quite unspecific . Structures of ascorbate. Dehydroascorbate is not readily determined by use of this approach. Some of these methodologies are very fast. around 0.2.8% under the same conditions in the case of smokers . The HPLC methods for detecting plasma ascorbate electrochemically give results similar to those using UV detection . There was a significant increase in the concentration of dehydroascorbate over time at low total vitamin C concentrations .Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. 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 . Recovery is better than 98% with . Postcolumn derivatization can be used to reduce dihydroascorbate so that it can be determined by use of an electrochemical detector. allowing high sample handling rates to be achieved through automation . the stereoisomer of ascorbate. creating greater sensitivity with retention of speed. C and E 29 However. its true concentration seems to be very low. the ascorbyl radical and dihydroascorbate. this procedure cannot be recommended if both compounds are to be determined separately.27–28]. can be determined simultaneously [25. A method for the simultaneous detection of ascorbate and uric acid by means of capillary zone electrophoresis (CZE) has also been described . since treatment with dithiotreitol is known to reduce dehydroascorbate to ascorbate. Results in determining plasma ascorbate in this way correlate well with those obtained by use of chromatographic methods .1% of the total plasma vitamin C in non-smokers when the sample has been handled carefully.
3% < 5% CV% DHAA interday < 6% ND ND Limit of detection 0. and since there is no generally accepted assay procedure . ex vivo LDL oxidation. Table 2. quite disparate results have been obtained with use of these techniques. These flaws have caused some journals to generally regard the method as being invalid as a lipid oxidation biomarker . irrespective of the concentration. 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 . This marker is highly controversial since it is variable both within and between laboratories. with or without calibration. to detect malondialdehyde. plasma lipid hydroperoxides. Typical performance of different methods for determination of plasma vitamin C Test method Reversed phase HPLC Capillary electrophoresis Automated colorimetry ND — not determined. The product can be determined spectrophotometrically. CV% AA interday < 4% 1. either directly or online.Lars O.5 mg/l 3 mg/l Speed (samples/day] 40–50 40–50 500 Reference   [38. Dragsted 30 use of the HPLC and CZE methods and good linearity is obtained even at low ascorbate concentrations. The assays employed to this end here are the following: plasma thiobarbituric acid reactive compounds (TBARS). Only randomised study designs are included in this review.3. In interlaboratory comparisons. The interday coefficient of variation (CV) for TBARS with use of HPLC is in the order of 10–20 %. since it may partially measure aldehydes deriving from endogenous metabolism. In another study an interlaboratory difference of about 15% was observed. which led to relatively larger relative errors being registered at low concentrations .3. antioxidant capacity markers.1 mg/l 0. depending on the method employed for hydrolysis.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. also within the same laboratory. The performance of different methods for the measurement of vitamin C in plasma is summarized in Table 2. . and plasma or urinary isoprostanes. whereas the intralaboratory variation was about 2 µmol/l. In a European study of laboratories carrying out population surveillance. The most commonly used marker is determination of TBARS. 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. following HPLC separation. 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.
In a larger study involving 56 smokers. 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 . 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. 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. acute doses. The method is disputed because the outcome depends on the antioxidants present in the LDL and these may be lost during LDL isolation. 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 . 25 males were exposed to vitamin C (500 or 1000 mg/d) and/or to exercise. 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 . in which 19 smokers participated for 4 weeks following a 2-week . In a smaller parallel study of vitamin C supplementation (1 g/d) versus placebo. in which isolated LDL is exposed to copper chloride or to a semistable radical such as AAPH. randomised double-blind crossover study the effect of vitamin C supplementation (six weeks. no change in whole plasma ex vivo oxidation was observed in this group . or a placebo in a parallel design. No effect of either treatment on plasma MDA was observed . but MDA to be unaffected by a slow-release formulation as compared with placebo treatment . In addition. In a third study of this sort. vitamin C plus 182 mg/d dl-α-tocopherol. Another controversial marker used by many laboratories is the ex vivo LDL oxidation assay. 30 mg beta-carotene and 100 µg organic selenium. 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 . 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. The interday CV for this latter assay is less than 10%. given in a normal formulation. 182 mg/d dl-α-tocopherol alone. In another counterbalanced design. No differences between groups in plasma malondialdehyde concentrations were observed either before or after supplementation . Oxidative stress induced by Zn deficiency was found to respond to 250 mg/d vitamin C. as compared with placebo treatment.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. given for 3 months. In a group of 48 middle-aged male and female participants in a 36-month intervention study receiving 500mg/d of either vitamin C. In another. the intake of 500 mg/d of vitamin C was found not to affect MDA as determined by HPLC . 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. by a decrease in plasma MDA concentration . The lag-time to oxidation and/or the slope of the oxidation curve are used as end points. the oxidative formation of conjugated dienes being followed spectrophotometrically at 234 nm . 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 .
the ferric reducing ability of plasma (FRAP) assay and the trolox equivalent antioxidant capacity (TEAC) assay which also correlated with ex vivo LDL oxidation [58. the size of the CVs depending on the exact wavelength used for determining the absorbance. Dragsted 32 ascorbate depletion period (≤ 30 mg/d). 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. A variety of antioxidant capacity markers exist for other blood compartments. 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 . Overall there seems to be no consistent effect of vitamin C supplementation on ex vivo LDL oxidation kinetics. These methods generally have interday CVs of less than 10% for repeated measurements of the same sample. In another group. The oxidizability of LDL is inherently an antioxidant capacity assay for this particular compartment. In a study with 30 coronary artery disease patients there was no evidence after 6 months that random assignment to vitamin C (1 g/d) together with vitamin E (800 IU/d). in which 0 mg. In a study without a control group. receiving only 60 mg vitamin C plus iron per day. At the same time. 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 . there was a significant increase in ex vivo LDL oxidation lagtime in the vitamin-supplemented group . or placebo. A borderline effect on LDL oxidation was observed in a similar study with only 18 participants after a shorter period of 12 weeks . 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 .Lars O. In a subsequent study of 30 young smokers. no effect was observed after 8 weeks supplementation by 1 g/d of vitamin C . decreased LDL oxidation or antibodies to oxidised LDL . 60 mg or 6 g of vitamin C was administered daily during 14 day-periods separated . usually one leading to a change in visual or UV absorption of the test matrix [52–55]. 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 .59]. They are all ex vivo oxidation systems composed of some oxidising system and some relatively simple marker of plasma oxidation. especially for whole plasma. There are important differences between the methods which call for caution when they are interpreted . no such increase was observed despite changes in plasma ascorbate . 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 . in a cross-over intervention study involving 48 non-smoking men and women. When applied to plasma samples some authors accordingly report poor correlation between methods  whereas others observed a high degree of correlation between some of the most commonly applied methods. the availability of a photometer with exact filters or one equipped with a narrow grid being required.
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.
In a very small study. 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.Lars O. The decrease was confined to individuals with a suboptimal intake of vitamin C. Vitamin C was found to decrease the exercise-induced increase in protein carbonyls in a dose-dependent manner . whereas no change was observed after supplementation of the known nutrients from the fruits and vegetables. the observed effect of vitamin C on this specific marker may reflect enzyme leakage from the cartilage rather than prooxidative stress. fluorescence or APCI-MS detection . involving seven divers. 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. A marginal increase in plasma protein AAS was observed (Dragsted. unpublished data). Use of this approach was found in one study to produce a significant time-dependent decrease in AAS during a 24-day period of fruit and vegetable depletion in the diet. the influence of taking 500 mg/d versus 1 g/d of vitamin C for 2 weeks prior to 30 min of hard exercise was evaluated in 12 males using a counterbalance design. Since vitamin C is a cofactor for the lysine oxidases that cross-bind cartilage in the body. Following protein isolation by a rapid sizeexclusion chromatographic step and complete acid hydrolysis. 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 . The level of protein carbonyls in the immunoglobulins was found to decrease after both 10 and 15 weeks of supplementation. the fluoresceinamine adduct could be detected by HPLC through the use of UV. Using the same method for protein carbonyls. No effect of fruit. 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. A third method employed for assessment of protein oxidation takes advantage of the reaction of protein aldehydes with fluorescein and reduction of the product by cyanoborohydride to form a stable adduct. The 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. Another line of evidence comes from studies of dietary fruit and vegetable depletion. None of the volunteers had suboptimal vitamin C levels before intervention. showing a rapid depletion of plasma ascorbate and a concomitant decrease in AAS. whereas no change in the total plasma sulfhydryls was detected . 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 . No effect of the supplement on protein carbonyls was observed . . including 150 mg/d vitamin C. The main disadvantage is the longer time required for analysis and the higher technical demands. indicating that the depletion of ascorbate might have an antioxidant effect. Dragsted 36 To assess oxidative stress by use of the simplest version of this assay.
γ. There are no studies currently available on the relationship between lipid or protein oxidation markers.and delta-tocopherols as well as the tocotrienols. no difference in the total vitamin E content of LDL was observed . Vitamin E Biomarkers for vitamin E in body fluids Vitamin E is a collective term for alpha-. Evidence for the prevention of stress-induced carbonyl formation by increasing the intake of vitamin C is controversial and needs further confirmation. Conclusions Total plasma vitamin C can readily be determined by automated colorimetric assays or HPLC. 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. of which RRR-α-tocopherol has the highest vitamin E activity. Various functional tests have also been suggested. since in a study comparing the delivery of RRR-α-tocopherol and all-rac-α-tocopherol to lipoproteins in humans following 8 weeks of supplementation by 1600 mg/d of either product. HPLC has the advantage of having lower detection limits. however. The relative vitamin E activity of the various tocopherols and tocotrienols in the rat together with their structures are shown in Figure 2. 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.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. Clear short-term pro-oxidant effects on lipid oxidation were observed following high-dose vitamin C infusion into the bloodstream. Since only the four R-forms of α-tocopherol (d-α-tocopherol) are recognised by the human vitamin E transporter in the body. permitting simultaneous determination of ascorbate and dehydroascorbate. The reference for the international unit (IU) is 1 mg of all-rac-α-tocopheryl acetate. 1 IU corresponding to 1. and possibly possessing higher accuracy. including the susceptibility of erythrocytes . C and E 37 Overall there appears to be certain but limited evidence for suboptimal vitamin C intake leading to an increase in protein oxidation in the plasma immunoglobulins. The vitamin E status in rats and possibly in other rodents can be determined as a function of all the active isomers. that of the formation of plasma isoprostanes.49 mg RRR-α-tocopherol. but in humans vitamin E status is preferably determined as the plasma.3. 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.and δ-tocopherols or of the tocotrienols are thought to have any vitamin E activity in humans as opposed to the rat. none of the four S-forms of α-tocopherol and none of β-. The handling and storage of plasma for vitamin C determination has a strong effect on accuracy. This is still controversial. serum or erythrocyte concentration of d-α-tocopherol. whereas the colorimetric assays have much higher throughput. vitamin C and chronic disease. gamma.
Fig. Dragsted 38 to lysis following an in vitro challenge with hydrogen peroxide [88–90]. and deletion of this transporter leads to neurological symptoms .3. . 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 . Redrawn from . Also. Structures of tocopherols and tocotrienols and their relative potency as vitamin E in the rat (TE. since these functional tests may be lipid oxidation markers. it seems more appropriate to evaluate the relationship of vitamin E supplementation to currently used lipid oxidation markers. or the exhalation of breath pentane .Lars O. 2. dl-α-tocopherol equivalents).
In another study. antioxidant capacity markers. ex vivo LDL oxidation. no effect on exerciseinduced oxidative stress could be shown by serum MDA . a decrease in serum MDA was found in the group given vitamin E . In a trial involving 80 men showing an increase in plasma lipid oxidation from exposure to either olive oil or menhaden oil. 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. 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. In a randomised 2-months intervention study of 59 healthy smoking males. In a study of the effects on 77 smokers of taking vitamins C (272 mg/d) and E (800 IU/d) or a placebo for 90 d. In a 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. MDA was found to increase significantly following daily doses of 200 mg vitamin E combined with 250 mg ascorbate. of a group of 14 runners assigned to either a placebo or a 1200 IU/d dose of vitamin E starting 4 weeks before and continuing during 6 days of increased running training. In a trial of lipid oxidation induced in 18 untrained men by three sessions of resistance exercises. 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. or isoprostanes (see desciption in the section on vitamin C above). In a subsequent study of 24 diabetic children by use of the same design. a significant decrease in plasma MDA was found in the vitamin-supplemented group as determined by HPLC . lipid hydroperoxides. In a three parallel groups of 16 middle-aged male and female participants. 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. giving a 900 IU dose of vitamin E was found to be no better than a placebo in decreasing MDA . 30 mg beta-carotene and 100 µg organic selenium.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. No change in plasma MDA either within or between the two groups could be shown . there was a significant decrease in erythrocyte MDA in the vitamin-supplemented group . 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 . In another study. no effect on the plasma MDA was detected . but to be unaffected by a slow-release formulation as compared with placebo treatment . who were given either 500 mg/d of vitamin C together with 182 mg/d dl-α-tocopherol. controlled intervention study. given in a normal formulation. 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 . In a double-blind. a reduction in plasma MDA occurred in the group assigned vitamin supplements . 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).
or a placebo in a parallel design. no effect on plasma antioxidant capacity was observed at 12 or at 36 months . . or placebo. 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 . 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 dose-response study of 40 healthy men assigned doses of 60–1200 IU of vitamin E for an 8-week period. LDL-dienes. 182 mg/d dl-α-tocopherol alone. 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. and the strength of this effect was correlated with the level of plasma α-tocopherol . A significant change in these groups in whole plasma ex vivo oxidation at 36 weeks was likewise observed . Dragsted 40 182 mg/d dl-α-tocopherol alone. In 49 diabetic patients given 504 mg/d d-α-tocopherol or a placebo for 6 months. In a blinded intervention study of 33 triathletes participating in a world championship. In a double-blind controlled intervention study of 56 patients with congestive heart failure. the effects of giving mixed supplements of 200 mg/d vitamin E. In a study without a parallel control group. 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 . 900 mg/d vitamin C and 18 mg/d beta-carotene for 6 months were compared with those of a placebo. or LDL-hydroperoxides induced ex vivo . who were given 800 IU/d vitamin E or a placebo for 2 months prior to the race. In a trial involving 80 men who had an increase in plasma lipid oxidation following exposure to olive oil or to menhaden oil. In a group of 45 randomised healthy males and females. 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 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. Non-induced lipoprotein oxidation ex vivo was found to be delayed in the group given the vitamin supplement. a 1000 IU/d vitamin E supplement for a 10 d period was found to reduce the exhalation of breath pentane . no difference was observed in LDL-MDA. vitamin E given at doses higher than 200 IU/d was found to affect the kinetics of ex vivo oxidation of LDL . 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 . including IL-6 . 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 . this together with 182 mg/d dl-α-tocopherol. 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 . 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.Lars O.
In a study without a control group. no effect on the excretion of F2-isoprostane could be observed for any of these interventions . 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 . but this was not changed by the vitamin supplements. the rest being given a placebo. Running as such was found to increase the plasma isoprostane level. or a placebo during the 6 weeks prior to the race. 200. or a placebo during a 28 d period on the plasma isoprostane concentrations induced by exercise. In a dose-response study. which may have caused the difference. 800. 400. This was not observed for the young men. In another double-blind placebo-controlled trial with a crossover design. In a study of the combined effects of giving either vitamin C (500 mg/d) together with d-α-tocopherol (400 mg/d). C and E 41 In another study. no effect on the plasma F2-isoprostane level of taking 1000 IU/d of vitamin E was found for 20 patients with endothelial dysfunction . 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. 10 of them were given 500 mg/d and 10 of them 1000 mg/d vitamin E for a period of 3 weeks. and the vitamin supplement was found to prevent this . 600 or 1200 IU/d of vitamin E for 3 weeks. The inflammatory markers were also affected by running. The study design could not control for period effects. vitamin C (500 mg/d) together with d-α-tocopherol (290 mg/d) and d-γ-tocopherol (130 mg/d). the adding of vitamin E was found to have no further effect on the urinary excretion of F2-isoprostanes . groups of 5 healthy individuals received either 0. No other significant differences were observed between the two groups . 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 43 hypercholesterolemic patients randomised to taking simvastatin. In a third study of the effects of vitamin E on oxidative damage induced by exercise. particularly in the males. simvastatin together with 600 mg/d vitamin E or a placebo for 2 months. no effect of either vitamin treatment was observed. followed by 8 weeks of washout. No significant change in the plasma F2-isoprostane level could be detected by GC-MS at any time point. In a small non-blind study of cirrhotic patients. 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 . 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 . 21 ultramarathon runners were given either a combination of 300 mg/d d-α-tocopherol and 1000 mg/d vitamin C.Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A. . 300. In a double-blind controlled intervention study of 56 congestive heart failure patients. 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. 1200 or 2000 IU/d of vitamin E for a period of 8 weeks. In a group of 33 sclerosis patients. In an intervention study of 46 healthy smokers given 0. No effect on urinary excretion of F2-isoprostanes was observed . irrespective of the treatment .
and beta-carotenes) in human plasma by isocratic liquid chromatography. 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. each with 30 men. Sperm counts and sperm motility are thought to be partially influenced by oxidative stress. . these proposed functional tests for vitamin E must be regarded as obsolete. alpha-tocopherol and carotenoids (lutein. References 1. 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. Breinholt V. 2nd ed. Since plasma vitamin E levels are not always correlated with the stability of the erythrocyte membrane or with the exhalation of breath pentane. chemistry. 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 . J Chromatogr B Biomed.14:125–30. Invest Ophthalmol 1975. Bawa AB. 2. New York: Raven Press Ltd.654:129–33. Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat.154:201–10. rapid fluorometric determination of retinol in serum. Swanson D. but this parameter was not assessed in the other study [126. In two randomised studies. The retinoids: biology. 179–209. Daneshvar B. In: Sporn MB. Goodman DS. 3. Vitamin E intervention has not been shown to decrease the oxidation of plasma proteins.131:1626S–30S. and medicine. Jakobsen J. Cancer Lett 2000. Futterman S. present day HPLC and GC-MS methodology has high precision and accuracy in the detection of vitamin E analogues in plasma. a significant increase in the binding ratio to the zona pellucida of the unfertilized oocyte in a competitive binding assay was observed. Simple determination of retinol. Kalina RE. Conclusions In conclusion. Bui MH. Vitamin E deficiency is also known to cause semen abnormalities and infertility in rats. all-translycopene. little effect of taking 600–800 mg/d of vitamin E for 2–3 months was shown. 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. J Nutr 2001. Furr HC.Lars O. In one study. 4. A new. Roberts AB. editors. p. No evidence was presented that this effect was related to antioxidation. alpha. 1994. Olson JA. Analytical methods. Dragsted 42 Effects on protein oxidation In a study without a control group. Lauridsen ST.Appl 1994. 5.127]. Innovative approaches to vitamin A assessment. Craft NE.
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2.3]). Not only is DNA mutation a crucial step in carcinogenesis. that metabolites of atmospheric oxygen. unless repaired prior to DNA replication. lipids and nucleic acids. though not generally classed as carcinogenic agents. Peter Møller1. Nicolaus Copernicus University. together with their mutagenicity in mammalian cells. Denmark University of Leicester. nevertheless. The presence of 8-oxoGua in the cells can thus result in point mutations. and partly carcinogens that occur naturally in the products. Rafa∏ Ró˝alski3. increasing attention has been directed at oxidative DNA damage in relation to cancer. which can be produced both in the biochemical utilization of oxygen and as a by-product of O2 metabolism in the mitochondria. Oxidative mechanisms have been shown to have a potential role in the initiation. such as in the cooking of meat. are human carcinogens. partly those generated in processing. 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.and purine-derived lesions being formed in the DNA (as reviewed in ). Cook2.8-dihydroguanine (8-oxoGua). 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 . oxidative DNA damage needs to occur at a sufficiently high frequency to exceed the cell’s capacity for DNA repair. can damage cellular molecules of different types — in particular proteins. ROS. corresponding to about 2000 oxidative modifications of guanine per cell daily. Lesions such as 8-oxodG are used as biomarkers of oxidative stress. It is quite possible. has been documented in aging cells and tissues. Some of these modified DNA bases have considerable potential for damaging the integrity of the genome (reviewed in [2. UK Collegium Medicum in Bydgoszcz. One of the most widely studied lesions of this sort is 8-oxo-7. This.5 nmol per kg per day. When mutations accumulate. so-called reactive oxygen species (ROS). An increase in somatic mutations. Marcus S.Vitamins and selenium: Antioxidant vitamins and cancer risk 51 2. has led to the proposal that they be used as intermediate markers . The presence of 8-oxoGua residues in DNA. 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. Antioxidant vitamins and cancer risk: is oxidative DNA damage a relevant biomarker? Steffen Loft1. Since the cumulative risk of cancer increases at a rate corresponding to the fourth power of age and is associated with accumulated DNA damage. leads to GC→TA transversions . which are implicated in the development of cancer. In order to produce mutations. this can be due to the individual’s overall lifetime exposure to endogenous and exogenous agents that damage the DNA. however. Ryszard Oliƒski3 1 2 3 University of Copenhagen. Food and beverages frequently contain carcinogens.
Marcus S. . Ultimately. together with the increase in DNA damage that occurs. although their suitability for this has yet to be verified in prospective studies of cancer risk. or (ii) there being reduced DNA repair . creates a selection pressure for the malignant phenotype seen in the case of cancer. the nuclei of the undifferentiated. Issues of this sort have to be addressed before a causal link between oxidative DNA damage and cancer can be established. Again. Rafa∏ Ró˝alski. For DNA mutations to arise through oxidative damage.2 lesions per 106 unaltered guanines and that reports of values outside this range should be viewed skeptically . 3. Ryszard Oliƒski 52 of a potential disease endpoint such as that of cancer. 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. 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.3–4. not only must the DNA of the target cells be affected. 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. Elevated ROS levels may activate transcription factors and the corresponding genes being permanently activated. Peter Møller. connected with the spurious oxidation of DNA during sample preparation. Although studies that indicate this support the hypothesis that oxidative DNA damage can be an important risk factor for carcinogenesis. 2. Oxidative DNA damage may be an epiphenomenon in relation to the ongoing pathophysiological process. the analytical procedures in current use are unable to quantify the lesion levels found in the most important ‘target’ cells. the levels of damage having no causal role in carcinogenesis. 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. In addition. 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 . but also the damage must be within a coding region of the DNA. it has been argued that the mere presence of 8oxodG in DNA is unlikely to be a necessary and sufficient condition for tumour formation. Cook. 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. such as a high level either of metabolism or of cell turnover. regarding the question of ‘cause or consequence’. This raises a number of different issues: 1. An elevated level may be due to some well-established characteristic of the tumour. proliferating stem cells have to be affected. Numerous studies have been concerned with the relationship between the level of oxidative DNA damage and cancer (see  for a review). In order for a mutation to come about. there are serious problems in the chromatographic measurement of 8-oxodG. Since tissue samples from both tumours and normal cells represent a heterogeneous mixture of differentiated and undifferentiated cells (the former being likely to predominate).Steffen Loft. This. At the same time.
. that DNA damage. One should bear in mind. respectively. this soon being followed by the performance of antioxidant trials in which urinary 8-oxodG excretion served as the key biomarker. 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. C and E or phenolic compounds. A plethora of descriptive epidemiological studies have concerned a possible protective effect of a diet rich in fruits and vegetables . the first of many antioxidant supplementation trials dealing with this lesion in WBC was carried out . nevertheless. Intuitively. but that it is impossible at present to determine to what extent oxidative stress is directly involved in carcinogenesis. oxidants are involved in each case. large-scale intervention studies have failed to demonstrate use of antioxidant vitamins to reduce the risk of cancer . supplementation trials would appear to represent the most relevant way of exploring antioxidant effects. the mode of action of dietary micronutrients is complex and is far from being fully understood. It is very difficult. These antioxidants are effective free-radical scavengers and should protect the DNA from oxidative damage. have become by far the most popular assays for use in conjunction with antioxidant intervention trials. therefore.12]. altered gene expression and mutations are necessary elements in the process of carcinogenesis.Vitamins and selenium: Antioxidant vitamins and cancer risk 53 Summing up. 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. At the beginning of the 1990s. used for the detection of DNA strand breaks (SB). Although these events may come about by way of different mechanisms. Reliable detection of urinary 8-oxodG excretion became possible at about the same time . since full development of the disease in response to exposure to a carcinogen may take 20–40 years. 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). The literature on biomarkers of oxidative DNA in WBC employed in small-scale intervention studies of antioxidant supplements has been summarized in a series of reviews [9. Nevertheless. 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. Subsequently. the comet assay. to establish directly that the DNA lesion responsible for a carcinogenic process is the lesion present in a tumor many cell generations later. At the same time. Both experimental and epidemiological data indicate vitamin C to protect against both stomach and oesophageal cancer . however. although sampling is usually restricted to use of such surrogate tissues as white blood cells (WBC) and urine. It is reasonable to assume that agents that decrease oxidative DNA damage should also decrease the subsequent development of cancer. as the possibility of detecting 7-hydro-8-oxo-2’-deoxyguanosine (8-oxodG) appeared.
Rafa∏ Ró˝alski. from use of a non-optimum design. Table 2. Peter Møller.Steffen Loft. 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. Table 2. 200 IU α-tocopherol. 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. and 6 mg β-carotene) for 6 mo Carotenoidsc for 3 wk 100 M (50S) 50—59 A decrease in ENDOIII sites (Comet) in WBC after 20 weeks 13 63 MF (S) 42±9 No difference in 8-oxodG in WBC (antibody-based detection) between the supplemented and the placebo group.6 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 . 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. Cook. Ryszard Oliƒski 54 Many studies suffer. but a decline in the course of the trial in both groups 32 MF (NS) 32±11 No effect compared with baseline. and 25 mg β-carotene) for 20 wk Multi-vitamin (250 vitamin C. 280 mg vitamin E. 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. however.6. Marcus S.
but not earlier 68 MF (13S) 18—45 A decrease in the 12-h urinary excretion of 8oxodG (HPLC) 27. 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. Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont.Vitamins and selenium: Antioxidant vitamins and cancer risk 55 Table 2.6. 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.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. 45 Two interventions of green tea with 300 ml for 7 d or 32 oz for 7 days Green tea extractk for 3 wk 317 16 M (8S) 20—31 No effect of supplementation on the 24-h urinary excretion of 8-oxodG (HPLC) but a decrease in excretion during the study in all groups 38 F (NR) NR A decrease in the excretion of 8-oxodG (ELISA) in the active group (statistical test not reported) 44 Soya-hypocotyl tea (> 1 l) for 1 mo 42 . No effect on the 24-h urinary excretion of 8-oxodG (HPLC). but a 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.
