Dietary Vitamins, Polyphenols Selenium and Pro Bio Tics

ECNIS is a Network of Excellence within the European Union’s Sixth Framework Programme,
Priority 5: Food Quality and Safety. It brings together some of the best European research groups
in a concerted effort to achieve improved understanding of the environmental causes of cancer,
of the potential of diet to prevent cancer and of the ways in which heredity can affect individual
susceptibility to carcinogens, with the ultimate aim of reducing the cancer burden in Europe.
ECNIS is coordinated by Prof. Konrad Rydzyƒski, Nofer Institute of Occupational Medicine,
ul. Âw. Teresy 8, 91-348 ¸ódê, Poland.
This review has been prepared as part of ECNIS Work Package 9: Mechanisms of modulation
of cancer by dietary factors.
© ECNIS, 2007
All rights reserved. No part of this book may be reproduced in any form without the permission
of the publisher.
Compiled and edited by Björn Åkesson and Per Mercke

Biomedical Nutrition, Pure and Applied Biochemistry, Lund University
PO Box 124
SE-221 00 Lund
Tel: +46 (0) 46 222 45 23
Fax: +46 (0) 46 222 46 11
ISBN 978-83-60818-02-2
Technical editor: Katarzyna Rogowska

Cover design, computer typesetting: Beata Grabska
Published by Nofer Institute of Occupational Medicine

Âw. Teresy 8, 91-348 ¸ódê, Poland
Tel.: +48 (0) 42 631 45 04
Fax: +48 (0) 42 656 83 31
E-mail: ecnis@ecnis.org
Website: http://www.imp.lodz.pl
Contents
Executive summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.1. Introductory overview and future prospects
Björn Åkesson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Appendix 1.2. Overview of possible anticarcinogenic food components
Per Mercke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2. Vitamins and selenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.1. Biomarkers of exposure to and effects of vitamins A, C and E
Lars O. Dragsted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2. Antioxidant vitamins and cancer risk: is oxidative DNA damage
a relevant biomarker?
Steffen Loft, Peter Møller, Marcus S. Cook, Rafa∏ Ró˝alski, Ryszard Oliƒski . . . . . . . . . . . . . . 51
2.3. Measurement of serum 25-hydroxycholecalciferol as marker of vitamin D status
Jakob Linseisen, Sascha Abbas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
2.4. Selenium and cancer — selenoproteins as biomarkers in relation
to selenium supplementation
Björn Åkesson, Katharina Bruzelius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
2.5. Anticancerogenic activity of selenium — molecular mechanisms
and epidemiological data
Jolanta Gromadziƒska, Edyta Reszka, Wojciech Wàsowicz . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
3. Bioactive components in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
3.1. Anticarcinogenic effects of flavonoids
Margaret M. Manson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
3.2. Biomarkers of dietary polyphenol intake for studying diet-cancer relationships
Jakob Linseisen, Sabine Rohrmann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

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

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, especially those of relevance
for the partners of the NoE ECNIS and its contacts. Since this is a vast area, only certain selected
topics, as outlined below, are considered in detail. The important links found between use
of a substance as a biomarker and its mechanisms of action led to a further aim, that of reviewing
the mechanisms of action of some of the most promising anticarcinogenic compounds.
Many compounds contained in the diet have been proposed as having anticarcinogenic
properties. These compounds are found in foods of many different types, although plant-
based foods appear to be the richest source. The degree to which these compounds are
present in plant tissue can vary markedly and be dependent on the plant variety,
the growth conditions and other factors. The microbial flora in the gut also play an impor-
tant role in their action and composition through bioconversion and the release of various
bioactive dietary components.
Biomarkers are a key tool for assessing nutritional status and dietary exposure to specific
substances. Markers differ considerably in their characteristics, some of them mainly
reflecting recent dietary intake and others indicating more the individual’s long-term
nutritional status, which to a large extent reflects the kinetics of metabolism and transport,
and pool sizes of the substance in question. Also, the confounding factors affecting a given
biomarker vary appreciably for different dietary components as well as in terms of the body
fluid or tissue considered suitable for sampling. The area of biomarkers has progressed
markedly with the advent of new analytical technology and the emergence of data showing
that hitherto neglected food components may have important health effects and can be
of strong interest. Nutritional biomarkers are essential for studies of the links between
dietary intake and the risk of cancer, which is a very complex field indeed in view of the
large number of cancer diseases, pathogenetic mechanisms, food components and food
preparation methods involved. The ECNIS project aims at addressing important problems
in this field and identifying knowledge gaps that exist. Particular attention is directed
at identifying dietary factors that modulate environmental and lifestyle factors related
to cancer risk. The present review focuses on two major groups of food components,
vitamins and selenium on one hand, and bioactive compounds, on the other.
Vitamins and selenium
Although the measurement of vitamins in body fluids is a classical area for nutritional
biomarker research, particular progress has been made here recently. Current research
Dietary vitamins, polyphenols, 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,
including cancer. The measurement of vitamin D metabolites presents a number
of problems including the occurrence of vitamin D in different forms, the variations
in measurement between different assays and different laboratories, epidemio-logic
variations in vitamin D status and its determinants, and the problems in defining the
appropriate threshold for vitamin D sufficiency. The determination of vitamin D status
is still a challenging task, and since metabolites of vitamin D have important biological
effects, strong research efforts in this area should be encouraged. 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.
Biomarkers of vitamins A, C and E are an important part of the diet-cancer field.
Serum retinol is still the most reliable indicator for evaluating vitamin A deficiency.
HPLC methods are those which are most sensitive, but they are not useful as yet for large
screenings. Simpler tests using fluorescence or immunological techniques have a high
level of accuracy and precision, but there is a need of developing simpler methodologies.
Concerning the functions of vitamin A, it appears to have only limited capacity as a direct
antioxidant in vivo. Vitamin C, which is water-soluble, can readily be determined
in plasma by automated colorimetric assays or by HPLC. The latter has the advantage
of possessing lower detection limits, probably higher accuracy as well as the possibility
it to determine ascorbate and dehydroascorbate simultaneously, but colorimetric assays
have a much higher throughput. The handling and storage of plasma for vitamin C deter-
mination has a strong impact on its accuracy. There is limited evidence that plasma
ascorbate is a functional antioxidant, as assessed by currently available markers of lipid
or protein oxidation in humans, and that the intake of high doses of vitamin C can be
used to decrease oxidative stress. In contrast, clear short-term pro-oxidant effects on lipid
oxidation have been observed following the infusion of high-dose vitamin C into the
bloodstream. No studies are currently available on the relationship between lipid or protein
oxidation markers and chronic disease. Regarding vitamin E, both HPLC and GC-MS
methodology have high precision and accuracy for its detection in plasma. 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. The effects that have been
postulated of high levels of vitamin E supplementation on exercise-induced lipid oxidation,
as determined by isoprostanes and by inflammatory markers, are controversial. Vitamin E
intervention has not been shown to decrease the oxidation of plasma proteins.
The antioxidant vitamins have been studied very much in relation to risk of cancer,
using oxidative DNA damage as a biomarker. Since DNA mutations are a crucial step
in carcinogenesis and elevated levels of oxidative DNA lesions have been noted in many
tumours, such damage is strongly implicated in the aetiology of cancer. Although it is
likely that severe oxidative stress is a consequence rather than a cause of the development
of many types of cancer, at present it is impossible to assess the quantitative involvement
of oxidative stress in the origin of cancer. Regarding intervention studies using anti-
oxidant supplementation, there are six investigations that have reported beneficial effects
Executive summary
7
of it on oxidative DNA damage, whereas there are 13 studies that have reported a null
effect. 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. Most studies have involved healthy
individuals, but the few well-controlled studies that have reported realistic appearing
levels of oxidative DNA damage in leucocytes isolated from oxidatively stressed subjects
do not lend much support to the notion that such a population benefits more from anti-
oxidant supplementation than a normal study population would. In the future greater
attention should also be directed at alternative chemopreventive mechanisms such as up-
regulation of DNA repair systems and the chemopreventive effects of antioxidants
on non-lymphatic tissue.
The relationship of low selenium status and selenium supplementation to cancer
disease is another important segment of focus in the diet-cancer field. Several epidemio-
logic case-control studies have indicated a protective role of selenium in helping prevent
the development of cancer. In these studies, use has been made of several biomarkers such
as the concentration of total selenium in different body fluids as well as different seleno-
proteins, primarily glutathione peroxidase. It is important to explore the possibility
of using other selenoproteins as biomarkers too. There is also a need of clarifying the me-
chanistic role of selenium in the etiology of cancer. Experimental studies have shown that
selenium compounds affect cell growth, the cell cycle, DNA repair and signal transduction.
Both organic and inorganic forms of selenium have been evaluated, methylated
selenocompounds having been found to have particularly strong chemoprotective effects.
Several large human intervention studies have indicated that selenium supplementation
can prevent the development of several different forms of cancer (prostate, lung, colon).
Further studies are underway. It is also important to determine whether there is an increa-
sed risk of certain forms of cancer after selenium supplementation. The optimal type
and dose of selenium supplements needs to also be defined.
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. Like other types of dietary chemopre-
ventive agents, flavonoids exhibit a wide range of potentially beneficial activities in terms
of cancer prevention. These are usually divided up into blocking and suppressing
activities. The blocking activities include antioxidant effects and the modulation of drug-
metabolising enzymes and multidrug resistant genes. The suppressing activities include
the inhibition of signalling pathways responsible for cell proliferation and survival,
and the induction of apoptosis, mainly through intrinsic pathways involving members
of the Bcl family, mitochondrial membrane depolarisation, release of cytochrome c and
the activation of caspases. Flavonoids can also induce cell cycle arrest by modulating key
components of cell cycle regulation, including cyclins, cyclin-dependent kinases
and inhibitors. Some recent mechanistic findings on the flavonoids apigenin, epigallo-
8
catechin gallate, genistein, resveratrol, quercetin, the chalone, xanthohumol and the
novel flavonol tricin have been summarised.
Also, the use of polyphenols as biomarkers of dietary intake has attracted
considerable interest, but their utilisation in this way is hampered by several factors. The
bioavailability of different compounds varies markedly and for many compounds
information on this point is scarce. 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 also applies to analyses of these compounds in urine. From an analytical
point of view, the measurement of polyphenols in body fluids is a difficult task.
Hydrolytic and clean-up steps are usually needed, 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.
For some compounds, such as isoflavones and lignans, the availability of immunoassays
is of great value. The more recently developed metabolomic techniques may offer new
possibilities in the future, but further study regarding this will be needed.
Olive oil is one of the foods at which special interest has been directed in the diet-cancer
field because of its containing a special mixture of agents that affect the initiation,
promotion and progression of multistage carcinogenesis. These include tocopherol and caro-
tenoid antioxidants, certain phenolic compounds (tyrosol, hydroxytyrosol, secoiridoids
and lignans), as well as squalene and β-sitosterol. These compounds have a number
of different mechanisms of action. It has also been pointed out that their efficacy
is dependent on their bioavailability. To clarify the matter, a number of recent studies
on the bioavailability of certain minor but important olive oil components (polyphenols,
lignans, squalene and β-sitosterol) are reviewed. An especially interesting matter here is that
intake of olive oil can increase the bioavailability of anticarcinogenic compounds.
The anticancerogenic properties of the glucosinolates have received a great deal
of attention, its becoming increasingly clear that it is their breakdown products that can
influence the initiation and progression of carcinogenesis. They also appear to influence
apoptotic responses to chemotherapeutic agents. A major impediment to our under-
standing of the chemopreventative mechanisms stimulated by glucosinolates is that
relatively little is known regarding the biological effects of glucosinolate breakdown
products other than the isothiocyanates and the indole-containing derivatives. It is
unclear whether the formation of thiocyanates, nitriles, cyano-epithioalkanes and
oxazolidine-2-thiones from glucosinolates, which occurs at the expense of their forming
isothiocyanates, is undesirable in terms of chemoprevention of cancer. It is also unclear
whether the activity of the epithiospecifier protein (ESP), which reduces the formation
of isothiocyanates from glucosinolates, is undesirable. In addition, relatively little
is known of the pharmacokinetic properties of glucosinolate breakdown products
in humans. Without such information, it is difficult to relate responses of cells in culture
to particular concentrations of phytochemicals to the situation in vivo. These are areas
in need of further investigation. Mammalian cells display marked dose responsiveness
to phytochemicals, at low doses of phytochemicals cytoprotective adaptive responses
Executive summary
9
being activated, whereas at higher doses cell cycle arrest and apoptosis occur. It is pre-
sently unclear how these different types of responses are coordinated by the cell and how
matters of whether adaptation, growth arrest or apoptosis is to occur are determined.
Identification of the mechanisms that control such outcomes would be very useful.
A growing number of in vitro and in vivo studies indicate that combinations of die-
tary chemopreventive agents can sometimes result in significant levels of activity
at concentrations at which any single agent is inactive. 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. The development of ideas regarding this has
also been stimulated by findings that dietary supplements of only a single compound may
have negative effects. Although our understanding of the molecular mechanisms behind
the observed combinational effects is still limited, it appears that many combinations
of complementary modes of action may be involved. In some systems, combinations
of green tea flavonoids are more active than single compounds of this sort, and green tea
flavonoids can also show increased activity when present together with other phyto-
chemicals. The synergistic effects of dietary phytochemicals need to be investigated
further. This can well contribute to cancer prevention. 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 chemo-
preventive compounds.
Many healthful diet effects are attributed to probiotic bacteria. The strongest
evidence for the anticancer effects these can have comes from animal studies, evidence
from human studies still being limited. 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.
Several possible mechanisms may explain how lactic acid bacteria can protect against
tumour development in the colon. Different strains of the bacteria may possibly target
different mechanisms. More work needs to be done to identify the specific strains and
strain characteristics responsible for particular antitumour effects and the mediating
mechanisms involved. 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, promising and as deserving of closer scrutiny.
As has been indicated here, considerable information of detailed character concerning
the mechanisms governing the effects of bioactive food compounds and of nutrients on
the cellular processes that relate to carcinogenesis has accumulated. It is a highly
challenging task to integrate this knowledge in such a way that useful hypotheses can be
formulated that ultimately can be tested in human dietary intervention trials. An
understanding of the mechanisms involved is a necessary basis for the development and
validation of new biomarkers of nutritional status. It is important here to have insight
into recent developments in analytical technology and thorough knowledge of biomarker
kinetics. Much hope is attached in this connection to the powerful techniques available
today within the field of nutritional genomics.
1. Introduction
1.1. Introductory overview and future prospects
Björn Åkesson
Biomedical Nutrition, Pure and Applied Biochemistry, Lund University
and Department of Clinical Nutrition, Lund University Hospital, Lund, Sweden
Dietary assessment methodology and the development and validation of biomarkers