4 mg). and 15 mg β-carotene. selenium (167 mg). The fruit capsules were made from juiced apple. Cook.16]. broccoli. or a carotenoid-rich diet. lutein (4. the question of whether multiple vitamin supplementation provide better protection against oxidative DNA damage than a single dose does cannot be answered unambiguously. however.5 mg). tocopherols (650 IU). orange. Containing vitamin A (240 mg). catechin (13. In two well-designed studies of the effects of administering a combination of antioxidant vitamins. possibly because of differences in the respective rates of bioavailability and elimination. 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.44 mg) per day. 4 mg β-carotene). The effect of a single dose of vitamin C seems to disappear after several hours. β-carotene (399 mg). Consisted of tablets containing vitamin antioxidants (400 mg vitamin C. Tablets containing micronutrients similar to those of 3 fruits and 3 vegetables (containing 107 mg vitamin C and 83 mg vitamin E). α-carotene (1. and papika carotenoids (2. Among studies in which multiple doses of single antioxidants are given. lutein (500 mg). The six groups received daily supplements of 1) 200 mg vitamin E. cabbage. Smokers (S) and nonsmokers (NS) are indicated in brackets. Also. beet. Consisted of β-carotene (20050 IU). parsley. The daily dose consisted of 200 mg vitamin C. papaya. 2.4 mg β-carotene. cranberry and peach. 4) 90 mg coenzyme Q10 in oil. spinach. being given rye crisp . and zinc (30 mg). 2) 500 mg plain-release vitamin C. 3) 500 mg slow-release vitamin C. 5) 30 mg coenzyme Q10 granulate. lycopene (100 mg).Steffen Loft. Rafa∏ Ró˝alski. lycopene (4. Accordingly. a protective effect could be shown after only 20 weeks of supplementation but no effect after 10 weeks (or 6 months of supplementation) [13. 18 IU vitamin E. 6) placebo. 150 mg vitamin E. Multiple administration of dietary antioxidants with assessment of oxidative DNA damage in white blood cells and urine — cont. 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. bixin (11.7 mg). vitamin E (400 IU). vitamin C (219 mg). and powder or extract of fruits and berries). rice milk. The vegetable capsules were made from carrots. whereas tocopherols and carotenoids exert their effects somewhat longer. vitamin C (330 mg). Ryszard Oliƒski 56 Table 2.14]. and pomegranate extract (5 mg). there are fewer that reveal protective effects than those that report only negative results [9.12]. Supplement consisting of β-carotene (6 mg). respectively [15. vitamin C (500 mg). or cow milk (1 l) for 4 wk 10 M (NS) 20—50 A decrease in ENDOIII sites (Comet) for soy milk Polyphenol-rich olive oils (25 ml) for 40 d 12 M (NS) 20—22 A decrease in the excretion of 8-oxodG (HPLC) in spot urinary samples following supplementation in a dose-dependent manner 21 43 a b c d e f g h i j k Number of subjects indicated as males (M) and females (F). and tomato. selenium (100 mg).5 mg/10 MJ). pineapple. 60 mg vitamin E. capsules (containing 90 mg vitamin C. vitamin E (1.4 mg). Supplement given per day Subjectsa Age (yr)b Effect Ref Soy milk. Peter Møller. Age is shown as range or as mean ± standard deviation. N-acetyl-1-cysteine (181 mg). Tablets containing β-carotene (12 mg). Marcus S. kale. Consisted of 1000 mg extract/kg body weight in meat patties (total phenolics 23.6. This suggests that the protective effect of a single antioxidant is relatively short.2 mg).12].2 mg).
Vitamins and selenium: Antioxidant vitamins and cancer risk 57 bread (76. number of subjects. An overall summary of the studies showed that six investigations reported beneficial effect of antioxidant supplementation. 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. .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. which showed the numbers of ENDOIII and FPG sensitive sites to be reduced . The other. a cross-over study of male smokers. 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. 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 24 studies of the effects of antioxidant supplementation on the urinary excretion of 8-oxodG in which a controlled design was employed [20. no appreciable difference was present in terms of duration of the intervention period.23–44]. 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. or power to detect a 50% change between these studies reporting beneficial effects and those reporting no effects. was negative with respect to effects on the ENDOIII and FPG sites . It can be speculated that the subjects suffered a slight. 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 FPG-sensitive sites. The only study showing consistent effects involving more than one endpoint was a study of the effects of kiwi fruit supplementation (1–3 kiwi fruits/d for 3 wk). Effect of antioxidant supplementation on 8-oxodG in urine Measurement of the urinary excretion of 8-oxodG in antioxidant intervention studies is connected with the idea that it decreases following a steady state ingestion of antioxidants due to the rate of generation of oxidative DNA damage in the body being decreased. unintentional intoxication of the gastrointestinal tract (some of those in the active groups complained of nausea. or to have any effect on the ENDOIII sites . The one investigation. assessment of the DNA damage occurring showed the levels of ENDOIII to be reduced . whereas 13 studies reported null effect. Anthocyanines have low bioavailability and the dose provided here was rather high. there in fact being a tendency in the group of subjects drinking black currant juice for the number of FPG sites to increase . Two studies concerned the effects of an intervention of providing vegetables and fruits. for example). 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. and their effects in the gastrointestinal tract are easier to comprehend.
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. cranberry.2 mg)) revealed a statistically significant difference between the active and the placebo group in the delta values obtained (i. that involved only male subjects.5 mg). A comparable study involving supplementation of a mixture of carotenoids (daily intake: α-carotene (1. bixin (11.0 mg). and vegetables. 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. orange. Taking capsules containing extracts of fruits and berries and eating diets rich in carotenoids were both found to lower the excretion of 8-oxodG . the Brussels sprouts supplementation was found to lower the urinary excretion of 8-oxodG . papaya. broccoli. A number of studies have involved supplying antioxidants in the form of berries. A positive effect of vegetable juice consumption on 8-oxodG excretion was also observed in subjects enrolled in a soccer summer training camp .34. 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 . parsley. whereas this intervention was found to have a pronounced period effect . In the one study a beneficial effect was found for drinking 300 ml/d for a week .31. the effect was less clear. β-carotene (6. pineapple. 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 . Rafa∏ Ró˝alski.Steffen Loft.39]. tea.35].44. the difference between results at the end of supplementation and at baseline) but no difference between the two groups at baseline . and peach) and of vegetables (carrot.41]. In the first study. fruits. cabbage.32. and paprika carotenoids (2. In the subsequent study. in which both sexes were included. Peter Møller. 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. No effect on the urinary excretion of 8-oxodG was found for the ingestion of capsules containing juices and powders of fruit (apple.e. neither supplementation of vitamin C or vitamin E or a combination of them was found to have any effect in healthy subjects [24. spinach. Three additional studies in this area concerned the effect of drinking green tea [see 27. and tomato) .45].38. In four additional studies.28. whereas in one of the other two studies .4 mg). lycopene (4. there being a tendency for only the male subjects to benefit from ingestion of Brussels sprouts.4 mg). beet. lutein (4. Mixed results concerning the effects of a dietary supplementation of Brussels sprouts (300 g/d) were obtained in two studies [40. Investigations of the effects of natural food products are distributed about equally between studies reporting beneficial and those reporting null effects. 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 . Cook. Further studies showed multi-vitamin tablet supplementation to provide no beneficial effect either in normal subjects [20. On the other hand.36].35. kale.37] or in subjects undergoing cold-weather field training at a moderate altitude [33. Marcus S.7 mg).
Paradoxically. adjustment of the data for a number of variables. therefore. such that iron catalytic to free-radical reactions (a so-called labile iron pool — LIP) is present in the blood plasma ). such as in the case of severe oxidative stress.Vitamins and selenium: Antioxidant vitamins and cancer risk 59 no effect of ingesting green tea extract in meat patties for 3 wk was obtained . It is worthy of note in this context that presumably healthy men may have the hereditary disease idiopathic haemochromathosis. 3. Although the unadjusted data of the third study indicated no beneficial effect of drinking green tea. and 3) the alterations in the urinary concentration could not be assessed due to insufficient information . 5. Indeed. including baseline 8-oxodG levels. such as those of regulating changes in gene expression. 2.45]. drinking soya hypocotyl tea was found to be associated with a lower urinary excretion of 8-oxodG. that the presence of oxidative stress. antioxidant vitamins may have biological activities. a positive correlation has been shown between LIP and the oxidatively modified nucleoside in human lymphocytes . which leads to iron overload. It is possible that a preventive effect of the vitamins can be only be seen when their basal levels are very low. 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. . 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. that are separate from their direct antioxidant effects . Experimental data suggest that supplementation of vitamin C to iron-overload subjects may increase the oxidative DNA damage that occurs . In still a further study. which might fail to be recognized. Interestingly. 4. the prooxidative properties of certain of the antioxidant vitamins (vitamins C and A) may take effect. vitamin supplementation of HIV-infected patients showing very low levels of antioxidant vitamins and significantly increased lymphocyte amounts of 8-oxoGua (as well as other base modifications) was found in one study to result in vitamin restoration to levels characteristic for the control subjects. 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 . revealed a statistically significant positive effect of drinking green for 4 months [27. The absorption of non-haem iron has also been found to be affected by ascorbic acid . 2) the putative active constituents (isoflavones) were not measured in the plasma. although it should be emphasized that the fate of the antioxidants in this investigations was inconclusive since 1) the plasma concentration of carotenoids decreased. Under some circumstances. could increase the likelihood of detecting a protective effect. It is possible. 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.
At present. which is considered to be the least effective form of antioxidant supplementation. and to other types of DNA damage. On an experimental basis. 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. Peter Møller. WBC studies provide little support for the idea that long-term antioxidant supplementation lowers the basic level of oxidative DNA damage. It should be noted. There is a tendency for supplementation with use of antioxidant-rich food to decrease the urinary excretion of 8-oxodG. It should also be borne in mind that the majority of studies involve healthy individuals. 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. many of the studies performed are of only limited value due to methodological problems related to the study design and the assays. Although single studies may possess considerable power due to specific aspects of their design such as use of repeated measurements. it is easy to understand the ingestion of antioxidants being associated with lower levels of oxidative DNA damage. no firm conclusions can be reached on the basis of antioxidant intervention studies. Marcus S. more attention should probably be devoted to alternative chemopreventive mechanisms such as the upregulation of DNA repair systems. whereas the protective effect of antioxidants may be more easily detected in subjects who are suffering from oxidative stress. Cook. yet the use of WBC as a surrogate tissue may not be particularly well suited for detection of this effect. Rafa∏ Ró˝alski. hundreds of subjects would be needed. 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. Also the chemopreventive effect of antioxidants on non-lymphatic tissue. particularly as regards the need of proper investigative designs and of the validity biomarkers. most studies encompassing far fewer subjects than this. and many of them lack the power of detecting 50% differences between two separate groups. should be addressed more thoroughly before the idea of antioxidants having clearly beneficial effects is abandoned. At the same time. Hopefully. . to obtain significant results. however. Ryszard Oliƒski 60 Comments There are numerous experimental studies published each year on the potential of antioxidants or antioxidant-rich foods for preventing the oxidation of DNA. In the future. such studies will benefit from the lessons learned from antioxidant intervention studies. that most of the studies reported have used supplements of vitamin E. The most likely effect ratio in healthy subjects involves a difference of less than 10%. which means that. This can be interpreted as antioxidants having an overall beneficial effect on the body as a whole. although there may be a beneficial effect in the first few hours after ingestion. which are of pivotal importance for such studies.Steffen Loft. which is not the case with use of single antioxidants. such as those involving bulky DNA adducts. which has been only sparsely investigated. a major problem is that many of these studies have too few subjects.
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since its conversion from 25-(OH)D is also tightly regulated. Since the time of first assays in 1971. Considerable interest has developed in assays for measuring 25-(OH)D. Sascha Abbas 64 2. As compared with 25-(OH)D. is converted by UV light from the sun (UVB 290–315 nm) into previtamin D3 which slowly isomerizes to vitamin D3. Such matrix effects can markedly diminish the validity of assays carried out .Jakob Linseisen. 25-(OH)D2 and 25-(OH)D3.25-(OH)2D. In addition. The biologically most active vitamin D metabolite is 1. its thus being more strongly affected by very recent sun exposure or dietary intake of vitamin D . In the blood vitamin D and its metabolites are bound to the vitamin D binding protein.2]. 7-dehydrocholesterol.g. or cholecalciferol. 25-(OH)D measurements are quite vulnerable to matrix effects. Extraction from the matrix (serum or plasma) is thus required for most analytical procedures (see Table 2. One of the major problems in measuring 25-(OH)D lies in the molecule itself. Because of its serum half-life time of about three weeks . the determination of circulating 25-(OH)D is not an easy task . with an even shorter serum half-life time of 4–6 h.25-(OH)2D or calcitriol) occurs primarily in the kidney (1α-hydroxylase). e.).25-(OH)2D whereas 25-(OH)D has only limited biological activity [1. Although such measurement is very accurate. this metabolite is a good indicator of the vitamin D-stores obtained both from exposure to sunlight and ingestion of vitamin D . is also not regarded as a suitable biomarker of vitamin D status . the circulating 1. Measurement by means of HPLC is considered by most to be the gold standard here [6. the half-life time of its precursor. to lipids. Because of its hydrophobic nature. Measurement of serum 25-hydroxycholecalciferol as marker of vitamin D status Jakob Linseisen and Sascha Abbas DKFZ. Further hydroxylation to 1.25-(OH)2D level does not provide valid information on the individual’s vitamin D status . strong efforts have been made to simplify them for routine use . There is overall agreement that 25-(OH)D is the appropriate biomarker to measure vitamin D-status in humans. Germany Vitamin D3. vitamin D (cholecalciferol).3. arising from the irradiation of ergosterol). Determination of 25-(OH)D As reflected in the recent scientific literature. Heidelberg. requires expensive equipment (HPLC) and is more time-consuming than other . Vitamin D is metabolised by hepatic 25-hydroxylase into 25-hydroxycholecalciferol (25-(OH)D). the human diet contains vitamin D in the form of vitamin D3 (in food of animal origin) and vitamin D2 (ergocalciferol contained in plant food.7. First of all it is a very hydrophobic compound and secondly it exists in two forms.8].25-dihydroxycholecalciferol (1. is rather short (24 h in the blood circulation). is synthesized in the skin. it has to be performed by experienced personnel. Plasma 1. Its precursor. Cancer Epidemiology. which is the major circulating metabolite.
however.7. as well as being fast. no yield determination required Calibrators and controls in the serum matrix. Table 2. There are important differences in the performance of the various detection methods. counting or microplate reading times. 10 min Uses vitamin D binding protein * Represents the initial starting volume of the sample. 50 µL Extraction detection (nmol/l) Acetonitrile Sensitivity Intraassay Interassay Assay time** 2 h. Radioimmunoassays (RIA). no yield determination required No radioactive waste. IDS Serum or plasma 50 µL Two-step reagent extraction 4—400 ≤5 5. HPLC — high-performance liquid chromatography. Sample type Manufacturer and volume* RIA.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 65 methods. 24. Biomedica Proprietary extraction reagent Unknown 6.4 20 min The laboratory must have an HPLC unit with a silica column CPB. ** Does not include extraction.2 8. .5 7. 0 min ELISA. requires luminometer Comments (nmol/l) CV (%) CV (%) ≤6 <8 <12 RIA. RIA — radioimmunoassay. DiaSorin Serum or plasma. 10 min Calibrators and controls in the serum matrix. IDK Serum 500 µL 17. ELISA — enzyme-linked immunosorbent assay.25 (OH2)D The primary antibody is the vitamin D binding protein from serum Fully automated.9 14 1 h. and are thus more frequently used. Table 2. 100% cross-reactivity with 24. 25 (OH2) D. IDS Serum or plasma 50 µL Serum or plasma 50 µL None 6—360 ≤5 <6 <9 3 h.plasma nostics or urine 500 µL 8—312 2.25-dihydroxyvitamin D. enzyme immunoassays (EIA) and chemiluminescence assays (CLPBA) are easier to handle.3—250 2. CV — coefficient of variation.6 11. Immundiag.2 75 min Acetonitrile and C18 cartridge extraction Acetonitrile 15—150 4.4 9 11 5 h.0 5. 0 min ELISA.5—300 18 6.9 3 h. 0 min ChemilumiSerum nescence. Serum.5 9. summarizes some of the performance characteristics of most of the assays that are available commercially. Commercially available 25-hydroxyvitamin D assays  Range of Test principle.7. 24. CPB — competitive protein binding. or plasma Nichols 20 µL Institute Diagnostics HPLC.
1 to 35.Jakob Linseisen.). Table 2. RIA — radioimmunoassay.). HPLC — high-performance liquid chromatography.5. 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. The means varied widely between laboratories from 17. . In their study.  recommended the use of RIA techniques for 25-(OH)D measurement until other methodologies have been developed further.8. Sascha Abbas 66 Validity of different assays Only few studies have compared the different commercially available assays [9–13]. After spiking of the serum samples with a defined quantity of 25-(OH)D.). Binkley et al.4. lists the methods and the normal range for 25-(OH)D measurement in the laboratories that participated.4. Mean (SEM) results for the serum 25-(OH)D concentrations for 10 subjects as estimated by six different laboratories (for abbreviations see Table 2. On the basis of their results.005) . the serum of 10 subjects was sent to six different laboratories.8.  reported recently not only immense variation between different assays but also variability between different laboratories in using a given assay. and the proportion of subjects below an arbitrary threshold of insufficiency (32 ng/ml) varied between 17 and 90%. Binkley et al. 2. The mean serum 25-(OH)D concentrations differed 2-fold between laboratories (Figure 2.8. Table 2.6 ng/ml (p < 0. Fig. a broad range (17–95%) of the expected concentration was found by the different laboratories  (Figure 2.
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 increment was less than anticipated and varied between laboratories (p < 0. several authors have called for international standardization of the vitamin D assays that are available and that are affordable for practicing clinicians. 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 . There has been discussion regarding the importance of the assays detecting 25-(OH)D2 as well as 25-(OH)D3. Both assays also underestimated the 25-(OH)D-concentration as compared with the HPLC-method . Each of the laboratories was given five serum samples quarterly. 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. shows the latest results of this external quality control project.4 to 44.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 67 Fig.7 to 18 ng/ml. Figure 2. finding discordant serum 25-(OH)D-values. as estimated by five different laboratories (for abbreviations see Table 2. In accordance with these findings.05). 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 .13]. 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) . 2. however. According to the latest DEQAS (Vitamin D External Quality Assessment Scheme) report. Another study compared the DiaSorin RIA assay with the Nichols chemiluminescence assay. Mean (SEM) results for the 25-(OH)D concentrations in the serum samples of 10 subjects spiked with the 25-(OH)D standard. especially those in the US. 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). Patients. These give the impression that only method 6 exceeded the limit. However.005) from 7.6 ng/ml (p < 0. 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 means varied widely between laboratories from 27.8). However.6. the greatest increment being detected by HPLC . 59% of the participating laboratories were found to have met the target [12.5.
In addition to the high degree of uncertainty that the divergence of the results for different techniques and different laboratories creates. Apart from the analytical problems. This is in contrast with most of the published studies. IDS EIA. competitive protein-binding assay.Jakob Linseisen. In a recent publication. and the composition of the population being studied. Populations at risk include nursing home residents and the elderly. A summary of studies on vitamin D status in the elderly is given elsewhere . around 25–40 nmol/L [1.18–23]. the serum 25-(OH)D concentrations in different populations vary with latitude. but there may also be a significant proportion of the population that exhibits asymptomatic subclinical vitamin D insufficiency. Nichols automated chemiluminescence assay. race. especially the home-bound elderly. Sascha Abbas 68 Fig. There is growing evidence that there is not only a specific seasonal decline in serum 25-(OH)D during the winter months. Method 1. method 6. DiaSorin RIA. method 2. Hollis defined 25-(OH)D levels below 80 nmol/L as being deficient. . suggested cut-offs for insufficiency range from 10 to 30 ng/ml (25–80 nmol/l).16]. Each column shows the mean deviation (% bias) and SE from the target value (ALTM) during the period of 2000–2004. due to the serious health risks encountered at levels less than this . age. that set the cutoff points at much lower levels. It is important to note that “normal” is not defined as the median population level. season. method 3. 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. Accuracy of 25-(OH)D methods used by the different DEQAS participants . Recent reports suggest European and North American populations to have a higher degree of insufficiency than had previously been thought [15. 2. method 4. HPLC. but the level of circulating 25-(OH)D sufficient to maintain good health and avoid clinical symptoms or consequences due to an inadequate vitamin D supply. The method means of samples known to contain 25-(OH)D2 were excluded. method 5.18]. In epidemiologic studies.6. as shown by the seasonal variation in vitamin D status that occurs [17. IDS RIA.15. epidemiological variability needs to also be taken into account. dietary intake.
Overview of Vitamin D Measurement and Methodologies. et al. Krueger D.89:3152–7. Goldswain PR. Gray RW. Potts JT. Myles M. The measurement of vitamin D: analytical aspects. Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D. This makes strong scientific research efforts needed. Lake E. Wilz DR. DeLuca HF. vitamin D3). Epidemiologic studies in particular can contribute knowledge concerning the association between vitamin D insufficiency and the risk of disease.Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol 69 Conclusions In conclusion. Immunodiagnostic Systems (IDS).46:1657–61. 6. 10. 8. J Clin Endocrinol Metab 1986. Cowgill CS. References 1. Zhou XY. et al. Clemens TL. Issues of methodology. including the risk of cancers at various sites. Lemann J. Hansen KE.46:756–65. Ann Clin Biochem 2003. Hollis BW. Smith GA. Binkley N. Review SeriesVolume 2. Nonetheless. the determination of vitamin D status is still a challenging task. J Clin Endocrinol Metab 1978. McGuiness M.63:656–60. 11. 9. 4. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. Hollis BW. Lindsay R. Endres D. Hart GR. J Clin Endocrinol Metab 2004. Clin Chem 2000. Taranto M. Hollis BW. 2. and problems in defining the appropriate threshold for vitamin D sufficiency.89–90:611–4. Lips P. Musk AA. J Clin Endocrinol Metab 2004. 3. Caldas AE.25-(OH)2-vitamin D3 in healthy adults. Editorial: The determination of circulating 25-hydroxyvitamin D: no easy task. Which circulating level of 25-hydroxyvitamin D is appropriate? J Steroid Biochem Mol Biol 2004. Noble JM. vitamin D metabolites have various important biological effects. Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects. Zerwekh JE. Comparison of commercially available (125)I-based RIA methods for the determination of circulating 25-hydroxyvitamin D. Ann Clin Biochem 2004. epidemiologic variation in vitamin D status and its determinants. Metabolism and excretion of 3H-1. Plum L.33:554–7. Glendenning P. Belsey R. May 2005. Several problems are important to bear in mind including the different forms of vitamin D (vitamin D2.40:546–51.135:317–22. the measurement variation introduced by different assays and different laboratories. J Nutr 2005. J Clin Endocrinol Metab 1971. 7. . standardization and metabolite recognition for 25-hydroxyvitamin D when comparing the DiaSorin radioimmunoassay and the Nichols Advantage automated chemiluminescence protein-binding assay in hip fracture cases.41:272–81. DeLuca HF. Competitive binding assay for vitamin D and 25-OH vitamin D. 5.89:3149–51.