of nutritional status are key scientific fields within nutritional science. They have
expanded markedly with the advent of new analytical technology and the emergence
of scientific findings showing that hitherto neglected food components may have interes-
ting and important health effects. A number of textbooks and reviews are available
within the field of nutritional biomarkers [see e.g. 1–6] dealing with the basic theories
and methodology, their physiological basis and the use of biomarkers in studies of dif-
ferent types. Various properties of biomarkers are summarised in Table 1.1.
Table 1.1. Some major issues regarding nutritional biomarkers
Markers of dietary intake vs. markers of nutritional status

Markers of differing kinetics (response times)
Markers of nutrients vs. 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. This is a very
complex field in view of the large number of different types of cancer, the pathogenetic
mechanisms involved, that role that various food components and food preparation
methods play in connection with this. The ECNIS project aims at addressing important
problems in this field and identifying various knowledge gaps. A main concern is to
identify dietary factors that modulate environmental and lifestyle factors that can affect
risk of cancer and which in the long run can also provide support for the development
of functional foods.
The 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 further aim is to examine and explore the mechanisms responsible for
the action for different promising anticarcinogenic compounds. A brief overview of food
components that appear to have an anticarcinogenic effect is provided in Appendix 1.2.
Björn Åkesson
12
Dietary components as biomarkers

Many biomarkers involve the measurement of a nutrient or some other dietary component
in the blood, plasma, urine or hair. 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, are in routine daily
use in clinical laboratories, whereas others are performed only in a few specialised research
laboratories. Biomarkers show temporal variation, 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. Various diet-related compounds in the plasma also differ consi-
derably in turnover times. The amount of a substance occurring in the erythrocytes, often
reflects much more the long-term intake if it than the plasma level does, since the
erythrocyte cells have a lifetime of about 120 days [3]. A large number of confounding
factors such as hormones, the gut flora, diurnal variations, interactions between dietary
or endogenous components or xenobiotics, variations in metabolism or in excretory
pathways, and inflammatory and other diseases can influence the levels of different
biomarkers [3]. The level of a biomarker often varies as well with age, gender and gene
polymorphism (as outlined below).
Functional biomarkers of nutrients
Since many nutrients are essential for the activity of enzymes (as coenzymes for example)
the levels of activity involved are sometimes used as nutritional biomarkers such as
in activation assays for thiamine and riboflavin [3]. Also, trace elements are coupled
to the levels of enzyme activities, for example zinc and copper to superoxide dismutase
activity and selenium to the activity of glutathione peroxidase and other selenoproteins.
As reviewed in this volume there is a strong link between selenium content and gluta-
thione peroxidase activity since it is covalently bound in the peptide chain. Also proteins
with a transport function as well as other functions are used as biomarkers, such as
the retinol-binding protein and ceruloplasmin. In addition, different nutrient-dependent
physiological tests can be employed [2].
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, the main function
of some markers being that of serving as indicators of malnutrition, and they may not
respond to overexposure of nutrients at all. 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. [3]. 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.
Introductory overview and future prospects
13
Although several of the biomarkers mentioned here are widely used, 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. For this reason, methods
for measuring nutrients or nutrient-dependent variables in cells in the blood (mainly
leucocytes), buccal mucosa, urine, faeces, adipose tissue or biopies from other organs have
been developed. Some of these samplings require much skill and effort and can only
be used in small studies. Their use may increase, however, if the analytical methodology
improves so that smaller amounts of sample suffice.
Also, 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, as exemplified in the present volume by Linseisen and his colleagues in con-
nection with vitamin D and polyphenols. There are particularly good reasons for using
the 25-hydroxy vitamin D as a biomarker in extended studies in the future. The two
contributions mentioned also illustrate the difficulty in defining valid cut-off points.
The assessment of dietary intake in epidemiological studies has been a major use
of biochemical markers of nutritional status. An overview of this matter is provided
in [7]. This topic is only taken up briefly in the present volume, although epidemiological
findings on the association between the selenium level and cancer are reviewed
by Gromadziƒska et al. Biomarkers in studies of that type have been used either alone,
together with an assessment of dietary intake by a food frequency questionnaire
or by some other methods, or for the calibration of the dietary assessment methods [8].
Although the use of the biomarker concept for these purposes is very attractive, it should
be borne in mind that there are very few (if any) “ideal” biomarkers that have been
validated for studies of different types. A number of statistical methods for the correction
and evaluation of such data are available [8].
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.
As summarised by Hunter [9], information on the interactions between dietary intake
and gene polymorphisms “can serve to 1) define susceptible subpopulations, thus
strengthening dietary associations; 2) help establish causality of food and nutrient
associations in epidemiology studies; 3) aid in distinguishing causal components of complex
dietary mixtures; and 4) eventually provide the basis for gene-based screening tests”. Folate
has served as a prototype here for demonstrating how genetic make-up can influence the
nutrient status of an individual. 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]. 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 [11,12]. These matters are outside the scope of the present review.
Björn Åkesson
14
Biomarkers and response to dietary supplements

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

Emerging “omics” technologies — transcriptomics, proteomics, genomics and meta-
bolomics — 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 cancer-
protective effects of different nutrients. The metabolomics approach involves the
multiparametric measurement of metabolites and provides a comprehensive account
of the metabolic profile of different biological systems, such as various body fluids
[20,21]. Genomics, finally, includes techniques for the global genotypic characterization
of individuals with the aim of discovering polymorphisms associated with
susceptibility [9]. SNP arrays are one powerful genomic tool enabling whole-genome
association studies of up to 500 000 SNPs (single nucleotide polymorphisms) to be
carried out in a single run. The “omics” technologies as a whole will surely play a key
role in future nutritional research regarding the protective role of diet in connection
with cancer. The use of results obtained by various of these techniques involves
complex ethical considerations [22].
Concluding remarks
The contributions the present volume contains clearly indicate that a deeper
understanding is needed of the mechanisms underlying the protective effects that
various food components can have in preventing or counteracting carcinogenesis.
Considering the links to the susceptibility the individual has inherited is becoming
increasingly important too. At the same time it is often difficult to single out the
mediating components in the protective food patterns emerging from epidemiological
studies diet-cancer relationships. It is important, therefore, to identify the compounds
that are most active in different experimental systems, and to study variations in their
activity when they are ingested as a part of different foods, together with the
combinatorial effects of phytochemicals and other food components. There is also
much uncertainty regarding the relevance of observed in vitro or short-term effects in
humans in relation to the long-term effects documented in chemopreventative human
studies [7,23].
The newly obtained findings in the molecular nutrition area can be used to make
more rapid progress in the development and validation of biomarkers. Such biomarkers
should reflect not only exposure to food components but also damage to biological
components (DNA, protein and lipids) and other targets of the diet-carcinogenesis
interaction [18].
Björn Åkesson
16
References
1. Hunter D. Biochemical indicators of dietary intake. In: Willett W, editor. Nutritional epidemio-
logy. 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 preven-
tion 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.
17
21. Brigelius-Flohé R, Joost H-G, editors. Nutritional genomics. Impact on health and disease.
Weinheim: Wiley-VCH; 2006.
22. Görman U. Ethical issues raised by personalized nutrition based on genetic information. Genes
Nutr 2006;1:13–22.
23. Collins AR, Ferguson LR. Nutrition and carcinogenesis. Mutat Res 2004;551:1–8.
Per Mercke
18
Appendix 1.2. Overview of possible anticarcinogenic

food components
Per Mercke
Biomedical Nutrition, Lund University, Lund, Sweden
There is a vast range of dietary compounds proclaimed to have anti-carcinogenic proper-

ties as investigated by the scientific community. These protective compounds are found
basically in all types of food, but plant-based foods appear to be their richest source.
Different plant families harbor different mixtures of compounds of this sort. Plant tissues
can also vary enormously in the store of such compounds, depending on the plant variety
and the growth conditions, for example. 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.
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,
a list of putative anticarcinogenic food components has been generated (Table 1.3.).
This information was collected with use of a PubMed search conducted in 2005.
Table 1.2. A step-wise search in the database PubMed for reviews linking biomarkers of nutrition and diet
to cancer
PubMed search No. of refs
Biomarker 305256
Biomarker AND diet 3351
Biomarker AND (diet OR nutrition) 4173
Biomarker AND (diet OR nutrition) AND cancer 869
Biomarker AND (diet OR nutrition) AND cancer /reviews/ 181
Biomarker AND (diet OR nutrition) AND cancer /reviews/last 10 years 146
The forty-seven reviews listed below were selected from among the 146 reviews
arrived at last in this search. The criteria used for the selection were their relevance
as adjudged from their title, emphasis on recent reviews, and avoiding of similar articles
by any given author.
Overview of possible anticarcinogenic food components
19
Table 1.3. An overview of putative anticarcinogenic food components as assembled

from 33 out of the 47 selected reviews that were selected
Class of compound Example of bioactive

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

from 33 out of the 47 selected reviews that were selected — cont.

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

from 33 out of the 47 selected reviews that were selected — cont.

Methylxanthines Caffeine Tea, coffee, Manson [18]

Theophylline cola, cacao
Theobromine
Monoterpenes Limonene Citrus fruits (peel) Ferguson [3], Kelloff et al. [16], Manson [18],
Perillyl alcohol Rafter et al. [8]
Geraniol
Menthol
Carvone
Other non-flavonoid Hydroxytyrosol Olive oil Adlercreutz [31], Bartsch et al. [1],
phenolics Curcumin Turmeric Branca et al. [2], Wiseman [33]
Resveratrol Grapes, strawberries,
raspberries, black-berries,
walnuts, pecans.
Lignans Cereals, olive oil
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sis: potential role of lipid peroxidation, dietary fat and antioxidants. Biol Chem 2002;383:915–21.
2. Branca F, Hanley AB, Pool-Zobel B, Verhagen H. Biomarkers in disease and health. Br J Nutr 2001;86
Suppl 1:S55–S92.
3. Ferguson LR. Prospects for cancer prevention. Mutat Res 1999;428:329–38.
4. Greenwald P. Cancer risk factors for selecting cohorts for large-scale chemoprevention trials. J Cell
Biochem 1996;25 Suppl:29–36.
5. Turini ME, DuBois RN. Primary prevention: phytoprevention and chemoprevention of colorectal
cancer. Hematol Oncol Clin North Am 2002;16:811–40.
6. Gill CI, Rowland IR. Diet and cancer: assessing the risk. Br J Nutr 2002;88 Suppl 1:S73–87.
7. Manson MM, Farmer PB, Gescher A, Steward WP. Innovative agents in cancer prevention. Recent
Results Cancer Res 2005;166:257–75.
8. 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.
9. Sanderson P, Johnson IT, Mathers JC, Powers HJ, Downes CS, McGlynn AP, et al. Emerging diet-
related surrogate end points for colorectal cancer: UK Food Standards Agency diet and colonic health
workshop report. Br J Nutr 2004;91:315–23.
10. Milner JA. Incorporating basic nutrition science into health interventions for cancer prevention.
J Nutr 2003;133 Suppl 1:3820S–6S.
11. Rafter JJ. Scientific basis of biomarkers and benefits of functional foods for reduction of disease risk:
cancer. Br J Nutr 2002;88 Suppl 2:S219–24.
Per Mercke
22
12. Vlastos AT, Schottenfeld D, Follen M. Biomarkers and their use in cervical cancer
chemoprevention. Crit Rev Oncol Hematol 2003;46:261–73.
13. Bowen P, Chen L, Stacewicz-Sapuntzakis M, Duncan C, Sharifi R, Ghosh L, et al. Tomato sauce
supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers
of carcinogenesis. Exp Biol Med (Maywood) 2002;227:886–93.
14. 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:S5–35.
15. Granado F, Olmedilla B, Blanco I. Nutritional and clinical relevance of lutein in human health.
Br J Nutr 2003;90:487–502.
16. Kelloff GJ, Crowell JA, Steele VE, Lubet RA, Malone WA, Boone CW, et al. Progress in cancer
chemoprevention: development of diet-derived chemopreventive agents. J Nutr 2000;130
Suppl:467S–71S.
17. Loft S, Poulsen HE. Cancer risk and oxidative DNA damage in man. J Mol Med
1996;74:297–312.
18. Manson MM. Cancer prevention — the potential for diet to modulate molecular signalling.
Trends Mol Med 2003;9:11–8.
19. Maruvada P, Srivastava S. Biomarkers for cancer diagnosis: implications for nutritional research.
J Nutr 2004;134 Suppl:1640S–5S.
20. Mayne ST. Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxida-
tive stress status in epidemiologic research. J Nutr 2003;133 Suppl 3:933S–40S.
21. Seifried HE, McDonald SS, Anderson DE, Greenwald P, Milner JA. The antioxidant conundrum
in cancer. Cancer Res 2003;63:4295–8.
22. Sharma RA, Farmer PB. Biological relevance of adduct detection to the chemoprevention
of cancer. Clin Cancer Res 2004;10:4901–12.
23. Wild CP, Andersson C, O'Brien NM, Wilson L, Woods JA. A critical evaluation of the application
of biomarkers in epidemiological studies on diet and health. Br J Nutr 2001;86 Suppl 1:S37–53.
24. Wagner KH, Kamal-Eldin A, Elmadfa I. Gamma-tocopherol — an underestimated vitamin?
Ann Nutr Metab 2004;48:169–88.
25. Konety BR, Getzenberg RH. Vitamin D and prostate cancer. Urol Clin North Am
2002;29:95–106.
26. Mason JB. Biomarkers of nutrient exposure and status in one-carbon (methyl) metabolism.
J Nutr 2003;133 Suppl 3:941S–7S.
27. Bianchini F, Vainio H. Isothiocyanates in cancer prevention. Drug Metab Rev 2004;36:655–67.
28. Zhang Y. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism
of action. Mutat Res 2004;555:173–90.
29. Duthie G, Crozier A. Plant-derived phenolic antioxidants. Curr Opin Clin Nutr Metab Care
2000;3:447–51.
30. Saleem M, Adhami VM, Siddiqui IA, Mukhtar H. Tea beverage in chemoprevention of prostate
cancer: a mini-review. Nutr Cancer 2003;47:13–23.
31. Adlercreutz H. Phytoestrogens and breast cancer. J Steroid Biochem Mol Biol 2002;83:113–8.
32. Atkinson C, Bingham SA. Mammographic breast density as a biomarker of effects of isoflavones
on the female breast. Breast Cancer Res. 2002;4:1–4.
23
33. Wiseman H. The therapeutic potential of phytoestrogens. Expert Opin Investig Drugs
2000;9:1829–40.
34. Ocke MC, Kaaks RJ. 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. Am J Clin Nutr 1997;65 Suppl 4:1240S–5S.
35. Vineis P. Biomarkers, low-dose carcinogenesis and dietary exposures. Eur J Cancer Prev
1997;6:147–51.
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toxic effects of urban air pollution. Mutat Res 1999;428:91–8.
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marker concept. Nutr Rev 1999;57:104–13.
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2001;55:46–67.
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41. Rivlin RS. Nutrition and cancer prevention: new insights into the role of phytochemicals.
Future directions. Adv Exp Med Biol 2001;492:255–62.
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43. Moyad MA. Lifestyle/dietary supplement partial androgen suppression and/or estrogen
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2. Vitamins and selenium
2.1. Biomarkers of exposure to and effects of vitamins A, C and E
Lars O. Dragsted
University of Copenhagen, Copenhagen, Denmark
Since antioxidant vitamins can affect an organism’s capacity for defence against
reactive oxygen species, biological markers of the dietary exposure to these vitamins
is of importance. 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. This chapter is concerned with
such markers, except for markers of DNA damage, which are dealt with elsewhere
in this volume.
Vitamin A
Biomarkers for vitamin A in body fluids

The vitamin A (all-trans-retinol and its esters) level was originally determined by bio-
efficiency assay, a technique that was later superseded by various chromatographic and
fluorescent techniques. Due to worldwide concern for vitamin A deficiency (VAD),
the development of fast and simple methods for the determination of vitamin A status
has long been given a high priority. Direct fluorescence methods for assessing the retinol
level in plasma or in dried blood are feasible because of the high intensity of retinol
fluorescence when it is bound to its transporter, the retinol binding protein (RBP) [1,2].
With the advent of high performance liquid chromatography (HPLC), these techniques
took over and today retinol can be determined in serum routinely by direct- [3,4]
or reversed-phase [5–7] liquid chromatography . The reversed-phase techniques are faster
and smaller sample volumes suffice but they are generally unable to discriminate between
the various isomers of vitamin A to the same extent as the direct-phase methods can,
although reversed-phase methods able to separate certain of the retinol isomers have been
published [8]. The direct-phase methods can also typically measure a large number
of other lipid-soluble vitamin isomers in the same run, such as pro-vitamin A carotenoids,
xanthophylls, tocopherols and tocotrienols, menadione and phylloquinones. The struc-
ture of vitamin A and pro-vitamin A carotenoids is shown in Figure 2.1. 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]. The possibility of using dried blood spots, which
is advantageous from a collection standpoint, has also been demonstrated [1].
Lars O. Dragsted
26
A comparison of the various tests in terms of price, speed of performance and coefficient
of intraindividual variability is shown in Table 2.1. Although the accuracy for each
of the methods taken up in Table 2.1. is good (less than 5% deviation from the stan-
dards) 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
Cost/sample Speed
Test method CV% (relative) (samples/day) Reference
Reversed-phase HPLC 4 20 25 [7]

Direct-phase HPLC 4 30a 20 [4]
a
Fluorescence <10 2a 50–100 [1]
ELISA (RBP) 9 1 150 [9]
a
Estimates from the author’s lab. This may change with new faster LC techniques.
Vitamins and selenium: Biomarkers of exposure to and effects of vitamins A, C and E
27
Biomarkers of vitamin A related effects in the eye

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

Indicator (cutoff) its level of importance
mild moderate severe
Functional tests
Night blindness (age-specific) > 0 to < 1% ≥ 1% to < 5% ≥ 5%
Biochemical markers
Serum retinol (≤ 0.70 Ķmol/l) ≥ 2 to < 10% ≥ 10% to < 20% ≥ 20%
Breast-milk retinal (≤ 1.05 Ķmol/l) < 10% ≥ 10 to < 25% ≥ 25%
Histological markers
Abnormal conjunctival impression cytology < 20% ≥ 20% to < 40% ≥ 40%
* Common biological indicators of subclinical VAD in children 6–71 mo of age. A public health problem is considered to exist when the prevalence
criteria of at least two of the above indicators of VA status are met (adapted from [1,12].
The dark adaptation tests measure the time needed to adapt to a defined level
of limited illumination. A fast adaptation procedure involving the ability to discriminate
between red and blue objects for field and for screening studies has been described [16].
When the eye shifts from cone-mediated day vision to rod-mediated night vision, the
Purkinje shift occurs, the retinal peak light sensitivity shifting from red towards blue, blue
objects being perceived then as lighter shades of grey than for red objects. Patients have
to be taught use of the test, which must be repeated afterwards several times. In some
studies the test results have been found to be more closely related to vitamin A intake
by dietary assessment than to the plasma retinol level [13].
Lars O. Dragsted
28
Biomarkers of oxidative stress after supplementation with vitamin A