Aksnes L. Jones J.60:658–9. MacFarlane GD. Grauer A. Seasonal variation of biochemical indexes of bone turnover: results of a population-based study. Hercberg S. Preziosi P. Makin HL. Davison KS. Engelsen O. Ersfeld DL. 18.7:439–43.7:327–35. Horowitz M.89:5387–91. Jones G. Brunvand L. Leidig-Bruckner G. Metab 2004. Prevalence and predictors of vitamin D deficiency in five immigrant groups living in Oslo. 22. 14. Kissling C. Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation. 23. J Clin Endocrinol Metab 1998. Berry J. Prevalence of vitamin D insufficiency in an adult normal population. J Steroid Biochem Mol Biol 2004. Sascha Abbas 70 12. Hanley DA. Alsaker E. Jones J. Proc Nutr Soc 2003. Norway: the Oslo Immigrant Health Study. et al. Jakobsen J. 17. Carter GD.Jakob Linseisen.135:332–7. Beard MK. Vitamin D status: effects on parathyroid hormone and 1. 20. with particular reference to European countries. Public Health Nutr 2004. . J Steroid Biochem Mol Biol 2004. Vitamin D2 is much less effective than vitamin D3 in humans. 16. J Clin Endocrinol. Siris ES. Vitamin D status of middle-aged women at 65–71 degrees N in relation to dietary intake and exposure to ultraviolet radiation. Carter GD. Brustad M.83:68–75.89–90:621–2. J Nutr 2005. Carter CR. 19. Scheidt-Nave C. Morris HA. Am J Clin Nutr 2000. Woitge HW. Gunter E. Binkley N. Khan A. Lund E. Haug E. Carter R.50:2195–7. Need AG. et al. Galan P. 15. Geographical differences in vitamin D status. Holvik K. Obstet Gynecol Surv 2005. Chapuy MC. Eur J Clin Nutr 2005. Ovesen L.59:57–63. Meyer K. Armas LA. Hypovitaminosis D in a normal. Vitamin D insufficiency in North America. Holick MF. Meyer HE. Fenske JS. Clin Chem 2004. 13. et al. Arnaud S.62:813–21. Andersen R. Osteoporos Int 1997. 21. Katzer JT.89–90:467–71.71:1577–81. et al. Prevalence of vitamin D inadequacy among postmenopausal North American women receiving osteoporosis therapy. Maamer M. Heaney RP. Hollis BW. Body JJ. Nordin BC. Sackrison JL. Miller AB. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. apparently healthy urban European population. 25-dihydroxyvitamin D in postmenopausal women.
the mechanisms of action involved. When ingested by humans or other organisms. Selenium and cancer — selenoproteins as biomarkers in relation to selenium supplementation Björn Åkesson and Katharina Bruzelius Biomedical Nutrition. and methods employed in assessing an individual’s selenium nutritional status — both generally. Selenite and selenate are inorganic forms of selenium used as dietary supplements. These include the forms of selenium present in the body and in the diet. analogous to cysteine in which sulphur is replaced by selenium) (Table 2. In the diet.). Lund. together with epidemiological findings on relations between the selenium status in the body and risk of cancer are reviewed. Lund University Hospital. In the chapter thereafter (Gromadziƒska et al. Sweden Introduction The relationship between selenium and cancer involves many different factors. as well as in connection with long-term trials for investigating the disease-preventive potential of selenium supplementation.). Different forms of selenium With few exceptions. A variety of issues connected with this are reviewed in two of the chapters. and the use of intervention trials to study the cancerpreventive effects of selenium supplementation. several protein-bound forms having been identified.4.9. The replacement of methionine by selenomethionine appears to be random and to be dependent on the relative concentrations of these amino acids . The present chapter concerns the various forms of selenium and their metabolism. Lund University. and in epidemiological studies of the risk of cancer. several low-molecular-weight selenium compounds have been shown to be present in different foods. Another selenium-containing amino acid is selenomethionine. their functions and mechanisms of action. but little is known of the role it has here.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 71 2. it is incorporated non-specifically into proteins as an analogue of methionine . nearly all of the selenium in animals. It is uncertain whether they occur naturally in foods to any major extent. Other proteins can also bind selenium as a ligand . In addition to the forms of selenium mentioned. some of these compounds being uncharacterized [4–6]. plants and microorganisms is bound within proteins. A major part of the selenium in mammals is specifically incorporated into proteins of defined biological function. selenoproteins are largely found in animal foods. so-called selenoproteins. containing the amino acid selenocysteine (Sec. and Department of Clinical Nutrition. the occurrence of different selenoproteins used as biomarkers of selenium status. Pure and Applied Biochemistry. . which is synthesized by plants and by yeast.
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. . The major selenium metabolite excreted in urine is selenosugar.7. Selenomethionine. At toxic doses. dimethylselenide or trimethylselenonium ions for excretion. Selenomethionine and selenocysteine can also be converted to hydrogen selenide. selenocysteine and selenite can be converted into the key metabolite hydrogen selenide (H2Se). Several compounds can be converted into methylselenol (from ).). Katharina Bruzelius 72 Table 2. a much lesser amount being excreted as trimethylselenonium ions . which is turn is the precursor of selenocysteine in selenoproteins and various excreted forms of selenium.9. 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).Björn Åkesson. Selenate and selenite are reduced by glutathione to hydrogen selenide. selenium is removed as dimethylselenide via exha- Fig.7. Metabolic pathways of selenium.
Several studies have suggested that methylated Se derivatives. are the selenium compounds most effective in cancer prevention . mainly in the gastrointestinal tract Reduction of phospholipid hydroperoxides Peroxide reduction. Selenoproteins A total of 25 selenoprotein genes were discovered in the human genome several years ago by sequence analysis.10. mutations in gene associated with muscular diseases Unknown An antioxidant and selenium transport protein Reduces methionine-R-sulfoxides A membrane protein. found in embryos and in the olfactory epithelium Unknown Unknown Unkown. Several of these compounds can be converted to methylseleninic acid or methylselenol which have anticarcinogenic effects in vitro . involved in many biological pathways Mitocondrial thioredoxin reductase. involved in the elimination of misfolded proteins from the ER Unknown Unknown.) [1.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 73 lation. The distribution and concentrations of selenoproteins Table 2. yet the functions of many of the proteins involved are still unknown (Table 2.10. such as Se-methylselenocysteine and selenomethionine. only found in the testes Can reduce peroxides using glutathione as an electron donor Catalyses the formation of selenophosphate Cytoplasmic thioredoxin reductase. involved in many biological pathways Thioredoxin/glutathione reductase found mainly in testes . a membrane protein Involved in protein folding in the ER Unknown. 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.9].
The current conception is that the mRNA and the SBP2-EFsec loops back towards the translation complex [13. Except for the SECIS element there are no features in the DNA-sequence that selenoprotein genes are known to have in common. which has two . There are no selenoproteins known to be present in yeast or higher plants.11]. 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 .8. . Biosynthesis of selenoproteins Translation of the codon UGA to selenocysteine (Sec) and its insertion into proteins is a complicated process (Figure 2. Many of the known selenoproteins catalyse redox reactions in which selenium is at the active site. and the recently discovered L30 protein. In the first step. serine is bound to tRNASec and then transformed to selenocysteine .). Katharina Bruzelius 74 in different tissues are also not well known. selenoproteins also differing markedly in the nucleotide sequence of their SECIS elements. This takes place when a special stem-loop structure called the selenocysteine insertion sequence (SECIS) is present in the mRNA. and the thioredoxin reductases. but the mechanisms involved have not yet been elucidated (Figure 2. but the catalytic ability of these latter proteins is much lower. one internal loop. that regulate thyroid hormones. one apical loop. additional factors are required for the insertion of Sec in eukaryotes such as the SECIS binding protein 2 (SBP2). Several of the selenoproteins have an homologue protein containing Cys instead of Sec. and the SECIS core. The cytosolic or cellular form of glutathione peroxidase (cGSHPx) was the first selenoprotein identified [10. these organisms tending to have homologue proteins containing Cys instead of Sec . This structure is located in the 3’-untranslated region of the mRNA in eukaryotes and immediately after the UGA codon in prokaryotes [14. All known eukaryotic selenoproteins have one SECIS-element.15]. The SECIS consensus sequence consists of two helices. Several other glutathione peroxidases containing selenocysteine have been found since then. catalysing the reduction of oxidised thioredoxin and other substates [1. except for SeP. the Sec-specific elongation factor (EFsec). In eukaryotes the Sec codon and the SECIS element are located at some distance from each other in the mRNA.12].17]. In addition to the SECIS element.8. which is a short sequence of non Watson-Crick paired nucleotides that appear to exist in all selenoprotein mRNA:s. The other two major groups of known selenoprotein enzymes are the iodothyronine deiodinases. It is found in almost all tissues and is believed to play a part in the body’s antioxidant defence.Björn Åkesson. The next major step is the interpretation of UGA as a signal to insert Sec instead of stopping the translation.).
. phGSHPx and cGSHPx with the 3’UTRs (3’-untranslated regions) of each mRNA and then to study the system during selenium deficiency. This hierarchy is particularly noticeable in the glutathione peroxidase family. The stability of selenoprotein mRNAs is affected by the amount of selenium present in the cell. but it is not yet known how this regulation operates [18. In rats and mice fed a seleniumdeficient diet. 2. where the order of stability is as follows: giGSHPx ≥ phGSHPx > cGSHPx = eGSHPx. at least there are factors in both the coding and the non-coding mRNA regions that influence its stability . thus contributing to the body’s defence against free radicals. there being a selenoprotein hierarchy. the brain and the testes retain selenium whereas the selenium concentrations in the liver and the kidneys decrease markedly .19]. causing a conformational change which leads to the release of Sec-tRNASec. Accordingly an overview of the individual selenoproteins is provided.8. cGSHPx could not be stabilised by replacing its 3’UTR with those from more stable glutathione peroxidase mRNAs.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 75 Fig. but not with that from cGSHPx. Five selenium-dependent glutathione peroxidases are known to date . This indicates that in the cGSHPx mRNA. A special feature is that transcripts of some selenoproteins are much more stable than those of others. It was shown that giGSHPx and phGSHPx containing each other’s 3’UTRs would remain stable. After association with the ribosome the SBP2 and the L30 switch places. There is also a tissue selenium hierarchy that controls the retention of selenium in the tissues and organs under selenium-deficient conditions. and hydrolysis of GTP . Individual selenoproteins The glutathione peroxidase family The glutathione peroxidases generally catalyse the reduction of peroxides using mainly glutathione as the electron donor. The links between selenium and cancer probably involve different selenoproteins. The SECIS binds to SBP2 which binds the Sec-specific elongation factor EFSec and its Sec-tRNASec. Hypothesised Sec translation complex in eukaryotes. The role of the SECIS for mRNA stability was studied by combining the coding regions of giGSHPx.
Phospholipid hydroperoxide glutathione peroxidase. which has led to speculations that this enzyme is a form of selenium storage to some extent . protective adapatations against paraquat and other challenges were shown to take place. which has thus far only been found in olfactory epithelium and in embryos. This is the only selenoprotein that has been identified in milk thus far. which consists of four subunits. pancreatic. which is found in most tissues. phGSHPx occurs in mitochondrial. Gastrointestinal glutathione peroxidase (giGSHPx). In mouse and rat. This enzyme has been implicated in inflammation and molecular signalling. Putative functions of this enzyme are those of protecting against ingested lipid hydroperoxides and reducing susceptibility to colon cancer . catalyses the reduction of phospholipid hydroperoxides and is expressed in a wide range of tissues. but knock-out of both cGSHPx and giGSHPx leads to colitis in mice . is mainly found in the gastrointestinal tract and also in the human liver and in mammary cells and tissue according to certain studies [23. catalyses the reduction of hydrogen peroxide and organic peroxides. the selenocysteine in this enzyme is replaced by cysteine . Other tissues such as liver. thyroid. The most recently discovered member of the glutathione peroxidase family is glutathione peroxidase 6. The postulated roles of eGSHPx include control of peroxide transport and of the extracellular ‘peroxide tone’ [18. Disruption of the phGSHPx gene is lethal embryonically. Extracellular glutathione peroxidase. amniotic fluid. there also being a number of negative effects such as increased obesity and insulin resistance . This glutathione peroxidase isoenzyme is also found in extracellular fluids such as blood plasma. In model systems with an over-expression of cGSHPx. milk. placental and mammary gland tissue produce this enzyme to a lesser 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.21]. Although the glutathione concentration in the plasma is very low eGSHPx can also use other electron donors such as the thioredoxin system .Björn Åkesson. and its functional significance is unclear. nonmitochondrial and sperm-nuclei-specific forms produced from the same gene  and has an important role in sperm functioning .24]. Katharina Bruzelius 76 Cellular (or cytosolic) glutathione peroxidase (cGSHPx). It consists of four identical subunits. phGSHPx. each containing one selenium atom. giGSHPx knock-out models show no particular changes in phenotype to occur compared with the wild type. It differs from the three glutathione peroxidases just mentioned by being a monomer and having a different substrate specificity. the level of cGSHPx in most tissues decreases considerably. without any obvious damages to the host organism. eGSHPx. . is a tetrameric protein produced mainly in the kidney and then excreted into the extracellular environment .27–29]. cGSHPx knock-out mice have been used to explore the effects of complete loss of cGSHPx activity. skeletal muscle. lung lavage and the aqueous humour [21. unlike disruption of cGSHPx or giGSHPx. Under conditions of severe selenium deficiency.
The third isoform.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 77 The thioredoxin reductase family The thioredoxin reductases (TrxR) catalyse the reduction mainly of thioredoxin. 2 and 3 (DI1. thioredoxin/glutathione reductase (TGR).40]. and expression of DI2 and 3 having been found in many tissues. TrxR 1 and 2 are ubiquitous being located in the cytosol and the mitochondria. There are indications that it also acts as an antioxidant in the extracellular space. Truncated isoforms of this protein have also been found. this property indicating the existence of a feedback step in the production of selenoproteins. such as vitamin C. also catalyses the formation of selenophosphate . It can contain from 1 to 17 selenocysteines.38]. including bovine mammary tissue [34–36]. can also form complexes with mercury and cadmium . The iodothyronine deiodinases have high priority in the selenium hierarchy. was designated “P” because of its being found in the blood plasma. which is not a selenoprotein. In some tissues. It is localised in the endothelium. but not in the thyroid. kidneys and thyroid. such as DNA synthesis and the regulation of apoptosis. SeP is expressed in most tissues. Selenoprotein P is the major form of selenium in the plasma and is involved in selenium transport [37. organs that normally are highly prioritised during selenium deficiency. unless they are rescued by a high-selenium diet . DI1 decreases during selenium deficiency. binding to heparin and related carbohydrates . DI1 is found in the liver. their levels remaining virtually unaltered in the case of selenium deficiency. particularly DI2 and DI3. There are three main isoforms of thioredoxin reductases. iodothyronine deiodinase 1. is expressed in small amounts in many tissues but is primarily found in the testis . Targeted disruption of the TrxR 1 or 2 genes in mouse models is embryonically lethal . It can reduce peroxynitrite and phospholipid hydroperoxides [39. which catalyse the removal of different iodine groups from the thyroid hormones. but a number of splice variants of TrxR 1 and 2 have also been reported. but in mammals they can also reduce other substrates. DI2. Additional selenoproteins Selenophosphate synthetase 2 (SPS2) catalyses the formation of selenophosphate. respectively . Selenophosphate synthetase 1. Thioredoxin catalyses the reduction of protein disulfides and is involved in a number of vital processes. . Selenoprotein P Selenoprotein P (SeP). depending on the animal species. the second selenoprotein to be discovered. but is produced primarily in the liver and is secreted then into the plasma. and can stimulate the survival of nerve cells in culture . thus activating or deactivating them. DI3). SeP knock-out mice exhibit low levels of selenium in the brain and testes. SPS2 is a selenoprotein. These mice die after weaning of the young. which is the selenium donor for the formation of selenocysteine from serine bound in tRNASec. The iodothyronine deiodinase family The iodothyronine deiodinase family consists of three enzymes. for example. The 15 kDa selenoprotein.
which also includes non-specifically bound selenium. When data from . the measurement of selenoproteins can be expected to provide information on specific selenium functions. Selenoprotein M (SelM) is structurally similar to the 15 kDa protein and is supposedly also involved in protein folding in ER . A number of other selenoproteins have been discovered in man but information regarding their function is very limited. such as rigid spine muscular dystrophy. since the turnover of erythrocyte selenium is slower. Another important conception is that plasma selenoproteins may not be suitable biomarkers under conditions of high selenium status. the determination of selenium in the hair. Selenoproteins as biomarkers The use of selenoproteins as markers of selenium status has only been exploited thus far in a few large epidemiological studies. The function of selenoprotein W (SelW) has not been elucidated fully but it has been implicated in white muscle disease . SelN is the only selenoprotein in which a mutation of it has been shown to cause a disease . Katharina Bruzelius 78 located in the endoplasmatic reticulum (ER). as compared with plasma selenium. To date. mutations in the gene have been associated with various muscular diseases. Selenium in the plasma or serum is the best known and most accessible index. and may thus be suitable as an indicator of short-term changes. SelW occurs mainly in muscle and brain and has been shown to act as an antioxidant. utilising glutathione to reduce peroxides . nails and urine has been employed. It has been identified in prokaryotes. and it responds rapidly to changes in selenium status. usually responding rapidly to changes in selenium status or in the dietary intake of selenium . the activity of GSHPx in plasma has been used by a variety of laboratories in selenium supplementation studies. one that has been associated with the process of eliminating misfolded proteins by transferring them to the cytosol  and also with inflammation . In contrast. the selenium levels in whole blood or the erythrocytes appear to primarily reflect the long-term intake of selenium . Indices of selenium status The most commonly used methods for assessing the selenium status in humans involve analysis of selenium concentrations in the blood or blood fractions. For instance. Selenoprotein N (SelN) is a 70 kDa protein located in the ER.Björn Åkesson. The responses to selenium intake obtained if other blood fractions are analysed can differ. In general. Although its catalytic function is still unknown. This is an important step in the regulation of biological processes and the management of oxidative stress in the cell. since above a certain selenium level they tend to reach saturation. also known as VIMP) is a membrane protein in the ER. Selenoprotein R (SelR) reduces methionine-R-sulfoxides. is believed to be involved in protein folding . In addition. eukaryotes and archaea . Selenoprotein S (SelS.
44) 1.11.2 mmol/l .11.12) 0. 2.52 (0.66—1.32) 3.74)b — — — 126—205 38 21 16 31 1.65) 3.43) — — eGSHPx (mg/l) — 352 (306.08.60.41 (0.15) 1.83. Table 2. mU/mg protein.188.8.131.52. 1. 0.47) 1.83) 0.38 (1.51 (6.54—1.21) 1. respectively.22) 0.38 (2. .10 (1.20 (1.16) 0. 0.u.24) 1. Selenoprotein P accounts for at least 40% of the plasma selenium . selenium and eGSHPx in different studies (from ref.73) Values are means (95% CI).27) 1.23 (1. Trial I baseline Finland.75—2.41 (1.07) 1.73 (0.56) 4.43.98. Study European countries Prior to LDL-apheresis After LDL-apheresis Finland.31) 2.39.91 (0. 1. ).25) Plasma selenium (µmol/l) 1. Median values (and range) are given.11.69. Plasma concentrations of SeP.51.63—1.33.18 (0. 0.13 (4.86) 0.14 (1. 0.91 (1. glutathione peroxidase and protein-bound selenomethionine are the major selenium fractions contained in plasma .88) 184.108.40.206 (0. b GSHPx activity.). 0.38 (0.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 79 different cross-sectional studies was combined eGSHPx was found to approach a plateau at a plasma selenium concentration of approximately 1 mmol/l . Supplementation by selenate resulted in glutathione peroxidase activity plateauing at a plasma selenium concentration of 1.86 (0. 1. 0.52) 0. 1.34. 346)a 6.16.20—4.30—1. U/l.03 (0.46—1.64) 0.51) 0.00 (0. Immunoassays for measurement of this protein have been developed [57–59].83 (0. a GSHPx activity.07 (0. 1.31—4. medium and high fish intake.47 (1. In the following various results from their use in the authors’ laboratory are summarized (Table 2.95 (2. 1.78 (1. 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.83) 1. 1.77 (1. c Data from subgroups having (top to bottom) low. 397)a 302 (259.55 (0.56.14) 0. 1. 1.66) 1.69 (0. Selenoprotein P.41. 1.85) 1.) 1. 4.69—5.
Considerable attention has been directed at two studies conducted in Finland regarding the use of different forms of selenium [67.). eGSHPx and plasma selenium as markers of selenium status. . In a recent study of Chinese subjects of low selenium status given different doses of selenite and selenomethionine. Considerable variation between different regions was found. 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. the selenoprotein P values increased after selenium supplementation. A summary of the responses shown by the different selenium indices in the first of these two studies is provided in Figure 2. Poland revealed an inverse relationship between the blood lead level and the level of selenoprotein P and of extracellular GSHPx . In the Malmö Food Study  the relationship between the intake of different foods and selenium status was investigated. in which the subjects were low in selenium status.9. total selenium and selenoprotein P than control subjects did . In a study of Latvian fisherman . significant relationships between milk intake versus selenoprotein P and urinary selenium. and also between fish intake versus serum and urinary selenium were found. Home parenteral nutrition patients with a variety of gastrointestinal diseases showed much lower levels of extracellular GSHPx. but that at normal selenium status selenoprotein P usually correlated more highly with plasma selenium than eGSHPx did. 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. The levels of selenoprotein P and eGSHPx were found to vary markedly for subjects from different regions and with different diseases. plateauing within two weeks  (Figure 2. Regarding the relationships between SeP. studies of lead-exposed children in Katowice.9. performed after the introduction of selenium-enriched fertilizers in Finland. In the first study.68]. Concerning the association of selenium status and exposure to toxic metals. For women. The scientists there compared the responses to oral supplementation of 200 mg selenium per day in healthy subjects on two occasions. no significant increase in selenoprotein P levels was observed after selenium supplementation and no differences were found between groups given different forms of selenium . Since the direction of causality was uncertain. and it is not possible to conclude that lead exposure produces a decrease in the selenoprotein concentration or that a poor selenium status increases susceptibility to high blood lead concentrations. however. 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 . the selenium status of the subjects thus being higher.Björn Åkesson. In the second study. There was a close correlation between selenoprotein P and plasma selenium values. with some indication of a plateau. Katharina Bruzelius 80 Selenoprotein P as a biomarker Selenoprotein P levels were measured in healthy adults from 17 European Regions .
The calculated degree of protection was found to differ between cancer sites [81. β-carotene and α-tocopherol reduced the total cancer mortality and stomach cancer rate in a Chinese population. Clark and associates . particularly among men [75–81].5–2 ppm) levels of selenium to the diet has a carcinostatic effect in animals treated with carcinogenic chemicals [71. 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. In a number of case-control studies.72]. .9. Interest in the preventive role of selenium supplementation in humans was stimulated markedly by results from two intervention studies. Full expression of selenoprotein P was not achieved at the highest dose of either form (66 µg/d).69]). This suggested that selenoprotein P is a better indicator of selenium nutritional status than glutathione peroxidase is . 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. Blot and coworkers  found that a mixture of selenium. Fig. in turn found selenium supplementation to reduce total incidence and mortality of cancer in an American study group (see below). lower prediagnostic plasma selenium was found for cases of cancer than for controls. Generally speaking. Percentual changes in selenium status variables after supplementation of Finnish subjects of low selenium status with 200 µg/day of selenium in different forms (from ref [67.). whereas in other studies no significant differences in this respect were found between cases and controls [82–86]. Biomarkers of selenium status and cancer Experimental studies have shown that the addition of high (0.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. 2.87]. The strongest associations between premorbid plasma selenium levels and risk of cancer were observed for cancer of the respiratory and digestive tracts. These matters are reviewed in greater detail in the chapter that follows (Gromadziƒska et al.
2. Smoking and biomarkers of selenium status Several factors other than selenium intake may affect biomarkers of selenium status. 2. . These were 6. the SeP levels for cancer of the respiratory tract were found to be significantly lower than in matched healthy control subjects. and 0. Bold contour: p < 0. 2. For increasing quintiles. respectively (p for trend = 0. 2.0. In Figure 2. 3. The circles represent selenium and the squares SeP. 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 individual references are cited in . Fig.01). 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.85.4. Smokers were found to have significantly lower levels of selenoprotein P than nonsmokers . in the lowest tertile as compared with the cases in the highest tertile.90].6 respectively.Björn Åkesson.3. The factors contributing to the lower selenium status in smokers are unclear. When cases were divided into subgroups according to cancer site.9.05.10.89]. lower values of plasma selenium [77. normal contour: p ≥ 0. plasma selenium has been used as a marker of selenium status. and toenail selenium [90–92] were observed in smokers than in non-smokers. digestive and urinary tract and for cancer of other sites. In addition. the case-control differences of plasma selenium and SeP are compared for major cancer sites in several different study populations.0. 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.0 and 1. the ORs (adjusted for smoking) in tertiles of the SeP level were calculated for the respiratory.10.2. the ORs (adjusted for smoking) were 5. erythrocyte selenium . whole blood selenium [89.2. The previously reported association of plasma selenium levels with cancer risk can very likely be explained by the corresponding association of SeP levels.05. 0. In other studies. This is probably due to SeP constituting at least 40% of the total selenium in human plasma . The association between the relative risk of getting cancer and the SeP concentration found was also estimated from quintiles of the SeP level. Percentage differences in the prediagnostic selenium and SeP plasma levels between cancer cases and controls (set at 0) for cancer at different sites.
in particular mortality due to stomach cancer . suggests that the selenoprotein P levels are reduced by inflammatory activity. The natural presence of cadmium in tobacco smoke may also contribute to the lower selenium status found in smokers. It has been reported that smokers eat less selenium than non-smokers do.74]. Patients with a history of skin carcinomas who . which together with superoxide can produce peroxynitrite .25-year period. both being acute-phase reactants. several intervention studies having indicated beneficial effects [73. Multiple regression analysis also indicated the blood cadmium to increase significantly with a decrease in the selenoprotein P level. in a meta-analysis of 51 published nutritional surveys of the relationship between smoking status and nutrient intakes. α-tocopherol and selenium given for a 5. smoking was found to be significantly associated with an unhealthy pattern of nutrient intake. although this association disappeared when lead was included in the model. whereas the concentration of cadmium in the blood is significantly higher in smokers . Multiple linear regression analysis of the data also suggested a depressive effect of cadmium on the concentration of selenium in the blood. Smoking may also increase oxidative stress since cigarette smoke is a rich source of reactive nitrogen species.91). 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. a small but significant reduction in total cancer mortality was obtained (RR = 0. This may also explain the slightly lower selenoprotein P concentration in smokers. The cadmium blood level was found to be negatively associated both with selenium in the blood and with selenium and selenoprotein P in the plasma. This agrees with findings of Dreher and co-workers  indicating that the human selenoprotein P promoter is rendered less active by cytokine treatment. whereas smoking alone did not serve as a true predictor of this effect. 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. which suggests a repression of selenoprotein P expression during an acute phase reaction. 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 . which could exacerbate the risk of cancer associated with smoking . It has also been shown that selenoprotein P plays a role in the defence against peroxynitrite . and that these correlations are higher (more significant) in smokers.Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers 83 The finding that selenoprotein P is positively correlated with albumin level and negatively correlated with α1-antitrypsin. a result that could possibly be explained by the covariance of lead and selenium in the blood . selenomethionine and glutathione peroxidase can scavenge peroxynitrite. In the Linxian trial involving supplementation of β-carotene. As reported by Sies and coworkers . In another study. which could probably in part explain their lower selenium level . In addition.