There seem to be no studies reporting on markers of oxidative stress or oxidative status
following intervention with use of increased doses of vitamin A. Neither retinol nor
beta-carotene supplements to blood samples in vitro have been found able to affect a num-
ber of markers for antioxidant stability of the plasma and erythrocytes [17], which
indicates that this vitamin may have a limited capacity to act as an antioxidant in vivo.
Conclusions
Serum retinol is still the most reliable indicator of vitamin A deficiency. HPLC methods
are the most sensitive, but they are not useful for large screenings or for field studies
in poor areas of the world where deficiency is a common problem. Although simpler tests
using fluorescence, as well as immunological techniques possessing good accuracy and
high precision exist, there is still a need of methodology which is simpler, faster and
cheaper yet and requiring no complicated sample treatment or use of complex equipment.
Vitamin A appears to have only limited capacity as a direct antioxidant in vivo.
Vitamin C
Biomarkers of vitamin C in body fluids

Vitamin C (ascorbate and dihydroascorbate, Figure 2.2.) levels have been determined in
plasma, serum, dialysates and other body fluids by colorimetric and fluorimetric
techniques, by enzymatically based assays and by HPLC with or without post-column
derivatisation. Since ascorbic acid is easily oxidised to dehydroascorbate, which can
subsequently be degraded to diketogulonic acid, initial treatment by stabilising acids such
as metaphosphoric and perchloric acids has to be performed quickly after isolation of
a sample for analysis. The effects of the anticoagulants used during sample collection has
been compared in one study, heparin being found to result in only a minimal loss, EDTA
in contrast giving rise to a significant loss of vitamin C within a 30 min period. Also,
oxalate and citrate were found to be less efficient in stabilizing ascorbate than other
anticoagulants were [18]. Storage time of the sample and storage conditions are
important factors determining the stability of vitamin C. In one early study, the
concentrations of ascorbate and dehydroascorbate were found to be unaffected in samples
stored in the laboratory at a temperature of 12°C for up to 6 hours [19], but in other
studies considerable time- and temperature-dependent losses were found already from the
first hour of storage onwards even after optimization of the collection conditions [18].
In another study the storage time and temperature were found to have no effect on loss
of vitamin C during 2–14 day storage at either –25°C or –75°C, but a 3.5% loss due to
freezing was observed [20]. In a third study, pre-treatment with metaphosphoric acid was
compared with treatment by dithiotreitol, a commonly used laboratory antioxidant. The
latter performed slightly better than the former since no loss of vitamin C was evident
after storage at –80°C for 6 years, whereas the standard procedure involving the addition
of metaphosphoric acid led to a small but significant mean loss of < 1% per year [21].
29
However, since treatment with dithiotreitol is known to reduce dehydroascorbate to

ascorbate, this procedure cannot be recommended if both compounds are to be deter-
mined separately. The normal range of plasma concentrations of dehydroascorbate is
controversial and the observation of this compound in plasma may be a result of metal-
catalysed oxidation following acidification [22]. If dehydroascorbate is physiologically
present, its true concentration seems to be very low, around 0.1% of the total plasma
vitamin C in non-smokers when the sample has been handled carefully, and about 1.8%
under the same conditions in the case of smokers [23], possibly reflecting higher leakage
of haem in this group. There was a significant increase in the concentration
of dehydroascorbate over time at low total vitamin C concentrations [23].
Fig. 2.2. Structures of ascorbate, the ascorbyl radical and dihydroascorbate, together constituting vitamin C.
The colorimetric and fluorometric methods are generally based on redox-reactions,

with ascorbate and dihydroascorbate leading to the formation of a chromophore or
a fluorophore, which can be photometrically measured by use of manual or automated
equipment. Most of these methods are quite unspecific [24], but a few of them use assay
blanks produced by adding ascorbate oxidase to the sample, creating greater sensitivity
with retention of speed. Some of these methodologies are very fast, allowing high sample
handling rates to be achieved through automation [25]. Results in determining plasma
ascorbate in this way correlate well with those obtained by use of chromatographic
methods [18]. Dehydroascorbate is not readily determined by use of this approach.
The HPLC methods for detecting plasma ascorbate electrochemically give results
similar to those using UV detection [26]. Postcolumn derivatization can be used to reduce
dihydroascorbate so that it can be determined by use of an electrochemical detector; the
stereoisomer of ascorbate, erythorbate, can be determined simultaneously [25,27–28].
A method for the simultaneous detection of ascorbate and uric acid by means of capillary
zone electrophoresis (CZE) has also been described [29]. Recovery is better than 98% with
Lars O. Dragsted
30
use of the HPLC and CZE methods and good linearity is obtained even at low ascorbate
concentrations.
In interlaboratory comparisons, quite disparate results have been obtained with use
of these techniques. In a European study of laboratories carrying out population
surveillance, a 13–20% interlaboratory variation was found using plasma samples in the
‘normal’ range of 36–94 µmol/l in a second round after corrections had been instituted
at several laboratories [30]. In another study an interlaboratory difference of about 15%
was observed, whereas the intralaboratory variation was about 2 µmol/l, irrespective
of the concentration, which led to relatively larger relative errors being registered at low
concentrations [21]. The performance of different methods for the measurement
of vitamin C in plasma is summarized in Table 2.3.
Table 2.3. Typical performance of different methods for determination of plasma vitamin C
CV% AA CV% DHAA Limit Speed

Test method interday of detection (samples/day] Reference
interday
Reversed phase HPLC < 4% < 6% 0.1 mg/l 40–50 [36]
Capillary electrophoresis 1.3% ND 0.5 mg/l 40–50 [37]
Automated colorimetry < 5% ND 3 mg/l 500 [38,39]
ND — not determined.
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. The assays employed to this end here are the following: plasma thiobarbituric
acid reactive compounds (TBARS), ex vivo LDL oxidation, antioxidant capacity markers,
plasma lipid hydroperoxides, and plasma or urinary isoprostanes. Only randomised study
designs are included in this review.
The most commonly used marker is determination of TBARS, with or without
calibration, to detect malondialdehyde. The method is based on the liberation of alde-
hydes from amino groups by acid or alkaline hydrolysis followed by a colour reaction
involving use of thiobarbituric acid. The product can be determined spectrophotometri-
cally, either directly or online, following HPLC separation. This marker is highly contro-
versial since it is variable both within and between laboratories, since it may partially
measure aldehydes deriving from endogenous metabolism, and since there is no generally
accepted assay procedure [31]. These flaws have caused some journals to generally regard
the method as being invalid as a lipid oxidation biomarker [32]. The interday coefficient
of variation (CV) for TBARS with use of HPLC is in the order of 10–20 %, depending
on the method employed for hydrolysis, but as already mentioned this relatively simple
method has an odd tendency of sometimes giving spurious results and of varying from
one analytical series to another, also within the same laboratory.
31
In a randomised 2-months intervention study of 59 healthy smoking males MDA was

found to increase significantly following daily doses of 250 mg ascorbate combined with
200 IU vitamin E, 30 mg beta-carotene and 100 µg organic selenium, given in a normal
formulation, but MDA to be unaffected by a slow-release formulation as compared with
placebo treatment [33]. In another, randomised double-blind crossover study the effect
of vitamin C supplementation (six weeks, 250 mg/day) was determined in 20 subjects
each showing normal (67 µmol/l) or below average (32 µmol/l) plasma vitamin C con-
centration at baseline. No differences between groups in plasma malondialdehyde
concentrations were observed either before or after supplementation [34]. In another
counterbalanced design, 25 males were exposed to vitamin C (500 or 1000 mg/d) and/or
to exercise. No effect of either treatment on plasma MDA was observed [35]. In a study
comparing 8 smoking women with 8 controls during a 14-day period in which 1 g ascor-
bate was administered daily plasma TBARS was found to not be affected [36]. In a larger
study involving 56 smokers, the intake of 500 mg/d of vitamin C was found not to affect
MDA as determined by HPLC [37]. In a third study of this sort, giving a combination
of vitamin C (272 mg/d) and vitamin E (800 IU/d) compared to placebo was found to not
affect plasma MDA in 77 smokers treated for 90 d [38]. Oxidative stress in 10 volunteers
as determined by increased plasma MDA induced by infusion of free fatty acids was also
found to be unaffected by high-dosage vitamin C infusion [39]. Infusion of large doses
of vitamin C (5g) in combination with EDTA treatment resulted initially in a marked
increase in plasma MDA but in an overall decrease in this parameter after 16 repeated
sessions [40]. Oxidative stress induced by Zn deficiency was found to respond
to 250 mg/d vitamin C, given for 3 months, as compared with placebo treatment,
by a decrease in plasma MDA concentration [41]. 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, acute doses.
Another controversial marker used by many laboratories is the ex vivo LDL oxidation
assay, in which isolated LDL is exposed to copper chloride or to a semistable radical such
as AAPH, the oxidative formation of conjugated dienes being followed spectrophoto-
metrically at 234 nm [42]. The lag-time to oxidation and/or the slope of the oxidation
curve are used as end points. The method is disputed because the outcome depends
on the antioxidants present in the LDL and these may be lost during LDL isolation.
A faster method applicable directly to a plasma or serum sample overcomes this problem
by using a peroxide-sensitive fluorescent probe with high affinity for LDL [43]. The inter-
day CV for this latter assay is less than 10%.
In a group of 48 middle-aged male and female participants in a 36-month intervention
study receiving 500mg/d of either vitamin C, vitamin C plus 182 mg/d dl-α-tocopherol,
182 mg/d dl-α-tocopherol alone, or a placebo in a parallel design, no effect was observed
at 12 or at 36 months in the vitamin C group in terms of susceptibility of isolated LDL
or VLDL to oxidation ex vivo. In addition, no change in whole plasma ex vivo oxidation
was observed in this group [44]. In a smaller parallel study of vitamin C supplementation
(1 g/d) versus placebo, in which 19 smokers participated for 4 weeks following a 2-week
Lars O. Dragsted
32
ascorbate depletion period (≤ 30 mg/d), there was a significant increase in ex vivo LDL
oxidation lagtime in the vitamin-supplemented group [45]. In a subsequent study
of 30 young smokers, no effect was observed after 8 weeks supplementation by 1 g/d
of vitamin C [46]. 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), or placebo, decreased LDL oxidation or antibodies to oxidised
LDL [47]. A borderline effect on LDL oxidation was observed in a similar study with
only 18 participants after a shorter period of 12 weeks [48]. No effect on LDL oxidation
lag-times of 500 mg/d vitamin C supplementation for 4 weeks as compared with placebo
was observed in 30 type II diabetics [49]. In a study without a control group, an increase
in ex vivo LDL oxidation lag-time during a 12 week-period was observed in 20 volunteers
receiving 260 mg vitamin C in combination with 14 mg iron/d. In another group,
receiving only 60 mg vitamin C plus iron per day, no such increase was observed despite
changes in plasma ascorbate [50]. Supplementation by 1 g/d ascorbate for 4 weeks
in 11 healthy volunteers failed to change the plasma LDL oxidation lag-time as compared
with 9 controls [51]. Overall there seems to be no consistent effect of vitamin C
supplementation on ex vivo LDL oxidation kinetics.
The oxidizability of LDL is inherently an antioxidant capacity assay for this particular
compartment. A variety of antioxidant capacity markers exist for other blood compart-
ments, especially for whole plasma. They are all ex vivo oxidation systems composed
of some oxidising system and some relatively simple marker of plasma oxidation, usually
one leading to a change in visual or UV absorption of the test matrix [52–55]. There are
important differences between the methods which call for caution when they are
interpreted [56]. When applied to plasma samples some authors accordingly report poor
correlation between methods [57] whereas others observed a high degree of correlation
between some of the most commonly applied methods, the ferric reducing ability
of plasma (FRAP) assay and the trolox equivalent antioxidant capacity (TEAC) assay
which also correlated with ex vivo LDL oxidation [58,59]. These methods generally have
interday CVs of less than 10% for repeated measurements of the same sample, the size
of the CVs depending on the exact wavelength used for determining the absorbance,
the availability of a photometer with exact filters or one equipped with a narrow grid
being required.
The infusion of 1000 mg ascorbate for a 1 h period in ten healthy volunteers was
found to result in an increased antioxidant capacity as measured repeatedly by two
different methods during both the infusion period and the hour following this [60].
Marked increases in antioxidant capacity in the plasma of elderly women during
the 4 h period after a dose of 1250 mg ascorbate was given were also observed using three
different antioxidant capacity measures [61]. However, only a limited response of this
sort was observed with use of the FRAP assay during the 24-hour period following a single
dose of 500 mg ascorbate with or without 400 IU vitamin E [62]. At the same time,
in a cross-over intervention study involving 48 non-smoking men and women, in which
0 mg, 60 mg or 6 g of vitamin C was administered daily during 14 day-periods separated
33
by 6 weeks wash-out periods, the plasma antioxidant capacity of these persons was found
to be significantly affected [63]. In a 14 d parallel study of 16 women who smoked, half
of them treated with 1 g ascorbate daily and half of them given placebo, no effect on the
antioxidant capacity of the plasma could be shown [36]. Neither was the plasma TEAC
found to be affected by a 150 mg/d vitamin C supplement in a 2-week study involving
18 volunteers [64]. No effect of antioxidants on TEAC could be observed either in 39 lu-
pus erythromatosis patients randomised to being given 500 mg vitamin C together
with 800 IU vitamin E a day or a placebo for a 12-week period [65]. In a group
of 48 middle-aged male and female participants in a 36-month intervention study
in which 500 mg/d of vitamin C, vitamin C plus 182 mg/d dl-α-tocopherol, 182 mg/d
dl-α-tocopherol alone, or placebo were given daily in a parallel design, no effect in either
of the vitamin C groups on the plasma antioxidant capacity as determined by the TRAP
assay was observed at either 12 or 36 months [44]. At the same time, the antioxidant
capacity in 6 moderately trained males was found to be significantly affected, following
2.5 h of strenuous exercise stress, by the intake of a drink containing vitamin C
(0.15%, approx. 200 mg ascorbate) [66]. In contrast, FRAP was not found to be affected
by 1h of hard exercise following the daily administration of 600 mg vitamin C
in combination with a range of other micronutrients for a 7-day period [67]. In these
various studies, the effect of vitamin C on antioxidant capacity measures could
not always be explained simply by an increase in the plasma ascorbate concentration.
It appears that, although vitamin C may increase the antioxidant capacity in normal,
healthy individuals, the effect is more evident short- term and may be partially be due
to indirect actions of unknown character.
Although plasma lipid hydroperoxides can be determined individually by HPLC
together with electrochemical detection [68,69] or collectively by means of hydro-
peroxide-sensitive photometric assays [70–72], in most published papers the determi-
nation of ‘lipid hydroperoxides’ is a euphemism for TBARS. The effect of ascorbate
supplementation on the plasma lipid hydroperoxide concentration in humans under
normal conditions has not been frequently reported. The formation of lipid
hydroperoxides in LDL was not found to be affected by a 150 mg/d vitamin C supplement
in a 2-week randomised study involving 18 volunteers [64]. The effect of an ascorbate
supplement in reducing the plasma hydroperoxide level following various conditions
of oxidative stress showed a significant effect on this marker. A 30% reduction in exercise-
induced total lipid hydroperoxides was observed after acute supplementation of vita-
min C [73]. Also vitamin C supplementation in conjunction with biweekly apheresis
for 6 months in dyslipidemic and uremic patients markedly increased the efficiency
of such treatment in reducing the plasma phosphatidylcholine hydroperoxide level as
determined by HPLC [74]. Thus, Vitamin C seems to affect lipid hydroperoxide
formation in some oxidative stress conditions.
The least disputed lipid oxidation methods are the isoprostane assays. The deter-
mination of plasma isoprostanes can be performed by gas chromatography/mass
spectrometry (GC-MS) using electron capture negative ionization (ECNI) or negative ion
Lars O. Dragsted
34
chemical ionization detection (NCI). The compounds need to be extracted from the
sample and derivatized. The extraction has not always been straightforward and has
involved multiple chromatographic steps, including thin layer chromatography, and has
led to a poor overall recovery [75]. Improvements have included a combination of solid-
phase extraction cartridges and HPLC [76,77], two sequential solid-phase extractions [78]
and most recently a single anion exchange cartridge [79]. The derivatization of the com-
pounds is most often done by use of pentafluorobenzyl bromide followed by a silylation
step employing bis-(trimethylsilyl) trifluoroacetamide [79]. The overall extraction
efficiency is now around 70%, the interday analytical CV% varying from less than 1%
to 5–8%, depending on extraction procedures. Since the interindividual variation is much
larger, this method has considerable power for detecting the factors causing biologi-
cal variation. An immunological method for the determination of 8-isoprostane F2α
having 5–15% analytical variation also exists [80].
The formation of isoprostanes in plasma was found to not be affected by 150 mg/d
vitamin C supplementation in a 2-week study involving 18 volunteers [64]. Using
the chromatographic method on plasma samples, no change was observed after
a daily dose of 272 mg vitamin C in combination with vitamin E (31 mg/d) and folate
(400 µg/d) for a 90 day period in elderly male smokers and non-smokers [38]. A dose-
response study of hospitalised women in which vitamin C (30–2500 mg/d) was
administered to them for 186 days following a short ascorbate-depletion period, gave
no evidence of change in the levels of isoprostanes in the urine or plasma [81]. In a study
of 30 coronary artery disease patients limited evidence was obtained after 6 months that
random assignment to vitamin C (1 g/d) together with vitamin E (800 IU/d) versus
placebo led to a decrease in plasma isoprostanes [47]. No studies of the effect of ascorbate
supplementation on oxidative stress-induced isoprostanes have been reported.
The performance of plasma lipid oxidation markers and the effects on them of vitamin C
being administered is summarized in Tables 2.4. and 2.5. Overall, the effect of vita-
min 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
Normal Speeda
Test method Analytical CV% variation CV% (samples/day) Reference
TBARS (HPLC) 15—20 25 50 [83]

LDL ex vivo oxidation 5—10 20 10/50 [42,43]
Antioxidant capacity <10 20—25 >100 [55]
Lipid peroxides <10 60—65 20 [72]
8-Isoprostane F2α EIA 5—15 20—50 50—100 [80]
Isoprostanes GC-MS <1—6 50 50 [78,79]
a
Estimates in the author’s laboratory with use of standard semiautomated equipment.
35
Table 2.5. Summary of effects of vitamin C on plasma lipid oxidation markers
LDL oxidation Antioxidant Lipid

Treatment MDA ex vivo capacity hydroperoxides Isoprostanes
Vitamin C — — +/— — —
Stress + vitamin C — — +/— + NR
NR — not reported.
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 micro-
environment 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.
Lars O. Dragsted
36
To assess oxidative stress by use of the simplest version of this assay, 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. Vitamin C was found to decrease
the exercise-induced increase in protein carbonyls in a dose-dependent manner [83]. Using
the same method for protein carbonyls, 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. No effect of the supplement on protein carbonyls was observed [38].
In a very small study, involving seven divers, no effect on the levels of plasma protein
carbonyls as a results of diving apnea or of a 1 g/d supplement of vitamin C for a week
preceeding a diving experiment was observed [84].
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. The level of protein carbonyls in the immunoglobulins was found to
decrease after both 10 and 15 weeks of supplementation, whereas no change in the total
plasma sulfhydryls was detected [85]. The decrease was confined to individuals with
a suboptimal intake of vitamin C.
A third method employed for assessment of protein oxidation takes advantage of the
reaction of protein aldehydes with fluorescein and reduction of the product by cyano-
borohydride to form a stable adduct. Following protein isolation by a rapid size-
exclusion chromatographic step and complete acid hydrolysis, the fluoresceinamine
adduct could be detected by HPLC through the use of UV, fluorescence or APCI-MS
detection [86]. 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. The main disadvantage is the longer time required for analysis and the higher
technical demands. 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.
A marginal increase in plasma protein AAS was observed (Dragsted, unpublished data).
Another line of evidence comes from studies of dietary fruit and vegetable depletion,
showing a rapid depletion of plasma ascorbate and a concomitant decrease in AAS,
indicating that the depletion of ascorbate might have an antioxidant effect. 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, whereas
no change was observed after supplementation of the known nutrients from the fruits
and vegetables, including 150 mg/d vitamin C. No effect of fruit, vegetables or nutrient
supplements was found in that study on the levels of total plasma carbonyls or immuno-
globulin carbonyls by the antibody approach in conjunction with Western blotting [85].
None of the volunteers had suboptimal vitamin C levels before intervention. Since
vitamin C is a cofactor for the lysine oxidases that cross-bind cartilage in the body,
the observed effect of vitamin C on this specific marker may reflect enzyme leakage from
the cartilage rather than prooxidative stress.
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.
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. HPLC has the advantage of having lower detection limits, permitting simul-
taneous determination of ascorbate and dehydroascorbate, and possibly possessing higher
accuracy, whereas the colorimetric assays have much higher throughput. The handling
and storage of plasma for vitamin C determination has a strong effect on accuracy. 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 supple-
mentation. There is only limited evidence that plasma ascorbate is a functional anti-
oxidant in the body as assessed by means of these markers and very limited evidence that
high dosages of vitamin C may decrease oxidative stress. 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, that of the
formation of plasma isoprostanes. Clear short-term pro-oxidant effects on lipid oxidation
were observed following high-dose vitamin C infusion into the bloodstream. There are
no studies currently available on the relationship between lipid or protein oxidation
markers, vitamin C and chronic disease.
Vitamin E
Biomarkers for vitamin E in body fluids