4. Mammalian selenium-containing proteins. β-carotene. In addition. Mukhopadhyay R. Behne D.11:2071–3. In another Chinese investigation. J Nutr 1988. Occurrence of low-molecular-weight and high-molecular-weight selenium compounds in fish. Food Chem 1994. References 1.102]. It is apparent from this review that selenium can play an important role in cancer prevention. DNA sequencing of a mouse liver protein that binds selenium: implications for selenium's mechanism of action in cancer prevention. Cook RG. Scott J. selenomethionine supplementation of subjects with mild to moderate esophageal squamous dysplasia showed there to be a nonsignificant trend toward an increased regression and decreased progression of dysplasia as well as a significant beneficial effect in the subgroup showing mild esophageal squamous dysplasia . Vahl M. Carcinogenesis 1990.37) cancer . Bansal MP. 5. Srikumar TS.S. The type of selenium supplements best employed is also in need of further investigations. J Anal Atomic Spectrom 2001.21:453–73. vitamin E. Sunde RA. but additional studies are needed to determine whether there is also an increased risk of some forms of cancer after selenium supplementation. Medina D. . Annu Rev Nutr 2001. Speciation of selenoamino acids.118:367–74. Mukhopadhyay T. colorectal (RR = 0.50) and in incidence of lung (RR = 0. Waschulewski IH. Experimental findings also indicate that in some experimental systems selenium can both promote and inhibit cancer . selenonium ions and inorganic selenium by ion exchange HPLC with mass spectrometric detection and its application to yeast and algae.42) and prostate (RR = 0.51:45–9. Katharina Bruzelius 84 were treated with 200 mg selenium per day showed significant reductions in total cancer mortality (RR = 0. Kyriakopoulos A. wheras later results showed selenium supplementation to be ineffective in preventing basal cell carcinoma and that it increased the risk of squamous cell carcinoma and of total nonmelanoma skin cancer . Recent results of the SUVIMAX study showed supplementation with vitamin C. 3. Hansen M.16:1403–8. Effect of dietary methionine on utilization of tissue selenium from dietary selenomethionine for glutathione peroxidase in the rat. A summary of results of the first generation of nutritional intervention studies to prevent cancer have been presented  and future directions and criteria for evaluating the efficacy of such interventions have been proposed [101. Fan T.54). 2. Larsen EH. selenium and zinc to reduce the rate of prostate cancer in men having normal levels of prostate-specific antigen in their plasma . A better understanding of the mechanisms by which selenium interferes with the carcinogenesis process is a necessary focus for future research also for the evaluation of selenium related biomarkers. concerning the purportedly positive effects of selenium in connection with the prevention of cancer provided certain evidence for permitting a qualified health claim regarding selenium and cancer .Björn Åkesson. Åkesson B. evaluation of health claims by the FDA in the U.
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which suggests that the neoplastic changes that occurred could be attributed to Se. Shapiro . in addition to the occurrence of different selenoproteins.  observed the development of tumours in male rats fed a diet containing 4. Nofer Institute of Occupational Medicine.3 ppm Se.3 ppm level. In the present chapter the mechanisms behind the role that selenium can play in cancer prevention.  observed that liver tumours occurred in rats fed protein-deficient fodder and grains originating from Serich (5–10 ppm) regions. including the forms of selenium present in the diet and in the body. suggested that the pathological changes that occurred reflected regeneration of the liver rather than a carcinogenic process. ¸ódê. the methods used in assessing the nutritional status of selenium — in general. and intervention trials to investigate the effect of selenium supplementation on the incidence of cancer are summarized. Of 53 animals that survived for 18 months when thus fed.1% bis-(4-acetamine)-phenylselenyl hydroxide. History Historically.05–0. induced thyroid adenoma. equivalent to inclusion of 103–207 ppm Se in the diet for a 10-day period. Researchers of the former Soviet Union  feeding Se (as selenite) to rats at a 4. Poland Introduction There are many different aspects of the relationship between selenium and cancer. that since no studies have been reported of animals administered an analogous compound that did not contain selenium. in response to selenium supplementation. whereas such tumours were found in less than 1% of the animals in the control group. it is quite possible that the organic selenium compound employed there had carcinogenic properties not linked with the element per se. and in epidemiological studies of risk of cancer — and the outcome of long-term trials concerned with the disease-preventive potential of selenium supplementation. It should be noted. . In the previous chapter the forms and metabolism of selenium. their functions and mechanisms of action.5. Two of the chapters deal with these and related matters. as well as epidemiological findings regarding the relationship between selenium level and risk of cancer are reviewed. Wojciech Wàsowicz Department of Toxicology and Carcinogenesis.  published the alarming finding that feeding rats 0.Vitamins and selenium: Anticancerogenic activity of selenium 91 2. nevertheless. however. and concern for it as a carcinogenic agent dates to 1943 . Anticancerogenic activity of selenium — molecular mechanisms and epidemiological data Jolanta Gromadziƒska. In 1963. their use as biomarkers of selenium status. observed the development of different forms of liver tumours. Tscherkers et al. Nelson et al. Edyta Reszka. 11 were found to develop liver tumours. interest in selenium (Se) as a toxic trace element dates back to 1937 . Seifter et al. however.
In the control group. Supplying Se was found to decrease the number of tumours induced (from 97 to 46%). Ip and Sinha  investigated the effect of various concentrations of Se on the development of breast cancer induced in rats by 7. the first tumours developed when the animals were 4 months of age. a strain characterized by the spontaneous development of breast adenoma in 80% or more of the cases. Another early observation of such an effect was that of Clayton and Bauman .4-dimethylaminobenzene (3’-MeDAB). in rats to which either of two different forms of Se were administered . in addition to this.1 to 1.Jolanta Gromadziƒska. 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 . when Wassermann et al. whereas in the group receiving the largest amount of Se in its drinking water (1. The animals were divided into several groups. 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 .5 ppm) reduced the incidence of cancer even more. to 3%. Se supplied during only one of these two phases had a much weaker effect . but they also decreased in number with an increase in the amounts of selenium provided. Edyta Reszka. (cf.  showed that adding 0. At the same time. In the groups given extra Se in their drinking water. Harr et al.0 ppm Se in their drinking water. ) managed to inhibit the development of placental tumours in mice by use of Se compounds. These results indicated that supplying Se in larger amounts protects animals from developing cancer or delays .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). Providing selenium at a still higher level (2. respectively.0 ppm) they first developed at 17 months of age. Wojciech Wàsowicz 92 The anticancerogenic effect of selenium in animals was first reported in 1911.12-dimethylbenzantracene (DMBA). a control group receiving a basal diet that contained 0. using female mice of the C3H strain. 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. An interesting study of the anticarcinogenic role of selenium was carried out by Schrauzer et al. a supply of 0. The maize oil content in the one case was high (25%) and in the other case low (5%). who reported that in rats a diet enriched with 5 ppm Se decreased the incidence of liver tumours brought on by 3’-methyl-1. These findings were confirmed in a study showing that the number of liver tumours induced in male Spraque-Dawley rats by 3’MeDAB decreased from 92% in rats fed simply a control diet to 46% and 67%. tumours not only developed later. Two groups of animals received a diet containing maize oil. 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]. . particularly when it was provided at both the initiation and the promotion stage of chemical carcinogenesis.15 ppm Se and the other groups receiving.1 to 0. In both groups the incidence of cancer was found to decrease with increasing level of Se in the diet.
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). resulting in more efficient carcinogen detoxification [24.30] (Tables 2. 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.). one should bear in mind that the results of animal studies cannot be directly extrapolated to humans. Regarding the mechanisms involved. The anticarcinogenic effect of Se has been found to depend on the chemical form in which the element is administered. The effects can occur at a systemic. 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 . and perhaps selenoprotein P and other selenoproteins as well. its dose and the agent that induces the development of cancer .25]. 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. Experiments in which no effect of Se was observed were rather rare. and inhibiting angiogenesis [29. in the majority of cases the reduction being quite significant . altering the metabolism of carcinogens. in which the metabolism.Vitamins and selenium: Anticancerogenic activity of selenium 93 the carcinogenic processes. it has been postulated that the chemopreventive effect is related to the toxicity of selenium and the oxidative stress it induces. Cells adequately supplied with Se have been shown to be less sensitive to effects of both endogenous and exogenous carcinogens . Some reports suggest that the action of selenium toward cells that have been transformed earlier and toward normal cells differ . however. viruses or transplanted tumours . since reactive oxygen species can promote apoptosis in vitro . can prevent mutations by serving as free radical scavengers [26–28]. including their providing protection against oxidative damage. and 2.12. The anticarcinogenic effects of Se depend upon the chemical form of the Se-compound involved and the nature and dosage of the carcinogen. Glutathione . cellular or nuclear level. It is also possible that selenium in the form of glutathione peroxidases. It has also been shown. In addition. high levels of selenium compounds in the diet can reduce both the extent to which DNA adducts are formed and the extent to which DNA damage by carcinogens occurs. enhancing immune responses. proliferation and differentiation of the cells can be affected. In several studies. that selenium compounds can induce cell death through a mechanism distinct from oxidant toxicity [22. At the same time. affecting the cell cycle. The activity of many cellular targets. depends on the cellular GSH/GSSG ratio.13.23]. the activity of xenobiotic-metabolizing enzymes in vivo has been reported to increase when selenium compounds are given. Thus a number of mechanisms for the anticarcinogenic effects of selenium and of many selenocompounds have been proposed. 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.
for example: — ROS-activation of protein kinases in the cytoplasm and nucleus. Selenodiglutathione p-XSC. Se and the selenoproteins play a regulatory role in the following processes. 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. Yet non-protein selenium metabolites may also be important in the regulation of intracellular communication and metabolism. PKA .12. p-XSC. — Controlling changes in the cell redox potential through inducing activation of the transcription factors and initiating de novo gene expression. ) Form of selenium Selenite Selenite Selenodiglutathione Selenite. BSC. BSC ACF COX-2 PKC. Table 2. DNA synthesis (in vitro) Cell cycle Apoptosis BSC and ist glutathione conjugates p-XSC. Wojciech Wàsowicz 94 peroxidases and thioredoxin reductases are involved in ROS scavenging. 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. Edyta Reszka. and changes in cell growth. This suggests that selenoproteins participate in the regulation of intracellular signal transduction . intercellular communication. — Regulating the expression of membrane and nuclear receptors responsible for cell maintenance. Se-methylselenocysteine. — Affecting apoptosis. RNA.Jolanta Gromadziƒska. necrosis and cell survival processes . — ROS-activated modification of the thiol and hydroxyl groups in the Cys and Tyr. The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis (ref.
selenium dioxide.Vitamins and selenium: Anticancerogenic activity of selenium 95 Table 2. BSC. selenite Se-methylselenocysteine Se-methyselenocysteine. selenic acid Ebselen P-XSC. BSC — benzyl selenocyanate. whereas these processes can be inhibited by selenate . BSC p-XSC. Form of selenium Selenite p-XSC Selenite. p-XSC — 1. ACF — aberrant crypt foci. PKA. Selenite activates the protein kinases involved in the pathways of cellular response to stimulation by inflammatory agents. NF-κB — nuclear factor κB.12. triphenylselenium chloride p-XSC PKC TK Parameter Outcome I I I I Dose dependent E/I I I I/E/NE I Phospholipid/Ca+2 dependent PKC PKC JNK DNA cytosine methyltransferase PKC (in vitro) Cell proliferation and cell cycle biomarkers (in vitro) PKC and isoprostane (in vivo) I — inhibition. E — enhancement. JNK — Jun-N-kinase. ) — cont.13. TK — thymidine kinase. W — weak. NE — no effect. The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis (ref. 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. AP-1 — activator protein 1.4-phenylenebis(methylene)selenocyanate. PKC — protein kinase A and C. Inorganic Se compounds such as selenite reduce the number of fibronectin receptors at the cell surface. Oxidation of the thiol groups . COX-2 — cyclooxygenase. cell detachment Yes ++++ ++++ S/G2-M ++++ Necrosis Late Fibronectin is an extracellular component that plays an important role in intercellular communication. Since this is an immediate action. it cannot come about through activity of the selenoproteins. Table 2.
fos. mainly in regions in which DNA binding to nuclear factors occurs . directly. The upor downregulation of the effector genes can modulate many different cell pathways.Jolanta Gromadziƒska. such as cytokines. degenerative and neoplastic diseases. and in cellular proliferation or differentiation . such as those of proliferation. 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 . The process the translocation of NF-κB and its binding to DNA is multiphasic and highly complex. the cell cycle. This process is made possible by intracellular reduction in the disulfide bridges (-S-S-). as well as such transcriptional and nuclear factors as NF-κB . NF-κB is a crucial target in the pathogenesis of various chronic inflammatory. This process in turn results in the activation of AP-1 and of such transductional proteins as ras/rac. Wojciech Wàsowicz 96 in the transductional proteins results in their being modified structurally. the immune response. 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 . growth suppression. myc and c-Jun N kinase [35. and repair processes .36]. The reduced thiol group in Cys is essential for maintaining the activity of numerous transcription factors . for example. intensifying the binding of NF-κB and AP-1 to the DNA. The breakdown of hydroperoxides by GSH-Px and the resulting decrease in the cellular hydroperoxide level inhibit the Se-activated kinase p38 . GSH-Px in particular can act as a suppressor of protein kinases. Se inhibits the expression of one of the activating zinc finger proteins that regulates the activation of the c-myc oncogene . Edyta Reszka. I-κB. differentiation and senescence. Thioredoxin (Trx) plays a key role in this process. selenite inactivates the c-myc oncogene and activates the c-fos genes that lead to the growth of normal cells . In human hepatoma cells. GSH-Px and SeP are considered to have a particular role in MAPK inactivation . Reactive oxygen species (ROS) activates the phosphorylation of one of the NF-κB subunits. as well as the genes involved in apoptosis activation. TrxR thus affects the redox regulation of a variety of enzymes and receptors. to the promotor regions. In human colon cancer cells. The phosphorylation of mitogen-activated protein kinases (MAPK) represents one of these phases. The strength of the binding of NF-κB to DNA is influenced by Se. Through acting as a protein disulfide reductase. The redox potential is an important factor in regulating the nuclear factor κB (NF-κB) and the activation of AP-1 . NF-κB is also an important factor in regulating activation of the genes involved in the control of cell necrosis and apoptosis. NF-κB activation involves changes in protein conformation and in the binding of numerous genes. 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. It regulates the effector genes in the promoter regions and can thus activate or repress gene expression. which can lead to their becoming activated. Oxidative stress induced by chemical or physical factors translocates Trx to the nucleus. MAPK inactivation inhibits the cellular proliferation occurring in the course of the carcinogenic process. GSH-Px overexpression inhibits the translocation of another NF-κB subunit and reduces the phosphorylation of I-κB [40–42].
p53.51]. ref-1. a key enzyme in the deoxyribose formation needed for DNA synthesis. affect cellular metabolism. Effect of different selenium compounds in cancer prevention The chemoprevention of cancer by selenium can involve the action of different selenometabolites. Se is also known to arrest several kinase pathways important in signal transduction. etc. It is important to note that different selenocompounds are essential for the growth and differentiation of both normal and neoplastic cells . whereas Trx/TrxR activates ribonucleotide reductase.49]. selenomethionine and Se-methyloselenocysteine can be transformed into selenides. . they react with oxygen to produce superoxide and hydrogen peroxide . 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 . but also its metabolites. reduction in the oxidative DNA damage that occurs. Selenite. [50. It is believed that the role of selenium in the inhibition of carcinogenic processes is associated with oxidative damages caused by the redox cycle of selenides . the concentration of Se in the tissues also being found to be higher after the administration of selenomethionine than after selenite supplementation. 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 . Trx inhibits certain kinases of the MAPK family (e.54. modulation of tumour angiogenesis. Selenite has been shown to be more effective than selenomethionine in the inhibition of cancer cell growth during chemically induced carcinogenesis [31.54]. high concentrations of selenides tend to also be generated. Trx limits DNA synthesis. In cells with a high Se level.g.55]. such as protein kinase A. diacylglycerol kinase and thymidyl kinase . the bioactivation of carcinogens. and the glucocorticoid receptors [34. selenodiglutathione. From this it can be concluded that a high concentration of Se is not crucial for the inhibition of carcinogenesis. but it is likely that one of Se metabolites is essential to this process. It has been found that both sodium selenite and selenomethionine (SeMet) suppress tumour growth in many animal models involving a dose-dependent response [20. The various chemical forms of Se have been shown to differ widely in their anticancer properties.Vitamins and selenium: Anticancerogenic activity of selenium 97 and AP1 . AP-1. Some low molecular weight Se compounds have been shown to influence the regulation of gene expression. Ca+2-dependent and -independent kinase C (PKC). When selenides are generated in sizeable amounts. . ASK1) directly. The anticancerogenic activities of various Se compounds are summarized in Table 2. TrxR also regulates gene expression by affecting the cellular redox status and activating numerous DNA-binding transcriptional factors: NF-κB.. At the same time. not only the selenocysteine incorporated into the protein structure. It was also observed that methylated Se derivatives can induce apoptosis .12. cellular growth.
7. 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. p53 regulates the activity of genes involved in DNA repair processes. In human cancer cells.  showed that the selenium compounds as selenite. an enzyme involved in DNA repair processes. Fiala et al. and of selenomethionine. only about 5% of it being found in other metabolites . it is the actions of sodium selenite (Se IV). which have been shown in in vitro experiments to affect apoptosis or to arrest the cell cycle (see Figure 2. Experimental studies have shown that the organic Se compounds induce apoptosis without DNA damage. Seo et al. SeMet oxidizes the thiols of the p53 molecules at 275 and 277 cysteine residues. Regarding the selenocompounds. which is not a DNA-damaging agent. 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. 1. Wojciech Wàsowicz 98 When Se is supplied in high concentrations.Jolanta Gromadziƒska. Two other Se compounds. sodium selenite and methylseleninic acid. Selenium compounds can also affect activation of the p53 gene since the main dietary selenium compound. modulates p53 activity. Over 90% of the circulating Se is incorporated into the structure of the selenoproteins. selenomethionine. It appears then that the anticarcinogenic action of Se is due to the combined effect of selenium and selenoproteins . suggested that methylated Se derivatives are the most effective selenium compounds for cancer prevention . In normal cells.13. They suggested that this pathway may be the major mechanism involved in the chemoprevention that selenium compounds provide after the initiation of carcinogenesis. Such methylated selenium compounds as methylselenocysteine and selenomethionine show powerful anticarcinogenic activity and also lack some of the toxic effects produced by other Se forms. It is twice as active as selenomethionine in suppressing mammary carcinogenesis in rodents . even at rather high concentrations (10–50 µM) . Ip and Ganther [59. in the previous chapter). Edyta Reszka. that have been investigated most frequently . however. p53 is often mutated and its functional activity suppressed.4-phenylenebis-(methylene)selenocyanate and benzyl selenocyanate inhibit the activity of DNA cytosine methyltransferase.  demonstrated that the Se concentration is a determinant of basal p53 activity. Selenomethionine has been shown to act together with the p53 tumour suppressor protein in affecting the redox status. Methylseleninic acid in a range of concentration of 0. The results of in vitro experiments on cells treated with selenite and methylated selenocompounds are summarized in Table 2.5–1 µM was found to promote DNA repair processes by increasing the expression of two proteins associated with the p53 . The metabolic pathways of Se-methylselenocysteine and selenobetaine involve the release of methylselenol or methylseleninic acid derivatives. were found to cause phosphorylation of the serines and threonines contained in the p53 molecule. any surplus of it is excreted from the body. Methylselenocysteine is one of the most important selenocompounds for chemopreventive activity . in connection with studies of Se metabolism they conducted. . responsible for single and double DNA strand breaks.
but that the induction of apoptosis is not a simple result of the regulation of p53 by selenium . which is a dominant mechanism of the cell death these compounds bring about . Blessing et al. selenomethionine protects the cells from DNA damage through the induction of DNA repair. Seo et al. The postsynthetic methylation of DNA is catalyzed by the family of S-adenosylmethionine-dependent DNA methyltransferases . One possibility is that a small fraction of the SeMet is metabolized to a low-molecular-weight compound . one of the enzymes involved in DNA repair processes.  described the incorporation of Se into the factors involved in DNA repair regulation. and 2-nitrophenylselenocyanate) were found to cause a dose-dependent decrease in the activity of formamidopyrimidine-DNA glycosylase (Fpg). ebselen. 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. They also affect the integrity of the XPA protein (xeroderma pigmentosum group A protein). This decreased GSH concentration in the cells induced apoptosis. 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.68].66].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 . such as benzo(a)pyrene and arsenic . 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. There is a need of explaining how triggering of the p53-dependent DNA repair process by two different Se compounds can be achieved when a 10–20-fold higher Se concentration is employed. The methylation of Se compounds to Se(CH3)2 and Se(CH3) results in a decrease in methyl donation. which has an essential role in recognizing DNA lesions in the nucleotides and in the excision of damaged nucleotides in mammalian cells . an important epigenetic mechanism that exerts control over gene expression . . It has been found that Se derivatives can activate the p53 gene. 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.65]. this preventing DNA methylation from occurring. These selenocompounds affect the DNA repair processes through oxidation of the zinc finger structures and the release of zinc from DNA. In investigating the ability of inorganic Se compounds to induce apoptosis. In vitro studies of a mammary cell line exposed to DMBA showed 1. Selenium can also affect DNA methylation. as well as damaging the integrity of the genes of the DNA repair enzymes [67.  showed that in normal human fibroblasts. and to be involved in the pathway connected with the p53-dependent activation of DNA repair processes . selenocystine. Reducible selenium compounds (phenylseleninic acid. Limitations in the DNA repair capacity appear to be a key determinant of predisposition to cancer [49.Vitamins and selenium: Anticancerogenic activity of selenium 99 gene. DNA methylation is one of the first steps in the carcinogenesis induced by certain chemicals.
Case-control studies tend to reflect the short-term Se concentrations in the organism.74]. reduction in the activity of T lymphocytes. however. due to the large amounts of unsaturated fatty acids present in their structure. such as the Se level associated with the dietary pattern at the time of sampling in cases and controls. Selenium and cancer risk — epidemiological results Several studies have shown the development of cancer in humans to be inversely related to the intake of specific dietary components. The anticarcinogenic effect of selenium is also partly based on its ability to produce antitumour metabolites (e. such as in connection with antibody production and specific cell immunity . Edyta Reszka.Jolanta Gromadziƒska. the results of epidemiologic studies .80]. Research over the last decade points to a significant protective role of Se in preventing the development of malignant neoplasms. 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. Low Se concentrations in the tissues and in human body fluids also appear to be associated with numerous changes in the immune system. but no such relationship was found for lung cancer . methylselenol).g.82]. Low plasma selenium concentrations are thought to be associated with increased morbidity and mortality from cancer. CTL activity has been found to be significantly increased in Se (SeIV) supplemented patients with cancer located in the head and neck region who were given standard anticarcinogenic therapy. To date. and impairment of the body’s ability to reject implants and to destroy neoplastic tumours [75. Cellular membranes of the T lymphocytes are particularly sensitive to Se deficiency. such as suppression of the host immune response to bacterial and viral infections. Insufficient dietary intake of selenium results in many types of defects of the immune processes. 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. In a Chinese study. NK cells and macrophages. Se can stimulate the expression of IL-2 receptors found on activated T lymphocytes and on NK cells . the inhibition of prostaglandin and immunoglobulin synthesis. An inverse association between selenium and risk of cancer has been reported both in case-control studies and in followup cohort studies. a statistically significant increase in morbidity from oesophagus and stomach cancer was noted in a population with low levels of selenium. micronutrients and phytochemicals [81. Selenium compounds can activate cytotoxic cells. including nutrients.76]. stimulate the expression of cytokine receptors and the proliferation of lymphocytes [73. Wojciech Wàsowicz 100 Effects of selenium on immune functions Certain anticancer properties of the selenocompounds may be associated with their effects on cellular immunity. 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. Selenium regulates the immune response by stimulating natural killer cells and activating antigens of various types to destroy the tumour cells .
30) in studies of Se intake based on use of a questionnaire. The protective effect of Se in connection with lung cancer could also be noted in studies examining Se concentration in the toenails.54–34.58–1. These finding confirm the assumption that the Se level in the toenails can be used as a marker of long-term Se concentration . 95% CI: 0. The significant.00 (95% CI: 0.24–0. undertaken by Zhou et al . whereas others have obtained null results [84. indicated selenium to have a certain protective effect.72 (95% CI: 0. whereas the value for groups with a low basic level of Se was RR = 0.94 and RR = 0.57–0. Of the studies considered in the meta-analysis. It is notable that of the women investigated within the Nurses Health Study. those with high toenail Se levels were found to have a particularly high relative risk of lung cancer (RR: 4.Vitamins and selenium: Anticancerogenic activity of selenium 101 have been rather inconsistent. In most of the reports.97) (Table 2. the mean value obtained for groups showing a high basic concentration of Se was RR = 0. but only in populations with a low mean Se level. to 0. representing a daily Se intake of 55 mg.22).50. The total RR values for lung cancer in patients showing high Se levels ranged from 0. dosedependent protective activity of Se was documented in Finnish and Dutch studies [86.17–0.44) and for the Dutch population (RR = 0. In the control group of non-cancer patients the mean serum Se concentration was 100 ng/ml blood serum. 11 of which had a prospective design.20. Interestingly.86 (95% CI: 0.33. the risk of lung cancer at high Se concentrations was found to be RR= 0.87] in which Se in both serum and toenail samples was analyzed. The findings for three major forms of cancer are summarized below. 95% CI: 0.46 (95% CI: 0.77–1. only the findings for the two Finnish populations (RR = 0.30–0. to 1. when the study population was divided up according to Se level. In a metaanalysis of 14 epidemiologic studies. 95% CI: 0. .09–0.41.89]. the protective activity of Se could be clearly discerned when the reference group consisted of subjects showing the lowest level of this trace element.60) after adjustments for smoking status were made .87) when toenail Se was used as a marker of the concentration of Se in the body. 95% CI: 0.14. 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. Some authors have reported there to be an association between cancer risk and Se status.).61–1.10) for the serum Se level.80 (95% CI: 0. whereas no association between selenium level and risk of lung cancer was found in two studies of non-European populations [88.14.74 (95% CI: 0.85]. A review of epidemiologic studies of lung cancer and of the selenium concentration found in biological material.).16).45–1.81) revealed a statistically significant protective effect of high Se concentrations.