Vitamin E is a collective term for alpha-, beta-, gamma- and delta-tocopherols as well
as the tocotrienols, of which RRR-α-tocopherol has the highest vitamin E activity.
The reference for the international unit (IU) is 1 mg of all-rac-α-tocopheryl acetate, 1 IU
corresponding to 1.49 mg RRR-α-tocopherol. Since only the four R-forms of α-tocopherol
(d-α-tocopherol) are recognised by the human vitamin E transporter in the body, none
of the four S-forms of α-tocopherol and none of β-, γ- and δ-tocopherols or of the toco-
trienols are thought to have any vitamin E activity in humans as opposed to the rat.
This is still controversial, however, 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, no difference in the total
vitamin E content of LDL was observed [87]. The relative vitamin E activity of the various
tocopherols and tocotrienols in the rat together with their structures are shown
in Figure 2.3. The vitamin E status in rats and possibly in other rodents can be determined
as a function of all the active isomers, but in humans vitamin E status is preferably
determined as the plasma, serum or erythrocyte concentration of d-α-tocopherol. Various
functional tests have also been suggested, including the susceptibility of erythrocytes
Lars O. Dragsted
38
to lysis following an in vitro challenge with hydrogen peroxide [88–90], or the exhalation
of breath pentane [91]. In one study no correlation was found between results of the
erythrocyte membrane stability test and plasma or erythrocyte vitamin E concentrations
[90]. Also, 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. Vitamin E has a physiological transporter in the human that
only binds d-α-tocopherol, and deletion of this transporter leads to neurological
symptoms [92].
Fig. 2.3. Structures of tocopherols and tocotrienols and their relative potency as vitamin E in the rat
(TE; dl-α-tocopherol equivalents). Redrawn from [100].
39
Vitamin E and markers of lipid oxidation

This review concerns only randomised, controlled studies in which the effects of supple-
mentation by vitamin E was investigated on peripheral blood samples using plasma
or serum thiobarbituric acid reactive compounds (TBARS), ex vivo LDL oxidation, anti-
oxidant capacity markers, lipid hydroperoxides, or isoprostanes (see desciption in the
section on vitamin C above). 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, a signi-
ficant decrease in plasma MDA was found in the vitamin-supplemented group as deter-
mined by HPLC [101]. In a subsequent study of 24 diabetic children by use of the same
design, there was a significant decrease in erythrocyte MDA in the vitamin-supplemented
group [102]. In 49 diabetic patients given either 504 mg/d d-α-tocopherol or a placebo for
a 6-month period a decrease in ex vivo induced TBARS in the erythrocyte membranes was
found for the supplemented group [103]. 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, a reduction in plasma MDA occurred in the group assigned vitamin
supplements [104]. In a double-blind, controlled intervention study, 56 patients with
congestive heart failure were given 335 mg/d of the natural d-α-tocopherol for a 12-week
period and no effect on the plasma MDA level was detected [105]. In a randomised
2-months intervention study of 59 healthy smoking males, MDA was found to increase
significantly following daily doses of 200 mg vitamin E combined with 250 mg ascorbate,
30 mg beta-carotene and 100 µg organic selenium, given in a normal formulation, but to
be unaffected by a slow-release formulation as compared with placebo treatment [33].
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, no effect on the plasma MDA was detected [38].
In a trial involving 80 men showing an increase in plasma lipid oxidation from
exposure to either olive oil or menhaden oil, giving a 900 IU dose of vitamin E was found
to be no better than a placebo in decreasing MDA [106]. In a trial of lipid oxidation
induced in 18 untrained men by three sessions of resistance 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 [107]. In another
study, of a group of 14 runners assigned to either a placebo or a 1200 IU/d dose of vita-
min E starting 4 weeks before and continuing during 6 days of increased running training,
a decrease in serum MDA was found in the group given vitamin E [108]. 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, no effect on exercise-
induced oxidative stress could be shown by serum MDA [109]. In another study,
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. No change in plasma MDA either within or between
the two groups could be shown [110].
In a three parallel groups of 16 middle-aged male and female participants, who
were given either 500 mg/d of vitamin C together with 182 mg/d dl-α-tocopherol,
Lars O. Dragsted
40
182 mg/d dl-α-tocopherol alone, or placebo, no effect on plasma antioxidant capacity was
observed at 12 or at 36 months [44].
In a double-blind controlled intervention study of 56 patients with congestive heart
failure, treatment with 335 mg/d of natural d-α-tocopherol for 12 weeks was found
to have no effect on the level of breath pentane exhalation [111]. In a trial involving
80 men who had an increase in plasma lipid oxidation following exposure to olive oil or
to menhaden oil, a dosage of 900 IU vitamin E was found to equal placebo in reducing the
level of lipid hydroperoxides in the plasma or the exhalation of breath pentane [112].
In a 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, there was
found to be a reduction in plasma lipid peroxides and in breath pentane exhalation in the
group to which a vitamin supplement was assigned [106]. In a study without a parallel
control group, a 1000 IU/d vitamin E supplement for a 10 d period was found to reduce
the exhalation of breath pentane [91].
In a dose-response study of 40 healthy men assigned doses of 60–1200 IU of vitamin
E for an 8-week period, vitamin E given at doses higher than 200 IU/d was found to affect
the kinetics of ex vivo oxidation of LDL [113]. In a group of 45 randomised healthy males
and females, the effects of giving mixed supplements of 200 mg/d vitamin E, 900 mg/d
vitamin C and 18 mg/d beta-carotene for 6 months were compared with those of a place-
bo. Non-induced lipoprotein oxidation ex vivo was found to be delayed in the group given
the vitamin supplement, and the strength of this effect was correlated with the level
of plasma α-tocopherol [114]. 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, this together
with 182 mg/d dl-α-tocopherol, 182 mg/d dl-α-tocopherol alone, or a placebo in a parallel
design, a significant increase in the susceptibility of isolated LDL or VLDL to oxidation
ex vivo was observed at 12 and at 36 months in the group given only vitamin E and in
group given the combined dosage. A significant change in these groups in whole plasma
ex vivo oxidation at 36 weeks was likewise observed [44]. 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, no difference
was observed in LDL-MDA, LDL-dienes, or LDL-hydroperoxides induced ex vivo [87].
In 49 diabetic patients given 504 mg/d d-α-tocopherol or a placebo for 6 months,
no change in the group receiving the supplement was detected in the antioxidant capacity
of the erythrocytes as determined on the basis of the glutathione concentration and the
glutathione peroxidase activity [103]. In a double-blind controlled intervention study
of 56 patients with congestive heart failure, treatment with 335 mg/d of the natural
d-α-tocopherol for 12 weeks was found to have no effect on the level of activity of plasma
glutathione peroxidase [115].
In a blinded intervention study of 33 triathletes participating in a world champion-
ship, who were given 800 IU/d vitamin E or a placebo for 2 months prior to the race, the
treatment with vitamin E was found to significantly increase the level of plasma
F2-isoprostanes as well as of several markers of inflammation, including IL-6 [115].
41
In another study, 16 young and 16 older male subjects were given 45 min of eccentric
exercise and were then given a 1000 IU/d supplement of vitamin E for a 12-week period
before being given a second round of exercises. 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. This was not observed for the young men. No other
significant differences were observed between the two groups [116]. The study design
could not control for period effects, which may have caused the difference. In a third
study of the effects of vitamin E on oxidative damage induced by exercise, 21 ultra-
marathon runners were given either a combination of 300 mg/d d-α-tocopherol
and 1000 mg/d vitamin C, or a placebo during the 6 weeks prior to the race. Running as
such was found to increase the plasma isoprostane level, particularly in the males,
and the vitamin supplement was found to prevent this [117]. The inflammatory markers
were also affected by running, but this was not changed by the vitamin supplements.
In a study of the combined effects of giving either vitamin C (500 mg/d) together with
d-α-tocopherol (400 mg/d), vitamin C (500 mg/d) together with d-α-tocopherol (290 mg/d)
and d-γ-tocopherol (130 mg/d), or a placebo during a 28 d period on the plasma
isoprostane concentrations induced by exercise, no effect of either vitamin treatment was
observed, although the treatment by g-tocopherol was found to affect the exercise-indu-
ced increase in plasma and muscle heat shock protein (HSP72) and also the expression
of HSP72 in muscle [118]. In an intervention study of 46 healthy smokers given 0, 300,
600 or 1200 IU/d of vitamin E for 3 weeks, no effect on the excretion of F2-isoprostane
could be observed for any of these interventions [119]. In a double-blind controlled
intervention study of 56 congestive heart failure 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 plas-
ma F2-isoprostane level [111]. In another double-blind placebo-controlled trial with
a crossover design, no effect on the plasma F2-isoprostane level of taking 1000 IU/d
of vitamin E was found for 20 patients with endothelial dysfunction [125]. In a group
of 33 sclerosis patients, 10 of them were given 500 mg/d and 10 of them 1000 mg/d
vitamin E for a period of 3 weeks, the rest being given a placebo. No effect on urinary
excretion of F2-isoprostanes was observed [121]. In 43 hypercholesterolemic patients
randomised to taking simvastatin, simvastatin together with 600 mg/d vitamin E
or a placebo for 2 months, the adding of vitamin E was found to have no further effect
on the urinary excretion of F2-isoprostanes [122]. In a small non-blind study of cirrhotic
patients, the 9 patients receiving standard medication together with 600 mg/d vitamin E
for a 30-day period showed lower urinary excretion of F2-isoprostanes than 5 controls
given only standard medication [123].
In a dose-response study, groups of 5 healthy individuals received either 0, 200, 400,
800, 1200 or 2000 IU/d of vitamin E for a period of 8 weeks, 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, irrespective of the treatment [124]. In a study without
a control group, isoprostane excretion was found to be decreased at the end of a 2-month
period in a group of 15 healthy individuals receiving 400 IU/d of vitamin E [125].
Lars O. Dragsted
42
Effects on protein oxidation

In a study without a control group, the plasma carbonyl content in 15 healthy individuals
was found to be unaffected by 400 IU/d of vitamin E for a period of 2 months [125].
Other markers related to vitamin E effect

Intervention trials involving ingestion of synthetic vitamin E either alone or in combi-
nation with vitamin C have been performed to assess their effects on various conditions
assumed to be caused by oxidative stress. Sperm counts and sperm motility are thought
to be partially influenced by oxidative stress. Vitamin E deficiency is also known to cause
semen abnormalities and infertility in rats. In two randomised studies, each with 30 men,
little effect of taking 600–800 mg/d of vitamin E for 2–3 months was shown. In one
study, a significant increase in the binding ratio to the zona pellucida of the unfertilized
oocyte in a competitive binding assay was observed, but this parameter was not assessed
in the other study [126,127]. No evidence was presented that this effect was related to
antioxidation.
Conclusions
In conclusion, present day HPLC and GC-MS methodology has high precision and accu-
racy in the detection of vitamin E analogues in plasma. Since plasma vitamin E levels
are not always correlated with the stability of the erythrocyte membrane or with
the exhalation of breath pentane, these proposed functional tests for vitamin E must
be regarded as obsolete. There is only limited evidence for a protective effect of vita-
min E supplements at levels of 200–2000 IU/d on markers of lipid oxidation,
and supposed effects of high levels of vitamin E supplementation either on exercise-
induced lipid oxidation as determined by isoprostanes or on inflammatory markers
are controversial. Vitamin E intervention has not been shown to decrease the oxidation
of plasma proteins.
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Vitamins and selenium: Antioxidant vitamins and cancer risk
51
2.2. Antioxidant vitamins and cancer risk:

is oxidative DNA damage a relevant biomarker?
Steffen Loft1, Peter Møller1, Marcus S. Cook2, Rafa∏ Ró˝alski3, Ryszard Oliƒski3
1
University of Copenhagen, Denmark
2
University of Leicester, UK
3
Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University, Poland
The role of oxidative DNA damage in the development of cancer
Cancer is a disease of the genes. An increase in somatic mutations, which are implicated
in the development of cancer, has been documented in aging cells and tissues. When
mutations accumulate, this can be due to the individual’s overall lifetime exposure to endo-
genous and exogenous agents that damage the DNA. Food and beverages frequently contain
carcinogens, partly those generated in processing, such as in the cooking of meat, and partly
carcinogens that occur naturally in the products. It is quite possible, nevertheless, that
metabolites of atmospheric oxygen, so-called reactive oxygen species (ROS), though
not generally classed as carcinogenic agents, are human carcinogens. ROS, which can be
produced both in the biochemical utilization of oxygen and as a by-product of O2
metabolism in the mitochondria, can damage cellular molecules of different types —
in particular proteins, lipids and nucleic acids.
The hydroxyl radicals that ROS creates result in a large number of pyrimidine- and
purine-derived lesions being formed in the DNA (as reviewed in [1]). Some of these
modified DNA bases have considerable potential for damaging the integrity of the
genome (reviewed in [2,3]). One of the most widely studied lesions of this sort
is 8-oxo-7,8-dihydroguanine (8-oxoGua). The presence of 8-oxoGua residues in DNA, unless
repaired prior to DNA replication, leads to GC→TA transversions [4]. The presence
of 8-oxoGua in the cells can thus result in point mutations.
In order to produce mutations, however, oxidative DNA damage needs to occur
at a sufficiently high frequency to exceed the cell’s capacity for DNA repair. 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 2.5 nmol per kg per day, corresponding to about 2000 oxidative modifications
of guanine per cell daily.
Not only is DNA mutation a crucial step in carcinogenesis, but the large numbers
of oxidative DNA lesions observed in many tumours strongly suggests that such damage
is frequently a cause of cancer [5]. Oxidative mechanisms have been shown to have
a potential role in the initiation, promotion and malignant conversion (progression) stages
of carcinogenesis. Since the cumulative risk of cancer increases at a rate corresponding
to the fourth power of age and is associated with accumulated DNA damage, increasing
attention has been directed at oxidative DNA damage in relation to cancer. Lesions such as
8-oxodG are used as biomarkers of oxidative stress. This, together with their mutagenicity
in mammalian cells, has led to the proposal that they be used as intermediate markers
Steffen Loft, Peter Møller, Marcus S. Cook, Rafa∏ Ró˝alski, Ryszard Oliƒski
52
of a potential disease endpoint such as that of cancer, although their suitability for this has
yet to be verified in prospective studies of cancer risk. At the same time, there are serious
problems in the chromatographic measurement of 8-oxodG, connected with the spurious
oxidation of DNA during sample preparation. On the basis of an extensive investigation by
the European Standards Committee on Oxidative DNA Damage (ESCODD) it has been
concluded that the true background level of 8-oxodG in mammalian cells is approximately
0.3–4.2 lesions per 106 unaltered guanines and that reports of values outside this range
should be viewed skeptically [6].
Numerous studies have been concerned with the relationship between the level of oxi-
dative DNA damage and cancer (see [5] for a review). Elevated damage levels have been
purported to arise as a result either of (i) the tumour having an environment low in anti-
oxidant enzymes and high in ROS generation, or (ii) there being reduced DNA repair [5].
Elevated ROS levels may activate transcription factors and the corresponding
genes being permanently activated. This, together with the increase in DNA
damage that occurs, creates a selection pressure for the malignant phenotype seen
in the case of cancer. Although studies that indicate this support the hypothesis that
oxidative DNA damage can be an important risk factor for carcinogenesis, it has been
argued that the mere presence of 8oxodG in DNA is unlikely to be a necessary
and sufficient condition for tumour formation. In 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 [5]. This raises a number
of different issues:
1. Oxidative DNA damage may be an epiphenomenon in relation to the ongoing
pathophysiological process, the levels of damage having no causal role in carcinogenesis.
2. Again, regarding the question of ‘cause or consequence’, 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. An elevated level may be due to some
well-established characteristic of the tumour, such as a high level either of metabolism
or of cell turnover.
3. For DNA mutations to arise through oxidative damage, the nuclei of the undifferen-
tiated, proliferating stem cells have to be affected. Since tissue samples from both tumours
and normal cells represent a heterogeneous mixture of differentiated and undifferentiated
cells (the former being likely to predominate), the analytical procedures in current use are
unable to quantify the lesion levels found in the most important ‘target’ cells.
In order for a mutation to come about, not only must the DNA of the target cells
be affected, but also the damage must be within a coding region of the DNA. Issues of this
sort have to be addressed before a causal link between oxidative DNA damage and cancer
can be established. 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. Ultimately, 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.
53
Summing up, 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, but that
it is impossible at present to determine to what extent oxidative stress is directly involved
in carcinogenesis, since full development of the disease in response to exposure to
a carcinogen may take 20–40 years. It is very difficult, therefore, to establish directly that
the DNA lesion responsible for a carcinogenic process is the lesion present in a tumor many
cell generations later. One should bear in mind, nevertheless, that DNA damage, altered
gene expression and mutations are necessary elements in the process of carcinogenesis.
Although these events may come about by way of different mechanisms, oxidants
are involved in each case.
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.
A plethora of descriptive epidemiological studies have concerned a possible protective
effect of a diet rich in fruits and vegetables [7]. Both experimental and epidemiological
data indicate vitamin C to protect against both stomach and oesophageal cancer [8].
Nevertheless, large-scale intervention studies have failed to demonstrate use of anti-
oxidant vitamins to reduce the risk of cancer [9]. At the same time, the mode of action
of dietary micronutrients is complex and is far from being fully understood. It is reasona-
ble to assume that agents that decrease oxidative DNA damage should also decrease the
subsequent development of cancer. 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, C and E or phenolic compounds.
These antioxidants are effective free-radical scavengers and should protect the DNA from
oxidative damage.
Intuitively, supplementation trials would appear to represent the most relevant way
of exploring antioxidant effects, although sampling is usually restricted to use
of such surrogate tissues as white blood cells (WBC) and urine. At the beginning of
the 1990s, as the possibility of detecting 7-hydro-8-oxo-2’-deoxyguanosine (8-oxodG)
appeared, the first of many antioxidant supplementation trials dealing with this lesion in
WBC was carried out [10]. Reliable detection of urinary 8-oxodG excretion became
possible at about the same time [11], this soon being followed by the performance
of antioxidant trials in which urinary 8-oxodG excretion served as the key biomarker.
Subsequently, however, the comet assay, used for the detection of DNA strand breaks
(SB), 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), respectively, have become by far
the most popular assays for use in conjunction with antioxidant intervention trials. The
literature on biomarkers of oxidative DNA in WBC employed in small-scale intervention
studies of antioxidant supplements has been summarized in a series of reviews [9,12].
54
Many studies suffer, however, from use of a non-optimum design. Table 2.6. provides
an overview of intervention studies involving optimal design in which the effects
of antioxidants or antioxidant-rich food supplements on oxidative damage to DNA
in WBC or in urine have been investigated.
Table 2.6. Multiple administration of dietary antioxidants with assessment of oxidative DNA damage
in white blood cells and urine
Supplement given per day Subjectsa Age Effect Ref

(yr)b
Multivitamin tablet (100 mg vitamin C, 100 M 50—59 A decrease in ENDOIII sites (Comet) in WBC after 13
280 mg vitamin E, (50S) 20 weeks
and 25 mg β-carotene) for 20 wk
Multi-vitamin (250 vitamin C, 63 MF (S) 42±9 No difference in 8-oxodG in WBC (antibody-based 14

200 IU α-tocopherol, detection) between the supplemented and the
and 6 mg β-carotene) for 6 mo placebo group, but a decline in the course of the
trial in both groups
Carotenoidsc for 3 wk 32 MF 32±11 No effect compared with baseline, but a post- 30

(NS) supplementation decrease in the urinary excretion
of 8-oxodG (ELISA) in the active group
Vitamin C (500 mg) for 3 wk 30 MF 17—49 No effect of vitamin C supplementation on 8- 24

(NS) oxodG (ELISA) in spot urine, but an increase in
excretion during the washout period
Six groups receiving combinations 116 M (S) 30—65 No effect on the 24-h urinary excretion of 8-oxodG 35
of vitaminsd for 2 mo (HPLC)
Vitamin C (1 g) and vitamin E (0.6 g) 13 M (NR) 30±3 Lower 24-h urinary excretion of 8-oxodG (HPLC) in 25
for 1 mo the active group of HIV-infected patients receiving
zidovudine therapy
28
2x2 parallel study of vitamin C (500 mg) 184 MF 58±14 No effect on the 24-h urinary excretion of 8-oxodG
and vitamin E (400 IU) for 2 mo (NS) (ELISA)
2x2 parallel study of vitamin C (500 mg) 48 M 45—69 No effect on the 24-h urinary excretion of 8-oxodG 34
and vitamin E 182 mg) for 12 mo (22S) (HPLC)
Multivitamin tablete for 2 wk 30 NR 22±1 No effect on the 24-h urinary excretion of 8-oxodG 33
(NR) (ELISA) in subjects undergoing cold-weather
training at a moderate altitude
Multivitamin tabletf for 21 d 39 MF 7±2 No effect on the excretion of 8-oxodG (ELISA) in 37

(NR) spot urine samples
Multivitamin tabletg for 24 d 40 M (NR) 18—40 No effect on the overnight excretion of 8-oxodG 36
(ELISA) in subjects undergoing cold-weather
training at a moderate altitude
Fruit and vegetable capsulesh for 7 wk 59 MF 50±6 No effect on the excretion of 8-oxodG (ELISA) in 29
(11S) spot urine samples
55
in white blood cells and urine — cont.