76 (0.080) µg/g female)g 0.1 (2.2 (24.537 (0.811 (0.44)e 0. Edyta Reszka.14.33 (0.  NCC Serum Garland et al.109) µg/g female) 227 (33. 2459 (0.49 NA Comstock et al.0) µg/day) 0.2 (2.94) 1.37–1.66 (0.27–1. g data from 370 individuals.48 >0.15) 0.  cohort Toenail All The Netherlands 3.001 0.80 (33.0 (13.0) µg/l USA 5—14 356 (119.3) µg/l)c Finland 8—12 153 (57.60) 0.30–0.3 317 (0.  NCC Serum 0.  NCC Serum Ratnasinghe et al.41 (0.11 Kromhout et al.897 (0.46 0.9) µg/l)d 216 (45 µg/l) 47 (0.7) µg/l)b 153 (61.01 0.6 (15.20 (0. f male subjects randomized in the 5th year.308) µg/g) 250 (0.206) µg/g male.7 (18.102 Table 2.20–1.6) µg/l) 356 (117.41) f 0.56 (0.08 0.  CC Diet Jolanta Gromadziƒska.61 (0.52 0.134) mg/kg) China 4—5 108 (46.40) 4.20 (0.09–0.19) 0.07 µg/day) 0.50 (0.47–1. Wojciech Wàsowicz a Nested case-control.81) 0. 0.550 (0.8 (16.98 (0.537 (0. controls (mean Se level) RR (95% CI) (high vs. cases (mean Se level) No.  Male Female China The Netherlands CC Diet All China — 25 — 1. low Se) 0.65 (0. ) Author All All All M.108 ppm) 515 (0.5) µg/l)b 145 (57.17–0.  cohort Diet Zhou et al.20) 63 (NA) 290 (36.60–2.0) µg/day) 870 (64. c data from 91 individuals.166) µg/g) 250 (0.1 (19.  NCCa Serum Goodman et al.20 (0.70–2.5) µg/l) USA 15—18 258 (0. d data from 177 individuals.54–34.126) µg/g male.41–1.0 µg/l) 120 (119.  NCC Serum Kabuto et al.02) 1.3) µg/l F) Male All Male Female USA Male Finland 5—8 3.88) 0.  NCC Serum Knekt et al.43) P for dose-response trend 0.30 (0.006 Hu et al.41–2.  NCC Toenail Hartman et al.575 (0.129) mg/kg) Japan 11—13 77 (113.  NCC Toenail Van den Brandt et al. . Epidemiological studies of the selenium level in the body and risk of lung cancer (according to Zhuo et al.5 47 (0.17 NA Knekt et al.36) 0. b data from 189 sets.10 (16.12) µg/day 227 (33. 112.110 ppm) Study Se index Gender Population Years of follow up No.77–1.0 (16.5 µg/l) Finland 16—19 77 (53.529 (0.2) µg/day) 290 (39. e male subjects randomized in the earliest in the trial.547 (0.
The findings of this review showed that the pooled RR of prostate cancer for a particular Se intake. the odds ratio (OR) for the development of cancer that was associated with a high level of Se intake was 0. to be associated (at a 13-year follow up) with a significantly reduced risk of prostate cancer. however. colorectal cancer patients with low Se concentrations were found to have a significantly lower survival time . 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. A lower Se concentration in the serum was found to be associated with a stronger tendency to be afflicted with a colorectal tumour . although a protective effect of a high Se level (fifth quintile) was found (OR = 0. In addition.16. 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) .61-0.74 (95% CI: 0.49 (95% CI: 0.39) in case-control studies.85).15.).84) in cohort studies and 0. A systematic review of sixteen studies (11 cohort studies and 5 case-control studies) was conducted recently to investigate the association between selenium level and risk of prostate cancer. In a US case-control study of the relation between the risk of colonic malignant or benign tumour and the serum Se level. 95% CI: 0.72 (95% CI: 0. the protective effect of high Se levels and high α-tocopherol concentrations in the plasma was only particularly strong when the γ-tocopherol level was high .61–1. A nested case-cohort study conducted on Japanese-American men (at a > 20-year follow-up) also confirmed the association between a high Se level in the plasma and a decreased risk of prostate cancer (OR = 0.25–0.9) .103]. 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. in which the Se concentration in the toenails served as a measure of long-term Se intake.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. which involved a 6. 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. A high prediagnostic Se level was found.96) . In the Netherlands Cohort Study. Several studies have shown that selenium can be of specific help in combatting prostate cancer . high Se concentration in the toenails was associated with an appreciably lower risk of prostate cancer .17–0. no protective effect of higher Se levels was found in either of the groups investigated . In two large case-control studies of male populations in Great Britain and Canada. indicating that the intake of selenium was able to reduce the risk of prostate cancer . defined as the average of the 1st and 4th quintiles or the 1st and 3rd quartiles. in the Physicians’ Health Study. Also.3 years’ follow-up period. 95%CI: 0.5.3–0. In the Health Professionals Follow-Up Study. was 0. especially men who had a PSA concentration equal to or higher than 4 ng/ml .
3 (20.10) 0.6 (19.00 (0.69 (0.18—0.104 Table 2.85) P for dose-response trend 0. Wojciech Wàsowicz a Nested case-control.106 (0.108 (0.38 (0.15.17—0.84) 0.  NCC Plasma 577 (0.622 µg/g) 13 586 (0. cases (mean Se level) No. Edyta Reszka.96 µg/g) USA 5—14 235 (114.581 Allen et al.547 (0.611 µg/g) 0.48—0.6 µg/l) 0.  (2000) Toenail USA (JapaneseAmericans) USA UK — 300 (0.82 µg/g) 181 (0.12 Knekt et al.16 0. Epidemiological studies of the selenium level in the body and risk of prostate cancer Author Serum Serum Toenail Toenail USA 1—6 117 (0.0 µg/l) The Netherlands 6.  NCCa Goodman et al.090) µg/g) NCC Van den Brandt et al.4) µg/l)b 46 (58.4) µg/l) Finland 8—12 46 (59. controls (mean Se level) RR (95% CI) (high vs.65—1.39 (0.6) µg/l) 456 (114.8 (19.77 µg/g) 233 (0.126) µg/g) 249 (131.5 (0.  Case-control Nails Jolanta Gromadziƒska.  Cohort 1211 (0.3 (14.3—0.99) 0.78 (0.13) 1.8) µg/l)b Study Se indicator Population Years of follow up No. b data from 51 sets.69 0. .40) 1.79 µg/g) USA 5 181 (0.05 0.3 540 (0.02 Nomura et al.018) µg/g) — 249 (128.9) 0.  NCC Yoshizawa et al.008 0.54—1.  Case-control Serum Li et al.530 (0. low Se) 1.60) 0.42—2.018) µg/g) 300 (0.  NCC Helzlsouer et al.24 (0.707 0.02 (0.73—2.
3 (18.30) 1.5 89 (0.91 (0.0 (18.3 (15.42—2.724 0.326 Garland et al.59—4.41—1.123) µg/g) The Netherlands 3.01 (0.04 (0. low Se) P for dose-response trend Knekt et al.43—1.10 (0.3) µg/l)c Finland 8—12 29 (63.535 (0. 105 .273 0.0 (18.863 (0.547 (0. b data from 32 sets. cases (mean Se level) No.643 0.  NCC Toenail Van den Brandt Cohort Toenail Vitamins and selenium: Anticancerogenic activity of selenium et al.3 Colon 112 1246 (0.  NCC Serum Wallace et al. controls (mean Se level) RR (95% CI) (high vs.8) µg/l) Finland 8—12 48 (64.578 (0.92) 0.  a Nested case-control.44—1.2) µg/l)b 0.82 (0.560 (0.  Van den Brandt Cohort Toenail et al.843 (0.12 0.3 (17.22) 0.16.77 (0.092) µg/g) Rectal 76 (0. Epidemiological studies of the selenium level in the body and risk of and colorectal cancer Author Male Female 1.3 Colon 121 1209 USA 3.106)) Rectal 36 (0.88—4.Table 2.9) µg/l)b 1.00) 0.5—5.575 (0.  NCCa Serum Knekt et al.411) µg/g) Female (0.9) All All USA — 25 (138 µg/l) 138 (134 µg/l) USA 1—4 276 (131.75) 0.50 NA Study Se indicator Gender Population Years of follow up No.58) 0.091)) The Netherlands 3.131 0. c data from 59 sets.58 (0.593 (0.18—5.7 (0.7) µg/l)c 48 (65.108)) 1.733 (0.186) µg/g) 2.76 (0.5 (19.146) µg/g) 47 (0.  Case Serum -control Female Male (0.  NCC Serum Nelson et al.41—2.7) µg/l) 276 (130.65) 29 (64.45) 0.
There is a need of clarifying the role of Se . A low level of selenium concentration in the body was shown to be associated with a higher risk of lung. in a recent study no differences between healthy individuals and individuals with adenomatous colon polyps or colorectal cancer were found in terms of Se level. would need further verification as well. Wojciech Wàsowicz 106 and a lower cumulative cancer-related survival rate than such patients with higher Se concentrations did . To gain further insight into the anti-carcinogenic potential of selenium.93) . the Polyp Prevention Trial.34–0. especially in males. However. A similar result was obtained in a 3. In a Canadian case-control study. and the like need to be taken into account. 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. However. gender.Jolanta Gromadziƒska. a variety of confounding factors such as geographical location.118]. After adjustment for age. environmental exposure. a significant inverse association was obtained between toenail Se level and risk of colon cancer (OR = 0.19–0. however.57. To sum up. although this result was only statistically significant in the case of the Polyp Prevention Study (OR: 0. The majority of studies on relations between selenium level and occurrence of colorectal cancer have yielded null results for both males and females. The selenium level was measured in blood specimens of 1763 trial participants. possibly due in part to the rather low proportion of women in the study population . and the preventive role of Se regarding cancer of this type. However.3-year follow-up study of a Dutch cohort in which toenail Se level was used as an indicator . a pooled analysis of data from three clinical trials of colorectal adenoma was conducted: the Wheat Bran Fiber Trial. selenoprotein P (SeP) concentration or glutathione peroxidase activity in the plasma . smoking status and study site. Colorectal cancer risk was also analyzed in a 41-month follow-up of the Nurses’ Health Study . to be associated with an elevated risk of lung and prostate cancer. as found in the Polyp Prevention Study . there are some reports of a significant inverse relationship between blood Se levels and the prevalence of adenomatous polyps [117. In contrast to prostate cancer.42. breast cancer was not found to be influenced by the selenium level [114–116].95) . a number of epidemiologic studies show a low Se level. and the Polyp Prevention Study. genetic susceptibility. The potential role of dietary Se in the early prevention of colorectal neoplasms would need to be confirmed. age. prostate or colorectal cancer. 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. gender. 95%CI: 0. Edyta Reszka.112]. 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. Summary Results of epidemiologic and laboratory studies have indicated selenium to have a protective role in counteracting or preventing the development of cancer.
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hesperetin). daidzein). Many flavonoids possess antioxidant or free radical scavenging potential. are summarised here. apigenin). epigallocatechin gallate (EGCG). tea. Several recent reviews have summarised the potential chemopreventive mechanisms for a number of flavonoids [1–4]. Such chemopreventive agents can be effective at different stages of the carcinogenic process. volunteers consumed quercetin-rich blueberry/apple juice for 4 weeks. tricin. pelargonidin. according to structure. UK Introduction Many thousands of different flavonoids are found in plant species. and anticancer activities. Anticarcinogenic effects of flavonoids Margaret M. Total daily intake can range from 50–800 mg. petunidin). narginin. Blocking mechanisms Possible ways in which initiation of carcinogenesis can be blocked include prevention of reactive oxygen species attack on DNA. epicatechin-3-gallate) and chalones (xanthohumol). vegetables. These polyphenolic compounds are classified. with major dietary sources including fruit. progression. xanthohumol and the novel flavonol. which led to a significant increase in antioxidant capacity of plasma. flavanones (myricetin. genistein. anthocyanins (cyanidin.1. Bioactive components in foods 3. . antiviral. angiogenesis. isoflavones (genistein. as flavonols (quercetin. inhibition of carcinogen uptake into cells and enhanced DNA repair. flavones (luteolin. Similar findings were reported by Wilms et al . An early study by Duthie et al  reported that quercetin protected human lymphocytes from hydrogen peroxide-induced DNA damage. catechins (epicatechin. ageing and cancer. Some recent data for the well-studied flavonoids apigenin. which varies depending on the hydroxylation status of the benzene rings. Manson Cancer Biomarkers and Prevention Group.3. anti-allergic. who also found that quercetin protected human lymphocyte DNA from bulky adduct formation following treatment with benzo[a]pyrene. University of Leicester. chocolate and soy. Those flavonoids which have been studied in most detail exhibit many properties which could be protective against heart disease. anti-inflammatory. kaempferol). Amongst their health-promoting properties are antioxidant. resveratrol. Examples include quercetin (a flavonol in vegetables. xanthohumol (a chalone in hops and beer) and genistein (an isoflavone in soy). apples and onions). Leicester. Also in this study. the chalone. both by blocking initiation and by suppressing the later stages involving promotion. invasion and metastasis. quercetin. altered metabolism of procarcinogens in favour of conjugation and excretion of reactive metabolites.
without affecting expression. have been shown to induce G2/M arrest in SW480 and CaCo2 human colon carcinoma cells . flavonoids can both up. In a subsequent study by the same group . was shown to inhibit the growth of breast tumour cells. the flavonols. and induction of apoptosis involving release of cytochrome c from mitochondria. Xanthohumol was also found to inhibit COX-1 and COX-2 activities. along with inhibition of superoxide anion radical formation and nitric oxide production . a novel flavonol in rice bran. p53. accompanied by upregulation of p21 and p27. They have also been shown to influence the multi-drug resistance phenotype acquired by many tumour cells. tricin decreased the number of intestinal adenomas in Apcmin mice by 33%.). namely inhibition of signalling through the EGFR family. Resveratrol.and down-regulate key molecules. quercetin and kaempferol. while the isoflavones. Manson 116 Flavonoids can interact with the aryl hydrocarbon receptor (AhR) as agonists or antagonists.2. in a manner which suggested that they interact at the substrate-binding sites. The latter led to a 34% reduction of PGE2 levels in small intestinal mucosa and blood. A range of tumour suppressing activities is shown for apigenin (Table 3. . depending on cell type and experimental conditions. Such interactions might influence bioavailability of anti-cancer drugs in vivo and could be considered for combination therapies . p21. During later stages of carcinogenesis additional useful mechanisms include inhibition of angiogenesis. NF-κB. Quercetin and silymarin were found to inhibit MRP1/4/5-mediated drug transport from intact erythrocytes with high affinity. The inhibitory effect of other flavonoids on COX-2 expression and activity has been reviewed by O’Leary et al. Such interactions influence the expression of drug metabolising enzymes such as cytochromes P450 . genistein and EGCG (reviewed in ) have a number of effects in common with those detailed here for apigenin and quercetin. p27. . AP-1. In another recent study . depending on structure and cell context. Xanthohumol possesses several useful properties to block carcinogenesis including modulation of enzymes involved in carcinogen metabolism and detoxification (inhibition of Cyp1A. include growth inhibition by induction of cell cycle arrest or apoptosis. However. including JNK.1. but not apoptosis . genistein and daidzein. alone and in combination. elimination of tumour cells. A significant number of flavonoids. and to be antiestrogenic . or even better. causing G2/M arrest. induction of cell cycle arrest involving a decrease in cyclin D1 and phosphorylation of Rb. and PI3K. cyclin D1. activation of caspases 3 and 9 and downregulation of Bcl family members. Suppressing mechanisms Mechanisms which result in suppression. invasion and metastasis. modulated intracellular drug levels by inhibiting function.) and quercetin (Table 3. cdc2. and pAkt. scavenging of ROS. reduced P-glycoprotein expression and function in multi-drug resistant human cervical carcinoma KB-IV cells.Margaret M. with inhibition of COX-1 and COX-2 activity. including hydroxyl and peroxyl radicals. induction of quinone reductase activity). Tricin.
PC-3.8. MMP9. ↑IκBα Apoptosis Effect Reference  PWR-1E. ↓VEGF. ↓NF-κB p50 & p65  Growth inhibition. ↓VEGF and in nude mice OVCAR-3 and A2780/CP70 ovarian cancer cells ↓Akt. LAN-5. altered Bax:Bcl2 ratio. p16. ↓HDM2 Growth and angiogenesis inhibition Angiogenesis inhibition   HCT 116 colon carcinoma cells ↑ERK & p38 phosphorylation and activity ↑phosphorylation of Elk & ATF2  HCT 116. ↓CDK2/4/6. HT-29 colon cancer cells ↓CK2. VEGF Apoptosis  DU145 prostate cancer cells ↓cyclin D1/2 & E. ↑cyt c release. ↑p21. cyclin D1. ↓ colony formation  PC-3 prostate cancer cells ↓p50. ↓p70S6K1. ↑Bax. ↓TNFα activation of NF-κB ↓Bcl2. ↓cyclin D1/D3 & cdk4. COX-2. ↑p53.↓HIF-1α. ↓Bcl2.9 and cIAP-2 ↓fatty acid synthase activity Apoptosis   Growth inhibition and apoptosis ↑p53.↓HIF-1α. SK-N-BE neuroblastoma cells Breast cancer cells ↑ROS. ↓HER2/neu phosphorylation. ↓TNF a-induced NF-κB activation Apoptosis. ↑cleavage of caspases 3. ↓Akt. ↓NF-κB-DNA-binding. G1 arrest. NOS-2. ↑cleavage of caspase 3  Apoptosis (p53-dependent) ↓Her2. ↑APAF-1. ↓p65. ↓IKKα actvity. p18.  . DU145 prostate tumour cells Breast and prostate cancer cells NUB-7. p27. ↑p27 Apoptosis. ↑p21. ↓p70S6K1. ↓IκBα degradation and phosphorylation. ↓PI3K-HER2 docking.Bioactive components in foods: Anticarcinogenic effects of flavonoids 117 Table 3. ↑HER2 degradation Apoptosis  A549 lung cancer cells in vitro ↓Akt.1. LNCaP.7. Chemopreventive suppressing effects of apigenin Tissue/cell type Jurkat T cells Mechanism ↓chymotrypsin-like activity of 20S and 26S proteosomes. ↑Bax. apoptosis HER2-overexpressing breast tumor cells ↓PI3K & Akt activity. ↑DFF-45 cleavage. ↑cleavage of caspase 3. ↑IκBα .
G2/M arrest Apoptosis    One recent report by Fenton and Hord  has suggested a novel chemopreventive mechanism for flavonoids. which is often mutated in colon cancer.or over-expression. Manson 118 Table 3. During the carcinogenic process. ↓Pgp Apoptosis Effect Reference  Inhibiting chymotrypsin-like activity of 20S and 26S proteosomes. In normal colon. ↑cleavage of caspase 9 Suppression by 4-fold. PC3 prostate tumour cells HT29. epithelial cells migrate to the apex of the crypt. naringin and hesperidin induced a greater migratory response in APCMin/+ cells compared to those expressing wild type APC. Such flavonoid-induced migration was dependent on matrix metalloproteinase activity. ↓phosphoAkt Growth inhibition and apoptosis  ↓β-catenin/Tcf transcriptional activity ↑cyclin B1. ↑Bax. H1299 human lung carcinoma cells PC3 prostate cancer cells ↓Sp1 interaction with AR. apoptosis ↑3-fold  MiaPaCa pancreatic tumour cells MCF7 breast tumour cells ↓phosphoFAK Decreased invasion  ↑PTEN. ↓Bcl2. both hypermethylation of the promoter regions of tumour suppressor genes and hypomethylation of oncogenes can occur. Chemopreventive suppressing effects of quercetin Tissue/cell type HL60 human myeloid leukeamia cells Jurkat T cells Mechanism ↑Bax. ↑p53. ↑IκBα Apoptosis  Breast and prostate cancer cells Colonic aberrant crypt foci ↓fatty acid synthase activity Growth inhibition and apoptosis  ↑Bax.Margaret M. ↑phosphoBcl2. ↑p21 ↓HSP70 ↓c-myc Growth inhibition. ↑c-jun Inhibition of androgen receptor activity  ↓ErbB2/3. a process involving the APC gene. SW480 colon cancer cells SW480 colon cancer cells A549. Both EGCG  and genistein  have been shown to reactivate a number of key genes. ↓Akt Growth inhibition and apoptosis  LNCaP. ↑phospho cdc2. ↑p27. ↓Bcl2.2. These authors reported that apigenin. ↑survivin. epicatechin. such as the cell cycle inhibitor p16 and the retinoic . resulting in under.
Bioactive components in foods: Anticarcinogenic effects of flavonoids 119 acid receptor (RARβ).3:768–80. and induction of apoptosis. nor quercetin stability were affected by ellagic acid. involved direct interaction with the enzyme. Duthie GG. Collins AR. including cyclins. while quercetin alone only induced p38 phosphorylation. Ellagic acid potentiated the inducing effect of quercetin on levels of p21 and phosphorylation of p53 at serine 15. Li YW. Nature Rev Cancer 2003. For example Mertens-Talcott et al. 2. References 1. synergistic or antagonistic effects of combinations of more than one chemopreventive agent. Trends Mol Med 2003. Combinations of flavonoids were found to have an inhibitory effect on the breast cancer resistance protein (ABCG2). Suppressing activities include inhibition of signalling pathways responsible for cell proliferation and survival. Cancer chemoprevention with dietary phytochemicals. Mut Res Gen Toxicol Environ Mutagenesis 1997.393:223–31. cyclin dependent kinases and inhibitors. Cell signaling pathways altered by natural chemopreventive agents. Manson MM. using MOLT-4 human leukaemia cells. mainly through intrinsic pathways involving Bcl family members. Eur J Cancer 2005. modulation of drug metabolising enzymes and multidrug resistant genes. Conclusions Flavonoids. Sarkar FH. Combined effects There is now accumulating evidence for the additive. They can also induce cell cycle arrest by modulating key components of cell cycle regulation. which. Manson MM. Their blocking activities include antioxidant effects. 3. suggesting the potential use of ‘flavonoid cocktails’ to reverse multi-drug resistance in treatment of this cancer . Dodson VL Quercetin and myricetin protect against hydrogen peroxide-induced DNA damage (strand breaks and oxidised pyrimidines) in human lymphocytes. exhibit a wide range of potentially useful activities for cancer prevention.9:11–8. . Cancer Prevention — the potential for diet to modulate molecular signalling. Neither the generation of ROS. in several different cancer cell types. like other types of dietary chemopreventive agents. . in the case of EGCG. 4. showed that quercetin and ellagic acid acted synergistically in inducing apoptosis. mitochondrial membrane depolarisation. Mut Res 2004. Inhibition of survival signalling by dietary polyphenols and indole-3-carbinol.555:53–64. cytochrome c release and activation of caspases.41:1842–53. 5. Surh Y-J. The mechanism proposed was through inhibition of DNA methyltransferase. Duthie SJ. Phosphorylation of JNK1/2 and p38 was increased by the combination.
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Pelling JC. Zhou Q. FASEB J 2005. Sedlak J. Bodo J. 22. Calderwood SK.25:2017–25. Neoplasma 2005. Nutr Cancer 2004. Davenport DM. Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells. Kim ES. Sulikova M.279:4479–89. J Nutr 2003. Knudson A. Jung KC. Jiang BH. Jones EL. Eng C. Gupta S. 34.285:G919–28.20:835–49.48:182–8. Lee LT. Gong AY. Human Mol Genetics 2005. Moussavi M. Biochem Biophys Res Comm 2005. but not apigenin or luteolin. 35. Zheng JZ. Kim HK. Sinden MR. Zhao MJ. Flavonoids promote cell migration in nontumorigenic colon epithelial cells differing in APC genotype: implications of matrix metalloproteinase activity. Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells. Am J Physiol 2003. Flavonoid quercetin. Park CH. Targeting of focal adhesion kinase by flavonoids and small interfering RNAs reduces tumor cell migration ability. Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells. thelial growth factor and angiogenesis in human lung cancer cells. Apigenin induces apoptosis through proteosomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway. ginseng and rutin). 25. Eivemark S. Chang JY. Parhar K. Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K and HDM2/p53. 5. Int J Hyperthermia 2004.26:793–801. Farah M. Yang CH. Stevenson MA. Yuan HQ. Kang NE. 31. Salh B. Fang J.68:635–43. Anticancer Res 2005.133:3800S–4S. 32. 33. Quercetin decreases the expression of ErbB2 and ERbB3 proteins in HT-29 human colon cancer cells. The 70 kilodalton heat shock protein is an inhibitor of apoptosis in prostate cancer. Way TD. Hahm ER.Bioactive components in foods: Anticarcinogenic effects of flavonoids 121 21. Shukla S. Bang MH.52:273–9. 24. 39:114–24. Mol Pharmacol 2005. 28.14:1457–63. Lee MT. Hord NG. Wargovich MJ. Xue Y. 26. Fang J. 27.19:342–53. a potent inhibitor against beta-catenin/Tcf signaling in SW480 colon cancer cells. Reed E. Mol Carcinogenesis 2004. induced apoptosis in human myeloid leukemia cells and their resistant variants.328:227–34. Lin JK. Phytoestrogen exposure elevates PTEN levels.279:55875–85. Duraj J. Kuo PC. Volate SR. Lee PPH. Carcinogenesis 2005. Liu HF. Apigenin inhibits expression of vascular endo. Park S. Cao ZX. 30. Kim WK. Carcinogenesis 2005. Muga SJ. Lin YS. Waite KA. . silymarin. 36. Young CYF. 29. Chao JI. Lui LZ. J Biol Chem 2004. Fenton JI.26:1450–6.6-dichloro-ribifuranosylbenzimidazoleand apigenin-induced sensitization of colon cancer cells to TNF-alpha-mediated apoptosis. Hu XW Shi XL. Zazrivcova K. Cho HJ et al. Kao MC. curcumin. J Biol Chem 2004. The chemopreventive bioflavonoid apigenin modulates signal transduction pathways in keratinocyte and colon carcinoma cell lines. Quercetin. Van Dross R. Modulation of aberrant crypt foci and apoptosis by dietary herbal supplements (quercetin. 23. Jiang BH.16:155–62. J Nutr Biochem 2005. Huang YT.
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however. followed . and components for which bioavailability is low. 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. Isoflavones and gallic acid are polyphenols that are absorbed to the highest extent. flavanones. these including the flavonols. Biomarkers of dietary polyphenol intake for studying diet-cancer relationships Jakob Linseisen and Sabine Rohrmann Division of Clinical Epidemiology. the hydroxybenzoic and the hydroxycinnamic acids. measurement error being independent of that contained in the corresponding questionnaire data.1. Even more problematic. Polyphenols Polyphenols are provided by plant-derived foods. The basic flavonoid structure and various examples of them are given in Figure 3. Heidelberg. the sugar moiety of the compound in question and its further metabolism by the gut microflora. Dietary measurements tend to suffer. Current evidence from bioavailability studies suggests that the bioavailability varies from about 0. flavones. isoflavones. whereas others are limited to only a few kinds of food (such as apigenin found in parsley and celery).2. however. lignans. is the fact that the polyphenols markedly differ from one another in their bioavailability and intestinal metabolism. The validity and reproducibility of nutritional biomarker measurements. Germany Introduction The primary aim of analytical cancer epidemiology is to detect associations between exposure variables and disease endpoints. for example. Nutritional epidemiology deals with dietary factors that are difficult to assess. The use of biomarkers for such compounds should overcome some of the methodological problems in nutritional epidemiology just referred to. flavanols. Flavonoids are classified in several different sub-groups. for example). phenolic acids consist of two main subgroups.3 to 43% (based on urinary excretion) of the dose administered. from imprecision. such as various secondary plant products. The analytical data obtained are objective and more precise. German Cancer Research Centre. needs to be demonstrated in advance of their use in epidemiological and etiological studies . Estimates of dietary intake estimations are hampered by incomplete or missing data in food composition tables. reaching plasma concentrations of 0–4 µmol/l at an intake of 50 mg aglycone equivalents .Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 123 3. Some of the polyphenols are found in a rather wide variety of foods (kaempferol contained in many vegetables. particularly concerning dietary components provided by only certain kinds of foods. Bioavailability is determined by different factors. including beverages. anthocyanins and proanthocyanins. Long-term dietary habits are usually explored in epidemiological studies by use of food frequency questionnaires (FFQ).
Flavonoids undergo extensive first-pass phase II metabolism in the intestinal epithelial cells and the liver. data on the phenolic acids are very limited.1. The plasma concentrations present in free-living (fasting-status) subjects are even lower than the abovementioned range obtained after intervention. . The polyphenol concentrations contained in biological specimens are estimated in most studies after hydrolysis of the glycosides involved. Sabine Rohrmann 124 by the catechins. Fig. The steady-state levels of the plasma polyphenols should only be achievable through regular intake of the foods in question. although the kinetics involved differ considerable. The basic structure of flavonoids and the chemical structure of selected polyphenols. 3. the aglycones being quantified. Measurement of the compounds found in urine samples. For many of the polyphenols. and glucuronidation. flavanones and quercetin glucosides. due mainly due to the higher concentrations of polyphenols — at least after extraction and enrichment — as compared with plasma samples. is a promising approach. Thus far. their half-life time in the plasma is short. ideally 24-hour samples.Jakob Linseisen. concentrations of them reaching baseline levels within 24 hours . the galloylated tea catechins and the anthocyanins are much less available. their being substrates for methylation. The proanthocyanidins. sulfation.
Clean-up procedures For human specimens.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. . a single mass-selective detector often fails to fulfil the requirements for sensitivity. Identification of compounds by means of mass fragmentation is used as a gold standard. the flavonoids are usually glycosylated and the phenolic acids are ester-bound. Accordingly. HPLC has become the method of choice. with the option of time-resolved measurement.3.5]. frequent use is made of enzymic hydrolysis by means of sulfatase and glucuronidase unless a study aims at determining the exact metabolites. Some techniques such as liquid chromatography-mass spectrometry (LC-MS). although the use of LC-MS is becoming increasingly common. The availability of antibodies for isoflavones and lignans has enabled the antibodybased assays with a high degree of sensitivity to be developed . Thus. such as plasma. do require only minor sample-preparation steps. LC-MS) to characterise clusters of polyphenolic compounds that can potentially be used as biomarkers. the respective aglycones and acids being subsequently detected and quantified. or urine samples.). sulfated and glucuronidated compounds. Separation and detection systems The two major separation techniques for the quantification of polyphenolics are HPLC and GC. Hydrolysis (acidic or enzymatic) is frequently used to simplify the analytical procedure. Hydrolysis In foods. flavonoids mainly undergo modification by means of phase II enzymes leading to methylated. For the structural characterization of compounds. the fluorescence emitted is recorded by means of a plate reader. In biological specimens. For the purpose of quantification. and the phenolic acids are genarally quantified by means of GC after derivatisation. serum. new developments include the creation of HPLC-ESI-MS-MS systems and similarly coupled devices. the clean-up procedures for this type of samples may be more complicated than required for many food systems. the scientific literature contains no report on use of the metabonomics techniques (NMR. although a part of them is excreted unmodified. However. Phenolic acids undergo further metabolism and degradation. Although in principle these methods can be applied to human specimens. 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. mass spectrometric techniques need to be used. For the flavonoids. Thus far.
) Tmax (h) Mean (SEM) Range Daidzin Daidzein Genistin Genistein Glycitin Hesperidin 6.26 42.14 0.0–5.21) Range 0.52) 1.25) 1.Jakob Linseisen. Phenolic acids Phenolic acids Flavonoids.7 7.8) 8.3 0.84 (0.8–29.57 (0.7) 7.6) 4.5 (0.46–4.88 (0. Phenolic acids Flavonoids. 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 . GC — gas chromatography.0 5.5 15.0–9.6 (1.8) Range 3.3.9 2.0 27. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al. even after the administration of polyphenol preparations or polyphenol-rich food corresponding to 50 mg aglycone equivalents.00) 1.0) 6.8 4. Differences between the various compounds and of classes of polyphenol are striking nevertheless.4 . Phenolic acids Isoflavones.36–3.50 Urinary excretion2 (% of intake) Mean(SEM) Range Elimination half-life (h) Mean (SEM) 5. (according to ).46 (0. Most of the studies concerned only one or some few compounds within a given subclass of polyphenols.21–0.3 (3.2 1.4.7–10.1) 5.1 6.0) 21.6 6.8 (0.4.92 (0.9 (1.3 5. Principal techniques and detection systems used for the identification and quantification of flavonoids and phenolic acids in human specimens (metabonomics/NMR excluded) Separation HPLC Detection system UV/VIS spectroscopy (Diode array detection) Mass spectrometry Electrochemical Fluorometric Substances class technique Flavonoids.4–8.8) 8.6) 4.04 1.1 (0.1 (0.26–4.0) 19.5 (0.0 7. Phenolic acids Flavonoids. Relatively low plasma concentrations were obtained in each case.3) 8.6) 5. Sabine Rohrmann 126 Table 3.87 8.5 (0.9 (12.5 42.0–55.76–3.7–9.56 (1.38) 0.3 (0. lignans GC LC Immunoassays Mass spectrometry Mass spectrometry Time-resolved fluorescence HPLC — high-performance liquid chromatography.0 3.27) 2. Phenolic acids Flavonoids.0) 3–24.4–9. LC — liquid chromatography.2 Cmax (µmol/l) Mean(SEM) 1.3 3. Kinetic data from the experiments in question are summarized in Table 3.00 0.6 (4. Table 3.0–6.8–7.4–62.50–2.6 4.4–5.3 (0.
2) 1.008–0.06 (0.0 1.70 Urinary excretion2 (% of intake) Mean(SEM) 8.20 (0.8 2.40 (0.5–2.03 All the data were converted so as to correspond to a supply of 50 mg aglycone equivalent.0 4. Rutin (Epi)catechin EGC EGCG Gallic acid Chlorogenic acid Caffeic acid Ferulic acid Anthocyanins Proanthocyanidins 1 2 Cmax (µmol/l) Mean(SEM) 0.31–6.1–61.5 (0.001–0.7–2.1 (3.6 (17.45) 0.7 27.1 (0. ) — cont.3–2.6–3.07–1.4–39.7 0.38 0.12 (0. EGC — epigallocatechin. Tmax — time to reach Cmax.2 0.09–1. Tmax (h) Mean (SEM) Range Naringin Quercetingluc.3 (0. flavanones or isoflavones) as biomarkers of polyphenol intake [9–16].0 1.0 19.1–55.1 1.5–5.02 (0.1) 1.7 5.1 Elimination half-life (h) Mean (SEM) 2.3 (0.03) 4.5 (0. A review of short-term intervention studies (n = 93) in which polyphenols were administered (either as isolated compounds or in the form of foods or food extracts) to human subjects was published recently .4 (0.5–2.0 1.50 (0.3) 6.1) 11.9–28.4) Range 1.03–0.6 (0.5 (0. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1 (according to Manach et al.70 37.5 0.6 0.03) 2.1–4.7) 11.5) Range 1.6) 3.6 0.6) 2.3 0.0 (0.4 (0.5 17.01) 0.80 0.30–2.13–1.45–1.3 (0.2 1.09) 1.02) 0.4) 2.9 (8.4 0.1–1.3) 1.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 127 Table 3.004–5.7–4.1–30.6–5.00 (0.2) 0.1 1.0–0.10 (0.09–0.5–2.7 (0.1 (0. The magnitude of the variation in the plasma polyphenol concentrations found between free-living subjects following their usual diet habits was .8–28.9 (2.0 4. 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.9 4.1) 2.1 2. AUC — area under the plasma concentration-time curve.46 (0. Usually represent 24-h urine samples.2–15.3 0.3–1.17) 2.2) 1.1) 1.8 (3.0 0.5 0.5 0.03 0.0 0.51–3.26 Range 0. although one study in particular failed to support this conclusion .52 0. The authors examined evidence relating to the effects of these polyphenols had on biomarkers used to test various pathophysiologically relevant hypotheses in vivo.7) 1.3) 18.57–4.50 0.40) 0.20 0.5 (5.4) 2.06) 0.3) 0.0 2.10 0.5 (1.7 (1.7–2.2) 3.3–9.1 0.57) 0.8 (0.96 (0.03 (0. 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.4.2) 10.35 10.0) 36.33) 1.4 (0. EGCG — epigallocatechin gallate.1) 1.26) 0.5 (0.
5) 1304.6 339.7 (15.6 (275.5) 908.8 82.0a 0. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet.1 6849. a precondition most likely to be fulfilled by compounds such as kaempferol that are widely distributed in plant foods. 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 0.0a 0.5 114.9 563.9) 140.9) 0. Sabine Rohrmann 128 found to be rather high (Table 3.2) 260.2 (17.0a 0.3 (67. close to of steady-state plasma concentrations of them can only be achieved if the substance in question is consumed regularly.5.0a 0.9) 267.6 460.4 104468.3 388. and that the correlations are much lower when intake calculations are based on data obtained either three or seven days prior to blood sampling.7 2290.2) 1031. 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 68.6 2542. 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.9 2641.6) 6990.3 1253.0 478.4) 107. 91.8 (77.8) 108.5 Mean (SD) Min.7 1348.5.8 0.4 2904.1 53.0 6103.6 413.9 7103. the intra-individual variation also being high .3 644.0a 0.1 (94. Table 3.8 .3 (42.0a 0.0 (3077.4) 10.8 26.6) 477.7) 277.0a 0.2 0.8 1121.9 4528. and if the bioavailability of the compound is not particularly low. (nmol/l) Max.6 (254.75.0a 78.1 (41.).3) 652.3 (153.0a 48.0a 0.1 1357.1) 520.Jakob Linseisen.6) 2160. Due to the short half-life time of most polyphenols.7 (78.7 561.1 (203. participating in a cross-sectional study (according to Bolarinwa and Linseisen .0a 58.8 158.1 (2.9 372.0 742.0a 19.4 59.7 (48.4 159.8 (15.0a 0.0 0.
3) 0. To give an example of the polyphenol concentrations found in 24-h urine samples of subjects on a habitual diet. respectively.0a 0. Compound Naringenin Luteolin Genistein Hesperetin Kaempferol Apigenin Isorhamnetin a Median 79.2 (4. Nielsen and coworkers  reported average (SD) concentrations of quercetin.8 5. Plasma and urinary polyphenol concentrations are not expected to reflect long-term or habitual dietary intake. 76(110).7 1356. In large-scale epidemiologic (etiological) studies of disease-related effects of dietary polyphenol levels.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 129 Table 3. are taken up.6 36.5 204.7) 545. several studies on associations with the risk of cardiovascular diseases and with cancer at different sites.9) 31. just as was indicated already for the plasma concentrations. Hertog and colleagues were the first to analyze commonly consumed foods in terms of their flavonol and flavone content by means of HPLC.1) 37.6).1 (24.0a 0.8) 157. Only few studies concerning the use of biomarkers of polyphenol intake were found to be available (Table 3. 122.1 27. Their work provided the basis for the estimation of dietary flavonol and flavone intake. the dietary intake estimates being based on FFQ data .0a 0.2 639. the correlation coefficients for selected flavonols and flavanones being between 0. naringenin. (nmol/l) Max.3 (1.28 and 0. .2 Mean (SD) Min.0 (7. except as regards the intake of phytoestrogens (isoflavones and lignans).0a 0.38 . In the following.1 2555. 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. some of which appear very promising. little use has been made of biomarker measurements.0a 0. 701(659). 1638(1316) µg/24 h.7 151.8 Below the detection limit.0a 533.9) 126.3 14.7 388. participating in a cross-sectional study (according to Bolarinwa and Linseisen  — cont.0a 0. and total flavonoids as being 25(23).24 and 0.8 (80. The urinary polyphenol excretion rates show a high degree of variability.1 (40. phloretin. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men on their usual diet. although this has not been investigated tested extensively.4) 9. 50(32). One study reported correlation coefficients of between 0.5 (21.0 108.8 52.74 for plasma isoflavone concentrations. kaempferol.5.
 Dai et al. Epidemiological studies of the risk of breast cancer risk in which biomarkers of dietary isoflavone and mammalian lignans intake were employed.7. NS — not significant. 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. isoflavones Urine. Urine. equol. isoflavones. 2002  Cohort 232/772 Urine.  Zheng et al.  Piller et al. lignans Plasma.6.  Murkies et al.7.  Cohort: nested CCS 97/187 114/219 Serum. genistein. Sabine Rohrmann 130 Table 3. Phytoestrogen Urine. enterolactone Urine. 2002  Type CCS N. enterolactone Serum. enabling the attainment of the requirement of sufficient statistical power. NS — not significant. One of the major reasons for the frequent use of biomarkers of phytoestrogen intake in epidemiological studies (see Table 3.  den Tonkelaar et al. 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. Author Ingram et al. cases/ controls 250/250 Specimen.  Pietinen et al.Jakob Linseisen. Table 3. citrus flavonoids Cancer site Breast Result NS Zheng et al. isoflavones. total phenols Breast NS Sun et al. Flavonoid Urine. enterolactone Urine. isoflavones Urine.) may be the ready availability of appropriate immunoassays suitable for measurements on large numbers of samples. enterolactone Result Significant inverse association NS Significant inverse association Significant inverse association Significant inverse association Significant inverse association NS 248/492 Plasma.  Type CCS CCS CCS CCS CCS CCS Cohort: nested CCS Cohort: nested CCS N. 1999  CCS 60/60 Urine. tea polyphenols Gastric and esophageal Significant inverse association CCS — case-control study. enterolactone for highest and lowest categories Significant positive association Grace et al. .  Hulten et al.
The higher values here are those for the MS-based techniques (coefficient of variation < 5–7%. the immuno-assays being located at the other end of the scale. However. such as HPLC-UV/VIS. Detection limits very close to 1 nmol/l of polyphenols have been found for techniques involving HPLC-ECD. . Reproducibility.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. GC-MS and HPLC with use of fluorometric detection is slightly lower. For all these methods. Accounts of analytical procedures that permit a wide variety of polyphenols to be determined in a single run were published recently [16. such as analytical time and costs). satisfactory figures concerning the analytical precision and accuracy are available. This differs from other methods. MS-based systems also have their problems in connection with concentrations close to the detection limit. In epidemiological studies in particular. HPLC-MS-MS. ECD (electron capture detection). Specificity and sensitivity In applying mass-selective detection methods at least to standard mixtures or biological specimens spiked with the compound in question. in which the sample volumes available are usually very small. and reports on the quality of the method in question are usually obtained clearly above the detection limit. working at the detection limit of a method is always a challenge. no systematic findings on repeated within-subject samples or on losses during sample storage are available. and TR-FIA (immunoassay). validity and reliability For each well-developed method. The sensitivity achievable with HPLC-MS. the degree of sensitivity a method provides can be decisive for whether it can be used or not (along with other factors. except for some few small studies . where the characteristic fragments may be absent. confirmation of their results by use of MS-based techniques is necessary. recovery 90–105%). MS-based systems also have their problems when the concentrations involved are very low. in which peak identity cannot be confirmed with sufficient certainty. however. having coefficients of variation close to or above 10% and recovery rates frequently at < 90%. To the best of our knowledge. the concentrations were found to be high enough to enable the characteristic mass fragments to be identified. or use of fluorescence detectors.20]. Antibody-based assays are also subject to failures in specificity due to cross-reactions with other matrix compounds. LC-MS. the analysis of polyphenol content is restricted in most cases to only a few compounds or classes of compounds. Sensitivity is a major issue with regard to analytical methods for determining polyphenols in biological specimens. As already mentioned.
Manach C. Measurement of food flavonoids by high-performance liquid chromatography: A review. Hollman PC. Kleemola P. Polyphenols: food sources and bioavailability. . 2. 16. Bioavailability and bioefficacy of polyphenols in humans.79:727–47. Alfthan G. I. Eur J Nutr 2002. Phenolic acids in foods: an overview of analytical methodology. Linseisen J.56:891–8. Key TJ. et al. 3. Manach C. 11. Morand C.41:203–9. Biochemical markers of dietary intake. Stumpf K. Appleby PN. J Agric Food Chem 2000. J Agric Food Chem 2003. Kelly IE. Free Radic Res 2004. Deighton N. II. J Chromatogr B Analyt Technol Biomed Life Sci 2005. Plasma concentrations of the flavonoids hesperetin. Manach C. 10. Zock PL. 1997. Burns J.38:771–85. Application of Biomarkers in Cancer Epidemiology. Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake. Morand C. Cancer Epidemiol Biomarkers Prev 2002. Am J Clin Nutr 1998. Lapcik O. Eur J Clin Nutr 2000. 142.823:143–51. naringenin and quercetin in human subjects following their habitual diets. 13. Kesaniemi YA. Crozier A. Am J Clin Nutr 2005. Linseisen J. Erlund I. Noroozi M. Morton MS. Merken HM. Remesy C. Review of 97 bioavailability studies. Br J Nutr 2004. Radtke J.91:447–57. 5. Flavonoids in human urine as biomarkers for intake of fruits and vegetables. Lyon: IARC. Bioavailability and bioefficacy of polyphenols in humans. 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. 15. Steroids 2000.Jakob Linseisen.51:2866–87. Scalbert A. Williamson G. Allen NE.54:143–9.68:60–5. 12. Riboli E. Bolarinwa A. 4. Hampl R. Am J Clin Nutr 2004. Kaaks R. In: Toniolo P et al. Prediction of dietary flavonol consumption from fasting plasma concentration or urinary excretion. van Staveren WA. Verkasalo PK. Scalbert A. Rantala M. 8. Cummings JH. Adlercreutz H. Remesy C. Pharmacokinetics and metabolism of dietary flavonoids in humans. Buysman MN. 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).81 Suppl 1:230S–42S. Lean ME. Uehara M. Williamson G. Al-Maharik N.86:415–21. Mutanen M. and diets high or low in fruit and vegetables. Review of 93 intervention studies. Jimenez L. Silaste ML. editors. et al. Fasting plasma concentrations of selected flavonoids as markers of their ordinary dietary intake. IARC Scientific Publications No. Davey G. Donovan JL. Am J Clin Nutr 2005. Manach C. Ritchie MR.. Beecher GR. Eur J Clin Nutr 2002. Wolfram G. Aro A. Meyboom S.81 Suppl 1:243S–55S.48:577–99. Br J Nutr 2001. 6.11:459–66. Plasma and urinary phyto-oestrogens as biomarkers of intake: validation by duplicate diet analysis. 14. Sinha R. de Vries JH. 7. Robbins RJ. 9. Nielsen SE.65:339–48. Freese R. Time-resolved fluoroimmunoassay of plasma daidzein and genistein. Sabine Rohrmann 132 References 1. Wang GJ. Blake A.
24.23:1497–503. Mennen L. Piller R. 27. Franke AA.Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships 133 17. Lee MJ. et al. Johansson R. Custer LJ. 23. High-throughput profiling of dietary polyphenols and their metabolites by HPLC-ESI-MS-MS in human urine. Den Tonkelaar I. An incident casereferent study on plasma enterolactone and breast cancer risk. Hebert JR.7:289–96. et al. Custer LJ. Taylor JI. Kolybaba M. Dalais FS. Cancer Epidemiol Biomarkers Prev 2001. Thijssen JH. Dai Q. Zheng W.11:815–21. Remesy C. Lopez D. Adlercreutz H. 22. Mannisto S. Adlercreutz H. Serum enterolactone and risk of breast cancer: a case-control study in eastern Finland. Menopause 2000. Cancer Epidemiol Biomarkers Prev 2002. Cancer Epidemiol Biomarkers Prev 1999. et al.22:241–3. et al. Healy DL. Hallmans G.350:990–4. Shu XO. Yang CS. Dai Q. Sun CL. Luben RN. 26. Jin F. Hulten K. Cancer Epidemiol Biomarkers Prev 2001. Yuan JM. Urinary phytoestrogens and postmenopausal breast cancer risk. Sanders K. Keinan-Boker L. Cancer Epidemiol Biomarkers Prev 2004. Urinary excretion of phytoestrogens and risk of breast cancer among Chinese women in Shanghai.13:698–708. .8:35–40. China. Burger HG. Kataja V. et al. Manach C. Adlercreutz H. Arts CJ. Jin F. Wahlqvist ML. Mulligan AA. et al. Gao YT. 25. Phytoestrogens and breast cancer in postmenopausal women: a case control study. Wen WQ. 18. Gonthiera MP. Lenner P. Pietinen P. Linseisen J. Low YL. Plasma enterolactone and genistein and the risk of premenopausal breast cancer.10:223–8. Lancet 1997. Case-control study of phyto-oestrogens and breast cancer.10:339–44. Eur J Nutr 2002. Grace PB. Urinary excretion of isoflavonoids and the risk of breast cancer. Winkvist A.15:225–32. Briganti EM. Morand C. 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. Carcinogenesis 2002. Ross RK. Murkies A. Shu XO. 21. Urinary tea polyphenols in relation to gastric and esophageal cancers: a prospective study of men in Shanghai.41:168–76. et al. Biofactors 2004. Ito H. Veer PV. 19. Uusitupa M. Stumpf K. 20. Ingram D. Eur J Cancer Prev 2006. Botting NP. Chang-Claude J.
Kyrtopoulos Institute of Biological Research and Biotechnology. the triterpene hydrocarbon squalene and the phytosterol β-sitosterol [1–3]. a number of simple and bound phenolics (tyrosol. These include tocopherol and carotenoid antioxidants. Athens. Figure 3.2. Thus. Anticarcinogenic compounds of olive oil and related biomarkers Theodore G. Kyrtopoulos 134 3. This indicates olive oil phenolics to maintain their antioxidant activities in vivo..8. secoiridoids and lignans). the aglycone of ligstroside. Sotiroudis and Soterios A. squalene and β-sitosterol). Epidemiological studies demonstrate rather conclusively that populations within Europe consuming this diet have a particularly low incidence of a number of common cancers [1. lignans. and a peak containing the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol [1–3] (Table 3. the excretion of both HValc and HVA also being significantly correlated with the dose of administered hydroxytyrosol. two secoiridoids (dialdehydes related to oleuropein and ligstroside but lacking the carboxymethyl group at C4).Theodore G. In addition. a dose-dependent decrease in the urinary excretion of the F2-isoprostane 8-iso-PGF2α. HValc in urine reflects the in vivo . The National Hellenic Research Foundation.2]. Oleuropein and its metabolites tyrosol and hydroxytyrosol. When olive oil samples containing increasing amounts of a phenolic extract of olive oil were administered to human volunteers. promotion and progression of multistage carcinogenesis. was observed. together with homovanillic acid — HVA) and isoprostane excretion. tyrosol. 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. considered as putative nutritional biomarkers in relation to cancer the incidence of cancer. 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.). 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. are dose-dependently absorbed in humans after the ingestion of realistic doses of virgin olive oil.3. which represent major antioxidants in olive oil. hydroxytyrosol. Sotiroudis. a biomarker of in vivo lipid peroxidation processes. a major metabolite of hydroxytyrosol. Greece Olive oil is an important ingredient of the Mediterranean diet. that have been thoroughly studied. Soterios A. oleuropein. A plethora of minor constituents in olive oil have been identified as being effective agents mitigating against the initiation.
An HPLC method for the simultaneous determination of oleuropein and of its metabolites hydroxytyrosol and tyrosol in human plasma has been developed [13.72  41.45 . that the concentration in human plasma of lycopene.83–4. mg/kg 27.65–4. After the ingestion of olive oil of low phenolic content plasma glutathione peroxidase activity was found to decrease postprandially.8. It was observed in this respect. 1. Hydroxytyrosol (A). 2. a biomarker of the intake of tomato-rich food and hypothesized to be responsible for reducing the risk of various . Table 3.2.71  103–205  27. oleuropein aglycone (B). but this was not observed after the intake of olive oils of moderate to high phenolic content . 38–65  Fig.53 .14]. Recent findings suggest that olive oil may also affect the bioavailability of other food bioactive components with a chemopreventive potential.42 . 3.75  14. (+)-pinoresinol (C). Concentrations of the major phenolic compounds found in virgin olive oil Compounds Tyrosol Hydroxytyrosol Oleuropein aglycone Total secoiridoids Lignans References  and .Bioactive components in foods: Anticarcinogenic compounds of olive oil and related biomarkers 135 concentration of hydroxytyrosol [5–11]. Structures of certain phenolic compounds detected in olive oil.
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. 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. very little is known concerning the postprandial metabolism of squalene. Kyrtopoulos 136 cancers. the squalene intake would be more than 200 mg/d . increased dramatically after the consumption of tomatoes cooked in olive oil. while virgin olive oil is a major source of phytosqualene.000 mg/kg. leading to a reduced farnesyl pyrophosphate availability for prenylation of the ras oncogene . anti-angiogenic. suggesting that dietary lignans may be important in the etiology of breast cancer. Recent findings suggest that enterolactone is more rapidly metabolized in human colon epithelial cells and/or excreted by them than enterodiol is. Phytoestrogens have an anticarcinogenic potential through the anti-estrogenic. Nevertheless. was found to improve the antioxidant activity of plasma . proapoptotic and anti-oxidant mechanisms established for them [17. whereas the findings of epidemiological studies are controversial . Nevertheless. the prostate and the colon. and that the epithelial cells in the colon may be responsible for this metabolism . Soterios A. 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 ).Theodore G. The consumption of tomato products prepared together with olive oil. Sotiroudis. a tendency for a lower risk of breast cancer to be associated with higher plasma concentrations of enterolactone. 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. 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. It has been observed that postprandial squalene metabolism is age dependent .18]. but not with sunflower oil. This triterpene hydrocarbon is found mainly in nonedible shark liver oil. restricted almost entirely to estrogen-receptor alpha negative breast cancer has been found. which expands the plasma pool of this metabolically active hydrocarbon . its content ranging from 800 to 12. that the phase II metabolism of enterolactone and enterodiol already may take place during their uptake in the colon. Experiments in vitro and animal models suggest squalene to play a tumour-inhibiting role. If virgin olive oil were the sole source of dietary fat. Plasma enterolignan concentrations can thus be considered to be good biomarkers of dietary lignan exposure and be used to evaluate the effects of lignans . Although animal studies have enhanced our understanding of the possible . their reaching a steady state. particularly in premenopausal women .
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.
9. condensation. Feeding experiments in animals have also suggested broccoli can protect against liver cancer . this variable elongation of amino acid side chains entails repetitive additions of methyl groups through a series of transamination. Anticarcinogenic effects of glucosinolate breakdown products John D. tyrosine and tryptophan . Glucosinolates are substituted β-thioglucoside N-hydroxysulfates. As shown in Figure 3. Brussels sprouts. greater health benefit may be obtained from raw as opposed to cooked vegetables . isomerisation and decarboxylation reactions . Kelleher1 and Ian M. phenylalanine. the oxime is metabolised to a thiohydroximate. Ninewells Hospital and Medical School. University of Dundee. The most common of these are the methylsulfinylalkyl glucosinolates glucoiberin and glucoraphanin. catalysed by members of the cytochrome P450 (CYP) 79 family . formed by the plant from any one of eight amino acids. isoleucine. Over 115 naturally occurring glucosinolates have been identified. United Kingdom Glucosinolates and their association with cancer chemoprevention Epidemiological studies have revealed that regular consumption of cruciferous vegetables. leucine. though certain broccoli . Furthermore. Michael O.12–15].4. Kelleher. principally valine. cabbage. the olefinic glucosinolates sinigrin. Much of the diversity amongst glucosinolates arises from the addition of different sized alkyl groups to the side chain of those amino acids. Each cruciferous vegetable contains a mixture of glucosinolates that varies according to the strain of the plant [8. Eggleston2 1 2 Biomedical Research Centre. and the aromatic glucosinolate gluconasturtiin (Table 3. kale. Eggleston 140 3.11]. methionine. though it can be influenced by environmental factors [16. Claverton Down. Dundee DD1 9SY Department of Pharmacy and Pharmacology.) [9. alanine. swede and turnip. Glucoraphanin has been reported to be abundant in broccoli . University of Bath.2].. phenylalanine and methionine.17]. Ian M. Michael O.3.John D. cauliflower. Bath BA2 7AY. lung cancer . the synthesis of glucosinolates proceeds through the conversion of elongated amino acids to their oxime derivatives. namely. and possibly prostate cancer . 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 [10.20]. Subsequently. which is in turn conjugated with glucuronic acid to form a desulfoglucosinolate before finally being sulfated to yield the glucosinolate . This endeavour is simplified to some extent by the fact that relatively few glucosinolates are present in the human diet. gluconapin.9]. The glucosinolate content is primarily under genetic control. valine. Cruciferous vegetables uniquely contain glucosinolates at approximately 20 µmol/g dry mass of vegetable [8. glucobrassicanapin and progoitrin. The task of establishing a link between the ingestion of particular glucosinolates and their possible health benefits is not straightforward. is associated with a reduced incidence of cancer [1. The types of neoplastic disease in man that these vegetables appear to protect against include colorectal cancer . Hayes1. Hayes. used in their biosynthesis. such as broccoli.
Brussels sprouts and cauliflower [9.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 141 strains also contain substantial amounts of glucoiberin .9. cauliflower and kale . and whilst not abundant it can elicit distinct pharmacological effects. Sinigrin has been reported to be the predominant glucosinolate in Brussels sprouts.22].3. 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. . The indolyl glucosinolate glucobrassicin is present in Savoy cabbage. The aromatic glucosinolate gluconasturtiin is present in watercress. Synthesis of glucosinolates. gluconapin is also found in high levels in Brussels sprouts . cabbage. 3. Substantial amounts of progoitrin are present in many cruciferous vegetables . Table 3. The R group is derived from the original amino acid and is highly variable.
The outcome of the reaction with myrosinase depends on the nature of the aglycone.4A. In addition.25]. Kelleher. suggesting that glucosinolates can be hydrolysed in the gastrointestinal tract during digestion of food [24. EC 3.). thiocyanates. as well as the reaction temperature.4B.John D. resulting in the formation of an epithioalkane. Hydrolysis of these phytochemicals is catalysed by myrosinase (β-thioglucoside glucohydrolase.147). 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. Eggleston 142 Production of isothiocyanates. or during chewing whilst eating. 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 . 3. Hydrolysis of glucosinolates. 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. At high or neutral pH the formation of isothiocyanates is favoured while at low pH the formation of nitriles is favoured.3. for example during harvesting. and 3. . Michael O. myrosinase is released from the “myrosin” cells and is able to hydrolyse glucosinolates within the damaged plant. Hayes. Fig.2. Upon wounding of the vegetable.4A. during freeze-thawing. the pH and the presence of ferrous ions (Figure 3. Ian M. 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. but rather it appears to be due to certain of their breakdown products. during food preparation. myrosinase activity may be present in human colonic microflora.
3-butenyl-ITC. Certain thiocyanates that are formed during a Lossen rearrangement. 3. 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 . thiocyanates or nitriles [10. are unstable and can undergo a relatively slow spontaneous conversion to their respective isothiocyanate . to form their respective isothiocyanates (ITCs). give rise to a cyano-epithioalkane . gluconapin and glucobrassicanapin) may. hydrolysis of glucosinolates with aliphatic or aromatic side chains gives rise primarily to isothiocyanates (ITCs). olefinic and aromatic glucosinolates undergo a Lossen rearrangement. respectively. glucobrassicanapin and sinigrin yield 3-methylsulfinylpropyl-ITC. in the presence of an epithiospecifier protein (ESP) and ferrous ions. with the elimination of sulfate. On the right-hand side of the figure. the thiohydroximate-O-sulfates formed by myrosinase from glucosinolates with a side chain containing a double bond (e.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 143 ESP — epithiospecifier protein. . Elemental sulfur is also formed in certain circumstances. glucoraphanin. Fig. Following damage to the plant tissue the glucosinolate sinigrin is hydrolysed by myrosinase resulting in the formation of four distinct compounds.4B. Thus. Hydrolysis of sinigrin. at pH 4 and in the presence of Fe2+ ions. The glucosinolates glucoiberin.23]. gluconapin. formed from sinigrin. 4-methylsulfinylbutyl-ITC (sulforaphane).g. At low pH. The thiohydroximate-O-sulfates formed from methylsulfinylalkyl. such as allyl-ITC. can convert spontaneously to form allyl isothiocyanate . At neutral pH. In this case. sinigrin. an arrow shows that allyl thiocyanate. 4-pentenyl-ITC and 2-propenyl-ITC (allyl-ITC).
Michael O.5-epithiopentane .5. it can be hydrolysed by myrosinase to crambene (1-cyano-2-hydroxy-3-butene) [33.34]. but rather gives rise to indole-3-carbinol and a thiocyanate ion (Figure 3. . Progoitrin. glucobrassicanapin can be hydrolysed to 1-cyano-4. as a consequence of spontaneous cyclization. 3. This reaction may involve ESP.5. this reaction probably proceeds Fig. Gluconapin can similarly be converted by the combined actions of myrosinase and ESP to 1-cyano-3.3-epithiopropane . the sulfur atom may be lost and a nitrile formed [37–39]. Upon hydrolysis by myrosinase. Examples of these include progoitrin. Likewise.John D. At neutral pH. a (2R)-hydroxy-3-butenyl glucosinolate. The best studied of these is glucobrassicin. ESP and Fe2+ ions to an epithionitrile  and in the case of epi-progoitrin ((2S)-hydroxy-3-butenyl glucosinolate).). Production of indoles from glucosinolates. and is diminished by heating . Eggleston 144 myrosinase and ESP convert sinigrin to 1-cyano-2. is also converted in the presence of myrosinase. Ian M.4-epithiobutane [30. hydrolysis of glucobrassicin by myrosinase does not generate an ITC. those aglycones from glucosinolates that contain β-hydroxylated sidechains form oxazolidine-2-thiones. Hayes. Kelleher.31]. A few glucosinolates produce thiocyanates though the mechanism involved is unclear . Two cDNAs for ESP have recently been cloned from Arabidopsis and broccoli and the purified proteins characterized following their heterologous expression in E. glucoconringiin and gluconapoleiferin .36]. 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. Production of indoles from glucosinolates The indolyl glucosinolates glucobrassicin and neoglucobrassicin are synthesised by the plant from tryptophan. If the aglycone generated by myrosinase is from a glucosinolate with a side chain lacking a double bond. coli [35.
44]. indole-3-carbinol condenses to form various compounds including indolo[3. There is a dose-dependency in these responses: generally.2-b]carbazole and 3. indolo [3. and oxazolidine-2-thiones. such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1). At acidic pH. and stimulation of apoptosis.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 145 through a Lossen rearrangement generating an unstable ITC intermediate . both in vivo and in vitro [45.3∂-diindolylmethane. both of which have potent pharmacological effects .). Induction of gene expression mediated by Nrf2 Isothiocyanates increase the expression of antioxidant and detoxication proteins. 3. It can also combine with ascorbic acid to form ascorbigen  (Figure 3. inhibition of CYP enzyme activity. induction of cytoprotective genes and inhibition of CYP activity occurs at relatively low concentrations of phytochemical.3’-Diindolylmethane (DIM). A major problem exists in interpreting experiments utilizing vegetable extracts because of uncertainty about attributing biological effects to specific phytochemicals. cyanoepithioalkanes.46].6.6. cell cycle arrest. hydrolysis of glucobrassicin yields indole-3-acetonitrile. Fig. nitriles. A challenge in evaluating the literature arises from the emphasis placed on certain glucosinolate breakdown products and the dearth of data relating to others. Thus. In the acidic environment of the stomach. 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 . when compared with the data about thiocyanates. 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.2-b] carbazole (ICZ) and ascorbigen (ASG). there is a relative abundance of information about isothiocyanates and indoles. These include induction of antioxidant and detoxifying genes. Structures of indoles produced from glucosinolates — 3. whereas activation of cell cycle arrest and apoptosis occurs at higher levels of phytochemical [43. Agents such as ITC that increase GST and NQO1 .
ferritin. 4) 3-methylsulfinylpropyl-ITC. suggests AREs are present in the promoters of at least 100 genes [53–55]. Sulforaphane. 3. The battery of genes regulated by Nrf2 includes those encoding aldo-keto reductase (AKR).). Importantly. Fig. Hayes. Eggleston 146 enzymes without increasing arylhydrocarbon hydroxylase activity. benzyl-ITC and phenethyl-ITC. 6) 8-methylsulfinyloctyl-ITC. The mouse nqo1 gene contains a functional antioxidant response element (ARE) in its 5∂-upstream region . 8-methylsulfinyloctyl-ITC. microsomal epoxide hydrolase. small intestine and liver of rodents [53. multidrug resistance-associated protein. Examination of nrf2–/– and wild-type mice. glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits. 3) Benzyl-ITC. heme oxygenase 1. are sometimes called mono-functional inducers . carboxyl esterase.7. Ian M. UDP-glucuronosyl transferase. NQO1.54. 7-methylsulfinylheptyl-ITC. Indeed. GST. 5) 7-methylsulfinylheptyl-ITC. metallothionein. catalysed by the phase I drug-metabolising enzyme cytochrome P450 (CYP) 1A1.55–58]. leading to its inhibition and inability to serve as a substrate adaptor . all genes that are induced by ITCs contain an ARE in their promoters and are regulated by Nrf2. Kelleher. as does the rat GSTP1 gene (called GPEI) .49] (Figure 3. as far as is known. It is thought that ITCs possess the ability to induce ARE-driven gene expression because they are thiol-active . thioredoxin reductase. Through this characteristic.John D. ITCs modify cysteine residues in the Cullin 3:Rbx1 E3 ubiquitin ligase substrate adaptor Keap1. thioredoxin. 3-methylsulfinylpropyl-ITC. feeding broccoli seed to mice increased the levels of GCLC. Many of these compounds induce GST P1-1 in the rat liver RL-34 cells . allyl-ITC. Structures of isothiocyanates which induce NQO1: 1) Allyl-ITC. induce NQO1 in the mouse Hepa-1c1c7 hepatoma cell line [48. Michael O.7. 2) Phenethyl-ITC. Many of these genes are induced by sulforaphane in vivo in an Nrf2-dependent fashion in the stomach. 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 . GST and NQO1 in the gastrointestinal tract in an Nrf2-dependent fashion .
cranbene was found to be an approximately equally potent inducer in the rat . Indole-3-carbinol is a modest inducer of Nrf2-dependent ARE-driven gene expression in the liver and small intestine of mice [52. this may not necessarily lead to an increase in Nrf2 protein levels. a fact that implies it works through Nrf2. but not CYP1A1. Interestingly. Amongst genes for drugmetabolising enzymes. Consistent with this view. The identity of this metabolite is not known. and rat UGT1A6 contain an XRE. and this may also contribute to gene induction .3∂-diindolylmethane can increase the stability of Nrf2 protein is not known.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 147 required for the ubiquitination of Nrf2 under homeostatic conditions [60–62]. rat ALDH-3. rat GSTA2. Although indole-3-acetonitrile has not been studied in the Nrf2 knockout mouse.2-b]carbazole and 3. However. allyl-ITC does not increase Nrf2 stability  and there may therefore be other factors involved in enzyme induction by these phytochemicals.69]. mouse. and thereby generate reactive oxygen species. Surprisingly. The promoters of other genes including rat and human NQO1. Examination of the dose of indole required to double XRE-driven reporter gene expression shows that indolo[3.3∂-diindolylmethane.2-b]carbazole and 3.3∂-diindolylmethane. indicating that AhR ligands could lead to an increased production of Nrf2 mRNA. this seems unlikely . rat and human CYP1A1 are prototypic XRE-regulated genes. The indole-containing glucobrassicin breakdown products can activate gene expression by several mechanisms. the mouse nrf2 gene promoter also contains an XRE . and it is therefore anticipated that activation of AhR by glucobrassicin-derived indoles will influence significantly the metabolism of xenobiotics (for a review. By comparison with sulforaphane.2-b]carbazole and 3.2-b]carbazole is a much more potent inducing agent than 3. exposure of RL-34 cells or HepG2 cells to sulforaphane causes stabilisation and rapid accumulation of Nrf2 [63. it is a good inducer of GST enzyme activity in mouse liver and small intestine . as does the mouse BAX gene.67]. This cytochrome has O-deethylase activity towards ethoxyresorufin. Whether indolo[3. . It has been found that administration of cranbene to Fischer 344 rats causes an elevation in hepatic GST and NQO1 enzyme activities. but unless they are metabolised by CYPs. 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. suggesting it is a mono-functional inducer . 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. western and northern blotting that CYP1A1 is inducible by indolo[3. rat UGT1A1. see ). indole-3-carbinol or ascorbigen [43. 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. and there is abundant evidence from enzyme assays.3∂-diindolylmethane .64]. Treatment of cells with benzyl-ITC causes a rapid increase in the level of reactive oxygen species.
76].3∂-diindolylmethane can induce expression of the transcription factors ATF3. Most importantly. prior treatment of the LS-174 cells with both indolo[3.John D. 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. c-Jun. GADD45 and GADD153 [75. Furthermore. Indeed. Bonnesen et al  have reported that treatment of human colon LS-174 cells with indolo[3. Inhibition of CYP can also be achieved by sulforaphane [80. recognition of this possibility has lead to considerable recent interest in the ability of HDAC inhibitors to act as both chemopreventive and chemotherapeutic agents. the ITCs with highest lipophilicity and low reactivity of their NCS group had the greatest ability to inhibit lung tumorigenesis . such as growth arrest and DNA damage (GADD) GADD34.81].84]. Michael O. It has been argued that induction of CYP1A1 by indoles is potentially deleterious to the cell because the cytochrome can activate polycyclic aromatic hydrocarbons to ultimate carcinogens. Also.2-b]carbazole and sulforaphane before exposure to benzo[a]pyrene was found to confer substantial protection against genotoxicity. DNA is tightly coiled around an octamer of histone proteins in a structure known as a nucleosome. c-Jun and NF-IL6 as well as genes involved in cell growth. the basic structural unit of chromatin. and this protection was greater than was achieved by either phytochemical alone . Each of the histone proteins contains an evolutionary conserved amino tail protruding from . This viewpoint is probably an oversimplification and does not take into account the multiple changes in gene expression that indoles affect. It is not however clear whether the genes for ATF3. p21 is induced by indole-3-carbinole . Kelleher.79]. Within the cell. GST T1-1. 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. 3.2-b]carbazole before exposure to benzo[a]pyrene provides a small measure of protection against DNA damage as measured by the comet assay. as are the drug metabolising enzymes AKR. sulfotransferase and UGT1 . other cytochromes are inducible. This function is likely to alter gene expression substantially. Ian M. Inhibition of carcinogen activation by glucosinolate breakdown products Certain isothiocyanates can block the activation of several carcinogens to their ultimate carcinogenic forms. such as CYP1B1 and CYP19 . Eggleston 148 In addition to induction of CYP1A1 by indoles. Inhibition of histone deacetylase by isothiocyanates The isothiocyanate sulforaphane has been shown to inhibit histone deacetylase (HDAC) [83. Hayes. NF-IL6 and the GADD proteins are regulated through XREs and whether the process is mediated by AhR. In the case of the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
Cell proliferation by ITCs may also be achieved by distrupting cytoskeletal structure and tubulin polymerisation [93. along with other dietary HDAC inhibitors such as diallyl disulfide . 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. 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. Sulforaphane has been shown to diminish HDAC activity with a concomitant rise in histone acetylation in prostate cancer cells . apoptosis and differentiation . 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 tail is also subject to many post-translational modifications including acetylation. respectively. Conversely. allowing transcription factors access to the regulatory regions of genes. resulting in a reduction in its transcriptional activity. ApcMin/+ mice treated with sulforaphane at either 300 ppm or 600 ppm in their diet have been reported to develop fewer and smaller polyps in their small intestine than ApcMin/+ mice on a control diet. However. this was associated with a higher level of apoptosis and lower proliferation in animals on the ITC containing diet . human embryonic kidney cells  and human colorectal cancer cells . Equally. Inhibition of HDAC may prevent the removal of acetyl moieties from histones thus allowing transcription of the tumour suppressor genes.Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products 149 the nucleosome. For example. removal of acetyl groups causes the tail to wrap tightly around the DNA thereby preventing interaction with the transcription machinery. the majority of the studies into mechanisms by which this class of chemical inhibit cell growth have focussed on sulforaphane and phenethyl-ITC. The link between inhibition of HDAC activity and the resultant increase in transcription of tumour suppressor genes has been reported for p21 [84.85]. mediated through activation of checkpoint kinase 2 (Chk2) . 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. which can determine the accessibility of the DNA to transcription factors. sulforaphane has been found to cause a G2/M phase delay with an increase in apoptotic cell fraction in a timeand dose. mammalian HDAC is capable of downregulating p53 function.94]. tumour suppressor genes are associated with deacetylated histones resulting in the inactivation of these genes. p53  and Bax [84. In pre-cancerous and cancerous cells.85]. In human PC-3 prostate cancer cells.dependent fashion . and was accompanied by its translocation from the nucleus to the cytoplasm .92]. In addition. The loss of Cdc25C was reported to be due to proteasomal activity. Thus the ability of sulforaphane. The addition and removal of the acetyl groups is carried out by HAT and HDAC. .
Various ITCs have been shown to activate c-Jun N-terminal kinase (JNK) [102. Consistent with the view that production of ROS is necessary for apoptosis. In human bladder cancer UM-UC-3 cells. which appears to be necessary for cell death [92. Michael O. 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 . In the case of CDK6. and this is mediated by extracellular signal-regulated kinases. Furthermore. Cell cycle arrest at G1 occurs as a consequence of indole-3-carbinol inhibiting both cyclin-dependent kinase (CDK) 2 and CDK6.101]. in HaCaT keratinocytes. within 1 h of exposure. Ian M. though this may be cell specific. in human leukaemia HL60 cells. Furthermore. which neutralize the antiapoptotic effects of Bcl-2. The levels of pro-apoptotic proteins Bak and Bax. caspase-8 plays a major role in apoptosis stimulated by phenethyl-ITC . ERK1/2 . treatment with 400 µM indole-3carbinol induces the CDK4/6 inhibitor p15INK4b mRNA and protein causing hypophosphorylation of Rb protein . Hayes. In the case of CDK2. benzylITC and phenethyl-ITC are more effective at activating caspase-9 than caspase-8 .97]. overexpression of catalase suppresses ITC-initiated programmed cell death. Besides increasing the levels of these pro-apoptotic proteins. Treatment with ITCs leads to a loss of mitochondrial membrane potential and release of cytochrome c from mitochondria . Kelleher. There is evidence that ITCs can activate both the intrinsic and extrinsic caspase cascades. addition of GSH subsequent to ITC treatment can block apoptosis [44.John D. and this may lead to induction of Apaf-1 [91.3∂-diindolylmethane rather than indole-3-carbinol as significant quantities of the indole spontaneously condense to the dimer in culture conditions . It therefore appears that cyclin D-CDK6 activity can be inhibited by dual mechanisms. the pro-apoptotic proteins Bok and Bim EL are also induced by ITCs.103]. as does pre-treatment with N-acetylcysteine .92. in PC-3 cells sulforaphane can increase Fas protein levels and activate caspase-8 whilst simultaneously targeting mitochondria and activating caspase-9 . are increased by phenethyl-ITC and sulforaphane in PC-3 prostate cancer cells. the mechanism by which JNK activates caspases remains unclear. Eggleston 150 Administration of ITCs to cells at growth suppressive concentrations results in the rapid generation of reactive oxygen species (ROS). indole-3-carbinol has been . For example. ITCs down-regulate the anti-apoptotic proteins Mcl-1 and Bcl-xL . 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 . though the effect is cell-specific . 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 . Furthermore. and this is thought to amplify the effects of Bak and Bax . By contrast. However.95]. This may in part be due to 3. expression of the gene is reduced because indole-3-carbinol attenuates recruitment of the Sp1 transcription factor to the CDK6 promoter .
cyanoepithioalkanes and oxazolidine-2-thiones. ESP should possibly . Also. 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. which reduces the formation of isothiocyanates from glucosinolates. Furthermore. nitriles. the reduction in CDK2 activity is accompanied by redistribution of the kinase in the 200 kDa complex from the nucleus to the cytoplasm. Treatment of PC-3 prostate cancer cells with 60 µM indole-3-carbinol inhibits the EGF-induced autophosphorylation of PI3K and Akt . These changes result in prevention of the CDK2-mediated G1/S transition . Specifically. In HCT-116 human colon cells. is undesirable from a cancer chemoprevention perspective. 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 . an increase in p53 protein levels.3∂-diindolylmethane through a reduction in phosphorylation of IκBα [114. such as tamoxifen . a TGF-β family member associated with pro-apoptotic activities . there are few data about chemopreventative activities of thiocyanates. such as MCF-10A. The reduction in CDK2 activity is attributed to a selective alteration in the size of the complex in which it is contained.3∂-diindolylmethane.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 . In cells that contain wild-type p53. and the larger complex also contains an additional 75 kDa cyclin E immunoreactive protein. at the expense of forming isothiocyanates. cyano-epithioalkanes and oxazolidine-2-thiones from glucosinolates. It is unclear whether the activity of ESP. If so. It is possible that down-regulation of this receptor represents an antiproliferative mechanism in prostate cells. treatment with 300 µM indole-3-carbinol or 30 µM 3. nitriles. but not 3. Indole-3-carbinol. Concluding comments It is becoming clear that glucosinolate breakdown products can influence the initiation and progression of carcinogenesis. This may also mediate the anti-tumour effects of indoles. is undesirable. They also appear to influence apoptotic responses to chemotherapeutic agents. Thus. induction of p21 . suggesting that indole-3-carbinol can influence the nucleocytoplasmic shuttling of the kinase . Indoles can affect apoptosis in breast and prostate cancer cells. the 90 kDa and 200 kDa complexes include forms of cyclin E that differ in size. from an active form within a 90 kDa complex to a lower activity form within a 200 kDa complex .115]. It is unclear whether formation of thiocyanates. the Akt/PI3K cell survival pathway appears to be targeted by indole-3-carbinol. indole-3-carbinol can induce nonsteroidal antiinflammatory drug-activated gene-1 (NAG-1). nuclear translocation of NF-κB is inhibited by 3.3∂-diindolylmethane has been found to result in activation of the ATM signalling pathway.
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Evaluating the effects of phytochemicals that act synergistically Carcinogenesis is an extremely complex multistep process in which numerous molecular mechanisms play an important role. Although relatively high doses of single bioactive agents may show potent anticarcinogenic effects.2]. de Kok. numerous bioactive compounds have been isolated and identified. 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. One of the key problems of research in this field. In the research conducted. University of Maastricht. Maastricht. which are believed to have cancer-preventive effects. Table 3. The Netherlands Introduction The relatively consistent epidemiological finding that the consumption of whole foods of different types such as fruits. 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. both in vitro and in vivo. In addition to characterizing the chemopreventive effects of individual compounds. de Kok and Simone G. summarizes various mechanisms by which phytochemicals can modulate the risk of cancer [1. 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.10. Isolated and purified compounds. and their potential health-promoting effects evaluated extensively.Theo M. Cancer-preventive dietary compounds may interfere at a variety of different levels with these processes. vegetables. Combined action of different dietary compounds preventive of cancer Theo M. and the like. however. 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. Some studies show no reduced incidence of cancer as a result of taking supplements of vitamin C  or β-carotene .5. whole grains. . therefore. 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. and there are even reports of increased occurrence of lung cancer in smokers receiving dietary β-carotene supplements [5. in contrast. In this chapter. Simone G. The combinations of phytochemicals that natural foods contain can reduce the risk of cancer by affecting different overlapping and complementary mechanisms. van Breda Department of Health Risk Analysis and Toxicology. vegetables and whole grains is strongly associated with reduced risk of cancer and of other chronic diseases has led to the hypothesis that particular phytochemicals are responsible for the preventive effects observed.6]. evidence that bioactive compounds act synergistically is reviewed. in contrast. van Breda 160 3. are far from certain. The intended positive effects of an increased intake of β-carotene or vitamin C as dietary supplements. evaluation of the effects of synergistically acting phytochemicals is needed.
 reported that the effects of tritium-labelled epigallocatechin gallate (EGCG) being incorporated into human lung cancer cells were enhanced by epicatechin (EC).10. 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. presents a selection of relevant studies concerning such synergistic effects. Epicatechin was found to promote the occurrence . Suganuma et al. . one without a galloyl moiety.11.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 161 Table 3. Synergistic effects of combinations of various polyphenols A number of studies report enhanced chemopreventive effects of mixtures of polyphenols from green tea or other dietary sources. Table 3. another green tea polyphenol.
 Green tea infusions and grape Reduced tumour cell growth or grape skin extracts Polyphenols. Selection of studies of the synergistic effects of mixtures of polyphenols and of combinations of these with other types of phytochemicals Combination of compounds Polyphenol mixtures EGCG and EC. EC. reduced levels of estrogen receptor-α protein and of serum IGF-I. trap-ping of lipid peroxyl radicals and regene-ration of vitamin E Zhou et al.11] Polyphenols and other phytochemicals Green/black tea and soy (SPC)* Green/black tea and soy (SPC) Inhibition of prostate tumours.  Zhou et al.  EGCG and EC Inhibition of cell growth and induction of apoptosis in gastric carcinoma cells Horie et al. Zhou et al.  Zhou et al.  hydroperoxides and malondialdehydes. enhanced apoptosis. EGC and ECG Modulation of CYP1A1 expression in human hepatocytes Williams et al. vitamin E. EGC and ECG or gallic acid and α-tocopherol Reduced oxidative stress in micelles and human LDL .11. -8 and -9. Extracellular production of oxygen species Antagonism of TCDD-induced transcription of human CYP1A1 via interaction with the Ah-receptor Suganuma et al. tumour weight and metastasis Inhibition of breast tumour cell growth Reduction in serum levels of testosteron and DHT Inhibition of tumour angiogenesis. were also enhanced in a dose-dependent way by EC. induction of apoptosis.  EGCG. A and ß-carotene Reduced oxidative stress Morré and Morré  Reduced formation of lipid Gorelik et al. de Kok. induced by a tea polyphenol having a galloyl moiety. reduced co-oxidation of vitamins E. EC.  EGCG.  Liu et al.Theo M. Inhibition of tNOX. reduced release of TNF-α Increased production of caspases-3. [10. Table 3. van Breda 162 of EGCG-induced apoptosis and to inhibit not only growth of PC-9 lung tumour cells but also the release of tumour necrosis factor-α. C and ß-carotene Reduction in α-tocopheryl radicals. 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. These effects. sulindac or tamoxifen Synergistic effect Mechanisms involved Reference Inhibition of growth of human lung cancer cells Enhanced cellular uptake of EGCG. Simone G.
the activity levels of caspases 3. and epicatechin gallate (ECG) — and it was not found to be improved further by the addition of other compounds . 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. Williams et al. . epigallocatechin (EGC). EC alone had virtually no effect on carcinomic cell growth or induction of apoptosis.  Van Breda et al. 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). After this combined treatment.11. being due instead to the effects of the complex mixture involved. suggesting that the reactive oxidative species and production of hydrogen peroxide play a part in the synergistic mechanisms here . EGCG. were also found in gastric carcinoma cells . both on the inhibition of cell growth and the induction of apoptosis.  demonstrated that complex green tea extracts exert mixed agonist/antagonist activity on the Ah-receptors.  Jørgensen et al.Bioactive components in foods: Combined action of different dietary compounds preventive of cancer 163 Table 3. carcinogens or cytotoxic substances . the modulation of their expression and activity by phytochemicals is a potential mechanism by which the risk of cancer can be reduced. The optimal degree of synergy was achieved by a combination of the four major tea catechins — EC. interpreted mainly in preventive terms No indication of specific synergetic mechanisms * SPC — Soy Phytochemical Concentrate. Various gastric cell lines were shown to differ in their susceptibility to EGCG treatment. but under some circumstances substrates may be activated to become mutagens. 8 and 9 became elevated. Enzyme induction usually enhances detoxification. whereas EGCG acts as a strict AhR antagonist. Selection of studies of the synergistic effects of mixtures of polyphenols and of combinations of these with other types of phytochemicals — cont. Interestingly. catalase was found to block the synergistic effects of EC and EGCG. peas and unions) Up.24] Different types of vegetables (cauliflower. carrots.and down-regulation of genes involved in carcinogenesis. [23. Since the cytochrome P450 (CYP) enzymes are responsible for the metabolism of many environmental carcinogens. Some of the cytochrome P450 genes are expressed constitutively. but it had a significant synergistic effect on the induction of apoptosis when combined with other catechins. Synergistic effects of green tea catechins. 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.
These results suggest that more effective cancer prevention and cancer therapy could be achieved by use of combinations of different phytochemicals. such as by phytochemicals. de Kok. increasing at the same time the amounts of vitamin antioxidants reaching the blood system. Recently.). Zhou et al. reduced expression of estrogen-receptor (ER) alpha and reduced serum levels of the insulin-like growth factor (IGF)-I. The modulation of these two different mechanisms may be an explanation of the synergistic effects of the combined phytochemicals . In the gastric fluid. This was accompanied by inhibition of tumour angiogenesis. 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 . Simone G. the latter a biologically more active metabolite of testosterone and a prerequisite for the development of benign prostatic hyperplasia and prostate cancer .11. whereas no activity was detected for extracts of grape seeds. 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. In line with this. the authors suggested that the antioxidant network in the stomach can decrease the level of hydroperoxides and other cytotoxic compounds. all of these being factors in breast cancer development. The strongest synergistic activity was found for ethanol extracts of grape skins. 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. Polyphenols and dietary antioxidant vitamins can also have synergistic inhibitory effects on lipid peroxidation and on the co-oxidation of dietary antioxidants. NOX proteins are located at the cell surface and are responsible for the increase in cell size that occurs following cell division . They cease to divide and eventually undergo apoptosis. are unable to enlarge. indicating that the effects were not caused by resveratrol. A phytochemical soy concentrate (SPC) and green and black tea infusions were employed (Table 3. van Breda 164 Synergistic effects of polyphenols and other phytochemicals In two different studies. Cells in which NOX activity is blocked. The authors indicated that the resulting synergistic increase .13]. vitamin C can enhance the activity of polyphenols through a synergistic antioxidant effect. 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. Morré and Morré  showed there to be an exceptionally strong (10-fold) synergy between grape polyphenols and tea catechins in the inhibition of tNOX.Theo M. though to a lesser extent) . In an immunedeficient mouse model in which MCF-7 human breast cancer cells were implanted. which is found predominantly in the seeds. SPC in combination with green tea showed a synergistic inhibition of tumour cell growth. 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 .
 demonstrated clearly that several green tea polyphenols (EC. 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 . On the other hand. In one study. plays a major role in enhancing the antioxidant efficiency of vitamin E. the synergistic effects of taking whole foods or complex mixtures of compounds have been reported. 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. 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 . the Trolox equivalent antioxidant capacity (TEAC) value and phenolic composition were determined for a large number of pinotage wines . Zhou et al. 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. By studying the kinetics of the reaction of α-tocopherolyl radicals with green tea polyphenols by means of stopped-flow electron paramagnetic resonance.22]. EGCG. . EGC.24]. the individual vegetables were able to modulate genes which were not significantly modulated by the mixture. and assuming their concentrations to be those typical for red wines. combined with an α-tocopherol regenerating reaction involving coexisting antioxidants.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. These green tea polyphenols were also found to trap the initiating radicals (ROO•) as well as the propagating lipid peroxyl radicals (LOO•). it was found that most of the genes that were differentially expressed before and after vegetables were consumed. In two recent animal studies on the effects of vegetable consumption on the modulation of gene expression. When the contributions of the separate phenolic compounds were calculated. 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. This. ECG and gallic acid) can effectively reduce α-tocopheroxy radicals so as to allow α-tocopherol to be regenerated. 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 of whole foods and complex mixtures of compounds In addition to the synergistic effects of several individual compounds on biomarkers of cancer prevention. Taking account of the different mixtures found of the 12 phenolic compounds that were present. 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 changes in gene expression could be interpreted as a cancer preventive effect [23. indicating that combinations of different foods containing different complex mixtures of phytochemicals can also have an antagonistic effect on gene expression.
it appears . Although our understanding of the molecular mechanism behind the observed combinatorial effects of these chemopreventive agents is still limited. 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 . particularly those of sulindac. Simone G. Jørgensen et al.Theo M. Various combinations of drugs have been proposed for further clinical development based on their synergistic activity as shown in vitro or in animal studies. Other synergistic effects In addition to investigating the synergistic effects of various phytochemicals. van Breda 166 indicated 16 to 23% of the antioxidant activity to be synergistic.  demonstrated. Conclusions An increasing number of in vitro and in vivo studies indicate that combinations of dietary chemopreventive agents can result in a significant level of activity being achieved at concentrations at which any single agent is virtually inactive. Use of retinoids in combination with such SERMs (selective estrogen receptor modulators) as tamoxifen or raloxifene [29. 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 . de Kok. This implies that. Administering multiple agents can increase the efficacy and potency of the chemopreventive measures taken and reduce toxic side-effects. the regeneration of quercetin from its phenoxyl radical by the presence of (+)-catechin. EGCG and sulindac were also found to synergistically enhance apoptosis. without a loss in treatment potency.30] are examples of this. Also. 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. 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.28]. 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 effects reported by Suganuma et al. The fact that many of these phytochemicals act synergistically may why some food items or diets show cancer preventive effects that cannot be explained on the basis of their separate bioactive ingredients alone. however. In that study. A review of the effects of various combinations of chemotherapeutic and chemopreventive approaches in dealing with cancer has appeared recently . its also reducing side effects. This suggests that the regeneration of phenolic compounds from phenoxy radicals is a mechanism that can contribute to the synergistic effects observed.
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Probiotics usually refer to highly selected lactic acid bacteria. Bifidobacterium spp. and Streptococcus spp. 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. that can modulate gut microflora and its metabolism.and prebiotics on cancer is derived from in vitro studies. relieving vaginitis to name but a few. Overall. Sweden Introduction An example of a functional food. This has led to intense interest in factors. Administration of L. although there are some on breast  and bladder cancer .g.Joseph Rafter 170 3. such as probiotics. that have the potential to improve host health’)  and synbiotics (combinations of pro. e. Evidence from a wide range of sources supports the view that the colonic microflora is involved in the etiology of CRC.. The list of healthful effects attributed to probiotic bacteria is extensive  and includes: alleviation of lactose intolerance symptoms. Evidence for protective effects of pro. 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. animal models. serum cholesterol reduction. acidophilus to eleven volunteers on a fried meat diet known to increase faecal mutagenicity.6. anticancer effects.or prebiotics alone. 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. Lactobacillus spp. The evidence from animal studies provides strongest support for anticancer effects and data from human studies (epidemiology and experimental) are limited. 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. Karolinska Institute. resulted in a lower faecal mutagenic activity after 3 days . with defined gut survival properties and associated biological activities and which can be ingested in fermented milk products or as a supplement. Huddinge.and prebiotics). Diet makes an important contribution to CRC risk  implying that risks of CRC are potentially reducible. epidemiology and human intervention studies. alleviating constipation. The vast majority of studies on the anticancer effects deal with colorectal cancer (CRC). 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. Probiotics and prebiotics — potential anticarcinogenic food components Joseph Rafter Department of Medical Nutrition.2].
11]. It will be important to define dose and time relationships and it would appear at present. it will become possible to perform studies in healthy volunteers. In a cohort study in the Netherlands. from animal studies. particularly in urine. Thus. did not provide evidence that intake of dairy products is associated with a decreased risk of colon cancer . Hayatsu and Hayatsu  also demonstrated a marked suppressing effect of orally administered L. In another case control study. colon cancer incidence was lower than in other countries because of the high consumption of milk. the opportunity to exploit genomics and proteomics in investigations of effects of pro/prebiotics on gene expression and post-transcription . An epidemiological study performed in Finland demonstrated that. although a weak non-significant inverse association with colon cancer was observed . 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. casei on the urinary mutagenicity arising from ingestion of fried ground beef in the human. there are few epidemiological studies addressing the association between fermented dairy products and colorectal cancer. 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. In summary. and other dairy products [10.15]. it was shown that the intake of fermented dairy products was not significantly associated with colorectal cancer risk in an elderly population with a relatively wide variation in dairy product consumption. High levels of mutagenicity also appeared in urine on days 2 and 3 of the fried meat and ordinary fermented milk dietary regimen. Once such markers are available.and prebiotics. the most profitable approach would be to use combinations of pro. As yet. the 1980–1988 follow-up of the Nurses’ Health Study and the 1986–1990 Health Professionals follow-up study. an increase in the number of faecal lactobacilli corresponded to a lower mutagen excretion. at-risk groups and patients. despite the high fat intake. data from human intervention studies are of paramount importance in providing evidence that probiotics. the urinary mutagenic activity on days 2 and 3 was significantly lower compared to the ordinary fermented milk period. prebiotics or fermented milk consumption are causally related to reduction in cancer risk. adjusted for potential confounding variables. an inverse association was observed for yoghurt  and cultured milk consumption . On the other hand. During L. yoghurt. two companion American prospective studies. an inverse relationship for yoghurt consumption with risk of large colon adenomas in men and women was reported .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) . 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 . There will also be. in the near future. In conclusion. In two population-based case-control studies of colon cancer. this is an area of high priority for future studies. acidophilus administration. it would appear that the case control studies indicate protective effects while the prospective studies do not. In most cases.
22] or DMH [23. while heat-treated L. Metabolically active L. Goldin et al. L. prevented MNNG-induced DNA damage. acidophilus also inhibited the formation of ACF. including colonic mucosal markers. http://www. acidophilus. 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. in gastric and colonic mucosa in rats. funded by EU. such as aberrant crypt foci (ACF). are being measured. End points used are the tumours themselves or early lesions.Joseph Rafter 172 events in colonic biopsies and to identify human groups responsive to pro/prebiotic intervention.2-dimethylhydrazine (DMH)-induced genotoxicity. adolescentis culture and its supernatant did not show an inhibitory effect in this study . using the comet assay. In this study. using these models. confusus. Among different cell fractions from L. 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. there have been many studies.syncan. placebo controlled trial of a food supplement containing Lactobacillus GG.  reported. Bifidobacterium Bb-12 and Raftilose Synergyl in adenoma patients. faecal water markers and immunological markers. to examine the effect of a synbiotic preparation on colon cancer risk biomarkers in humans (SYNCAN project. casei subsp. acidophilus cells. L. Consumption of B. In recent years. gasseri. Although B. It involves a twelve-week randomised. that Lactobacillus acidophilus. acidophilus was not antigenotoxic. all of the ”state of the art” colon cancer risk biomarkers. Streptococcus thermophilus. It will also be particularly important to use data on mechanisms of action to develop hypothesis based intervention studies in humans. which clearly demonstrate a protective effect of dietary supplements of lactic acid bacteria against colon tumour development.24]. feeding of bifidobacteria suppressed the ACF formation induced by AOM [21. Of relevance here is a clinical trial which is presently ongoing i. as well as an acetone extract of the culture. induced by chemical carcinogens. Certain strains of lactic acid bacteria have also been found to prevent putative preneoplastic lesions or tumours induced by carcinogens. and involving 8 research centres in Europe. Overnight cultures of L.be). longum were antigenotoxic toward N’-nitro-N-nitrosoguanidine (MNNG). Bifidobacterium breve and B. Oral administration of lactic acid bacteria has been shown to effectively reduce DNA damage. Pool-Zobel et al. longum or inulin was associated with a decrease in AOM-induced colonic small ACF in rats and combined administration . induced by azoxymethane (AOM) . These bacteria were also protective toward 1.  showed that a specific strain of L. the peptidoglycan fraction and whole freeze-dried cells were antigenotoxic. ACF are putative preneoplastic lesions from which adenomas and carcinomas may develop. 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.e. double blind.
which are acid resistant in contrast to most bacteria. enriched with oligofructose. 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. 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. longum administered in the diet to rats inhibited liver. Biothree®. the results from a previous report from our laboratory. induced by the food mutagen 2-amino-3-methyl-3H-imidazo(4. Ingestion of B. mindful of the fact that the composition and metabolic activities of the intestinal flora of experimental animals are significantly different from those of humans . used in this study contained Streptococcus faecalis T-110. Recently. In addition. the results indicated that the prebiotic decreased AOM-induced carcinogenesis.33]. Goldin and Gorbach  showed that dietary supplements of L. Feeding of fermented milk increased the survival rate of rats with chemically induced colon cancer .5-f)quinoline (IQ). The mechanisms by which they act are less clear. which do not survive contact with gastric acid. we exploited human flora associated (HFA) mice to test the effects of a probiotic mixture on a parameter of relevance for colon carcinogenesis i.Bioactive components in foods: Probiotics and prebiotics — potential anticarcinogenic food components 173 significantly decreased the incidence of large ACF . while a possible protective effect of probiotics was observed. Reddy and Rivenson  reported that lyophilised cultures of B. longum resulted in a significant suppression of colon tumour incidence and tumour multiplicity and also reduced tumour volume induced by AOM in rats . infantis strain ATCC15697. lower proliferative activity and a variation in the expression of some enzymes involved in the pathogenesis of colon cancer. longum also significantly inhibited AOM-induced cell proliferation. breve reduced the number of ACF with four or more crypts/focus and crypt multiplicity which are reliable predictors of malignancy . but the data presented suggested that they may act through a combination of mechanisms involving an increase in short-chain fatty acid (SCFA) production. Clostridium butyricum TO-A and Bacillus mesentericus TO-A. five intralesional injections resulted in 70% tumour regression in the mice. and that the additional colonisation 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. there was a report on the antitumourigenic activity of the prebiotic inulin. More recently. ornithine decarboxylase activity and expression of the ras-p21 oncoprotein. colon and mammary tumours. demonstrated that human intestinal microflora had different effects than mouse microflora concerning DNA adduct formation after exposure to mutagens . acidophilus not only suppressed the incidence of DMH-induced colon carcinogenesis but also increased the latency period in rats. In addition. Sekine et al. Dietary administration of a lyophilised culture of B. in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis in the AOM-induced colon carcinogenesis rat model .e.  using whole peptidoglycan isolated from B. The authors concluded that. It has been . DNA adduct formation . Indeed. reported that a single subcutaneous injection significantly suppressed tumour growth. The probiotic mixture.
difficile and C. It is effective for the improvement of symptoms caused by abnormal intestinal flora. . It has also been reported that B. faecalis T-110 and C. Salmonella typhimurium. the use of lactic cultures for human cancer suppression is interesting. i. such mechanisms might include: alteration of the metabolic activities of intestinal microflora. It is possible that different strains target different mechanisms. 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. production of antitumourigenic or antimutagenic compounds.3-dihydroxyazetidine [39. binding and degrading potential carcinogens. butyricum TO-A . enhancing the host's immune response. 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. faecalis T-110 and C.Joseph Rafter 174 reported that S. 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. The strongest evidence for anticancer effects of probiotics comes from animal studies and evidence from human studies (epidemiology and experimental) is still limited. C. mainly originating from in vitro and animal experiments and some of them even have some support from human clinical studies. Also. Vibrio parahaemolyticus. Conclusion Many healthful effects are attributed to the probiotic bacteria and some of these effects have more scientific support than the anticancer effect. bile acid-metabolizing bacteria). All of the mechanisms have various degrees of support. mesentericus TO-A stimulated the growth of Bifidobacterium by producing 3. However.g. effects on physiology of the host. Biothree® is used as a clinical therapy in Japan. However. there are several possible mechanisms which might explain how lactic acid bacteria might protect against tumour development in the colon.3-b]indole (2-amino-alpha-carboline. even with the above reservations in mind and mindful of the limited number of human studies available. as mentioned above. alteration of physicochemical conditions in the colon. holds promise and certainly deserves more scrutiny. butyricum TO-A showed strong symbiosis with each other and the growth of enteropathogens (enterotoxigenic Escherichia coli. AAC). given by gavage. Thus. An important goal for the future should be carefully designed human clinical trials to corroborate the wealth of experimental studies.e. Interestingly. quantitative and/or qualitative alterations in the intestinal microflora incriminated in producing putative carcinogen(s) and promoters (e. 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.40]. diarrhoea and constipation. botulinum) was inhibited in mixed cultures of S.
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Âw. SE-22100 Lund. Strada Della Carita 10.uk . email@example.com Johannes Gutenberg-Universität Mainz (JOGU) Germany.dk Karolinska Institutet (KI) Sweden.org Deutsches Krebsforsforschungszentrum (DKFZ) Germany.mi.se Institute of Cancer Research (ICR) United Kingdom.ki.se Fondazione ISI (ISI) Italy. david.antsz. taioli@policlinico. oesch@uni-mainz. firstname.lastname@example.org Finnish Institute of Occupational Health (FIOH) Finland. skyrt@eie. mkirschv@vub. Viale Settimo Severo 65. 1050 Brussels.ac. giuseppe.pl Genetics Research Institute & Ospedale Policlinico (GRI) Italy. Collegium Medicum in Bydgoszcz Poland. 85-067 Bydgoszcz.Nair@dkfz.hu Nicolaus Copernicus University. steffen.ac. Saarstrasse 21. 10133 Turin.email@example.com@tbiokem. Gyáli út 2-6.uk National Institute of Environmental Health (NIEH) Hungary.firstname.lastname@example.org. Jagielloƒska 13.gr University of Leicester (ULEIC) United Kingdom.lth. Pleinlaan 2. 55099 Mainz. SE-171 77 Stockholm. H-1097 Budapest. pbf1@leicester. University Road.Annex ECNIS participants Nofer Institute of Occupational Medicine (NIOM) Poland. DK-1017 Copenhagen. Vassileos Constantinou Avenue 48. LEI 7 RH Leicester. Teresy 8.segerback@biosci. Paradisgatan 5C. coordinator.fi Vrije Universiteit Brussel (VUB) Belgium. 11635 Athens. schoketb@okk. dan. Neuenheimer Feld 280.Hirvonen@ttl.ac. bjorn. 91-348 ¸ódê.loft@farmakol. Topeliuksenkatu 41 a A. Norregade 10. J. 69-120 Heidelberg. 20135 Milan. 00250 Helsinki.be Lund University (ULUND) Sweden.de University of Copenhagen (UC) Denmark.it The National Hellenic Research Foundation (NHRF) Greece. SW7 3RP London. Ari. Old Brompton Road 123.
Dietary vitamins. Institute of Risk Assessment Science (IRAS-UU) The Netherlands. cagonzalez@ico. Lurup 4. Oude Markt 13. 6200 MD Maastricht. Leuven R&D Department. DD1 4HN Dundee roland. K.unimaas.ki. London.be Maastricht University (UM) The Netherlands. p. lennart. Dr Gernot Grimmer Foundation (BIU) Germany. 3000 Leuven.moller@csb. B-3000 Leuven. Krzysztofowicz Sp.nl Biochemical Institute for Environmental Carcinogens Prof. (NETIX) Poland.ac. Nethergate. Groot Begijnhof 58-59.Kromhout@iras.vlaanderen. Heidelberglaan 8.uk .casteleyn@lin. Universiteitssingel 50.uk International Agency for Research on Cancer (IARC) France. Selagarden. 08907 L'Hospitalet de Llobregat. Barcelona. Gran Via S/N Km27. polyphenols. 00-193 Warszawa. H.uu.pl Leocordia AB (Leocordia AB) Sweden.email@example.com@imperial. Exhibition Road.se Imperial College London (ICL) United Kingdom. albrecht. ludwine.es Utrecht University. firstname.lastname@example.org. 69372 Lyon. Stallarholmen.wolf@cancer. Stawki 2. selenium and probiotics: biomarkers of exposure and mechanisms of anticarcinogenic action 180 Katholieke Universiteit Leuven Belgium.de Institut Catala d'Oncologia (ICO) Spain. 3508 TC Utrecht. mask@netix. U. J.fr NETIX Skrzypczyƒski.nl University of Dundee (UNIVDUN) United Kingdom.email@example.com. 22927 Grosshansdorf. Cours Albert Thomas 150. f.
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