(yr)b
Antioxidant rich diet or tabletsi for 5 wk 55 MF 71±6 No effect compared with baseline, 32
(NR) but a post-supplementation decrease
in the 24-h urinary excretion of 8-oxodG (ELISA)
in the active group
Rye crisp bread (76.5 g) or placebo 12 F (NR) NR No effect on ENDOIII sites (Comet) in WBC 17
(fiber-free bread) for 2 wk
Flavonol (quercetin) rich diet for 2 wk 10 MF (3S) 60±7 No effect on ENDOIII sites (Comet) in WBC 16
in crossover design among type
2 diabetes patients
Vegetable/fruit (500 g) in crossover design 22 M (S) 33±11 No effect on ENDOIII sites (Comet) in WBC 19
for 3 wk with 2 wk washout
Vegetable/fruit (600 g) or tablets 43 MF 27±6 No effect on ENDOIII and FPG sites (Comet) 20
with the same concentration (NS) in WBC. No effect on the 24-h urinary excretion
of antioxidants/minerals for 24 days of 8-oxodG (HPLC), but a decline in all the groups
Cruciferous and legume sprouts (113 g) 18 MF 21—45 No effect on FPG sites (Comet) in WBC 15
for 2 wk (NR)
Kiwi fruit (1—3 pieces) for 3 wk 14 MF 26—54 A decrease at the ENDOIII and FPG (Comet) sites 22
(NS)
Brussels sprouts (300 g) for 1 wk 10 MF NR No effect on the 24-h urinary excretion 40

(NS) of 8-oxodG (HPLC)
Brussels sprouts (300 g) for 12 d 10 M (NS) NR A decrease in the 24-h urinary excretion 41
of 8-oxodG (HPLC) in the active group
Fruit juice (480 ml)j for 4 d 11 M (NR) 21±1 A decrease in the 12-h urinary excretion 26
of 8-oxodG (ELISA) in the active group
Parallel study of blackcurrant juice or antho- 57 MF 19—52 No effect on ENDOIII and FPG sites (Comet) in 18
cyanin drink (475—1000 ml) for 3 wk (6S) WBC
Green tea or black tea (4 cups) 120 MF (S) 18—79 A decrease in the excretion of 8-oxodG (ELISA) in 27,
for 1—4 mo spot urine samples in the green tea group after 4 45
mo, but not earlier
Two interventions of green tea 68 MF 18—45 A decrease in the 12-h urinary excretion of 8- 317
with 300 ml for 7 d or 32 oz for 7 days (13S) oxodG (HPLC)
Green tea extractk for 3 wk 16 M (8S) 20—31 No effect of supplementation on the 24-h urinary 44
excretion of 8-oxodG (HPLC) but a decrease in
excretion during the study in all groups
Soya-hypocotyl tea (> 1 l) for 1 mo 38 F (NR) NR A decrease in the excretion of 8-oxodG (ELISA) in 42
the active group (statistical test not reported)
56
in white blood cells and urine — cont.

(yr)b
Soy milk, rice milk, or cow milk (1 l) for 4 wk 10 M (NS) 20—50 A decrease in ENDOIII sites (Comet) for soy milk 21
Polyphenol-rich olive oils (25 ml) for 40 d 12 M (NS) 20—22 A decrease in the excretion of 8-oxodG (HPLC) in 43
spot urinary samples following supplementation in
a dose-dependent manner
a
Number of subjects indicated as males (M) and females (F). Smokers (S) and nonsmokers (NS) are indicated in brackets.
b
Age is shown as range or as mean ± standard deviation.
c
Supplement consisting of β-carotene (6 mg), α-carotene (1.4 mg), lycopene (4.5 mg), bixin (11.7 mg), lutein (4.4 mg),
and papika carotenoids (2.2 mg).
d
The six groups received daily supplements of 1) 200 mg vitamin E; 2) 500 mg plain-release vitamin C; 3) 500 mg slow-release vitamin C;
4) 90 mg coenzyme Q10 in oil; 5) 30 mg coenzyme Q10 granulate; 6) placebo.
e
Tablets containing β-carotene (12 mg), vitamin E (400 IU), vitamin C (500 mg), selenium (100 mg), and zinc (30 mg).
f
Tablets containing micronutrients similar to those of 3 fruits and 3 vegetables (containing 107 mg vitamin C and 83 mg vitamin E).
g
Consisted of β-carotene (20050 IU), vitamin C (330 mg), tocopherols (650 IU), selenium (167 mg), catechin (13.2 mg), lutein (500 mg),
lycopene (100 mg), N-acetyl-1-cysteine (181 mg), and pomegranate extract (5 mg).
h
The fruit capsules were made from juiced apple, orange, pineapple, papaya, cranberry and peach. The vegetable capsules were made
from carrots, parsley, beet, broccoli, kale, cabbage, spinach, and tomato. The daily dose consisted of 200 mg vitamin C, 60 mg vitamin E,
and 15 mg β-carotene.
i
Consisted of tablets containing vitamin antioxidants (400 mg vitamin C, 150 mg vitamin E, 4 mg β-carotene), capsules (containing 90 mg
vitamin C, 18 IU vitamin E, 2.4 mg β-carotene, and powder or extract of fruits and berries), or a carotenoid-rich diet.
j
Containing vitamin A (240 mg), β-carotene (399 mg), vitamin C (219 mg), vitamin E (1.44 mg) per day.
k
Consisted of 1000 mg extract/kg body weight in meat patties (total phenolics 23.5 mg/10 MJ).
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 [9,12]. The effect of a single dose of vitamin C
seems to disappear after several hours, whereas tocopherols and carotenoids exert their
effects somewhat longer, possibly because of differences in the respective rates of bio-
availability and elimination. Among studies in which multiple doses of single
antioxidants are given, however, there are fewer that reveal protective effects than those
that report only negative results [9,12]. This suggests that the protective effect of a single
antioxidant is relatively short. In two well-designed studies of the effects of administering
a combination of antioxidant vitamins, a protective effect could be shown after
only 20 weeks of supplementation but no effect after 10 weeks (or 6 months of supple-
mentation) [13,14]. Accordingly, the question of whether multiple vitamin supplemen-
tation provide better protection against oxidative DNA damage than a single dose does
cannot be answered unambiguously.
Antioxidant-rich foods
Ingestion of a diet rich in flavonols (including quercetin) and of one rich in cru-
ciferous and legume sprouts (113 g/d for 2 wk) was not found to alter the frequency
of ENDOIII and of FPG sites, respectively [15,16]. Also, being given rye crisp
57
bread (76.5 mg/d for 2 wk) as a source of lignans was not found to be associated with
any increase in the plasma enterolactone concentration, or to have any effect on the
ENDOIII sites [17]. 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, and their effects in the gastrointestinal tract are easier to comprehend.
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 FPG-sensitive sites,
there in fact being a tendency in the group of subjects drinking black currant juice
for the number of FPG sites to increase [18]. Anthocyanines have low bioavailability
and the dose provided here was rather high. It can be speculated that the subjects
suffered a slight, unintentional intoxication of the gastrointestinal tract (some
of those in the active groups complained of nausea, for example). Two studies
concerned the effects of an intervention of providing vegetables and fruits. The one
investigation, a cross-over study of male smokers, showed no effect on the ENDOIII
sites of ingesting 500 g/d of such food for 3 wk [19]. The other, 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, was negative with respect to effects on the
ENDOIII and FPG sites [20]. 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. 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; assessment of the DNA damage
occurring showed the levels of ENDOIII to be reduced [21]. 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), which showed the numbers
of ENDOIII and FPG sensitive sites to be reduced [22].
An overall summary of the studies showed that six investigations reported
beneficial effect of antioxidant supplementation, whereas 13 studies reported null
effect. There is little support for the notion that ingestion of antioxidant-rich foods
is associated with lower spontaneous level of oxidative DNA damage in WBC than
intake of single antioxidants.
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. For 24 studies of the effects of antioxidant supplementation on the
urinary excretion of 8-oxodG in which a controlled design was employed [20,23–44],
no appreciable difference was present in terms of duration of the intervention period,
number of subjects, or power to detect a 50% change between these studies reporting
beneficial effects and those reporting no effects.
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 [23,35,38,39]. A comparable study invol-
ving supplementation of a mixture of carotenoids (daily intake: α-carotene (1.4 mg), β-caro-
tene (6.0 mg), lycopene (4.5 mg), bixin (11.7 mg), lutein (4.4 mg), and paprika carotenoids
(2.2 mg)) revealed a statistically significant difference between the active and the placebo
group in the delta values obtained (i.e. the difference between results at the end of sup-
plementation and at baseline) but no difference between the two groups at baseline [30].
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. In four additional studies, neither supplementation of
vitamin C or vitamin E or a combination of them was found to have any effect in healthy
subjects [24,28,34,35], whereas in still another study a beneficial effect of the supplemen-
tation of high doses of vitamin C (1000 mg/d) and vitamin E (600 mg/d) was found for HIV-
infected patients treated with zidovudine [25]. Further studies showed multi-vitamin tablet
supplementation to provide no beneficial effect either in normal subjects [20,32,37]
or in subjects undergoing cold-weather field training at a moderate altitude [33,36].
Investigations of the effects of natural food products are distributed about equally
between studies reporting beneficial and those reporting null effects. Ingestion of olive
oils with a high content of phenolic compounds was found to be associated with a lowe-
ring of the urinary excretion of 8-oxodG [43]. A number of studies have involved
supplying antioxidants in the form of berries, fruits, tea, and vegetables. Taking capsules
containing extracts of fruits and berries and eating diets rich in carotenoids were both
found to lower the excretion of 8-oxodG [32]. A positive effect of vegetable juice
consumption on 8-oxodG excretion was also observed in subjects enrolled in a soccer
summer training camp [26]. On the other hand, 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, whereas this intervention was found to have a pronounced
period effect [20]. No effect on the urinary excretion of 8-oxodG was found for the
ingestion of capsules containing juices and powders of fruit (apple, orange, pineapple,
papaya, cranberry, and peach) and of vegetables (carrot, parsley, beet, broccoli, kale,
cabbage, spinach, and tomato) [29].
Mixed results concerning the effects of a dietary supplementation of Brussels sprouts
(300 g/d) were obtained in two studies [40,41]. In the first study, that involved only male
subjects, the Brussels sprouts supplementation was found to lower the urinary excretion
of 8-oxodG [41]. In the subsequent study, in which both sexes were included, the effect
was less clear, there being a tendency for only the male subjects to benefit from ingestion
of Brussels sprouts, but the results were uncertain because of the only small number
of subjects tested and of one of the male subjects showing an unexpectedly high
urinary 8-oxodG excretion level [40]. Three additional studies in this area concerned
the effect of drinking green tea [see 27,31,44,45]. In the one study a beneficial effect
was found for drinking 300 ml/d for a week [31], whereas in one of the other two studies
59
no effect of ingesting green tea extract in meat patties for 3 wk was obtained [44].
Although the unadjusted data of the third study indicated no beneficial effect of drinking
green tea, adjustment of the data for a number of variables, including baseline 8-oxodG
levels, revealed a statistically significant positive effect of drinking green for 4 months
[27,45]. In still a further study, drinking soya hypocotyl tea was found to be associated
with a lower urinary excretion of 8-oxodG, although it should be emphasized that the
fate of the antioxidants in this investigations was inconclusive since 1) the plasma
concentration of carotenoids decreased, 2) the putative active constituents (isoflavones)
were not measured in the plasma, and 3) the alterations in the urinary concentration
could not be assessed due to insufficient information [42].
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 da-
mage creates:
1. It is possible that a preventive effect of the vitamins can be only be seen when their
basal levels are very low, such as in the case of severe oxidative stress. Indeed, vitamin
supplementation of HIV-infected patients showing very low levels of antioxidant
vitamins and significantly increased lymphocyte amounts of 8-oxoGua (as well
as other base modifications) was found in one study to result in vitamin restoration
to levels characteristic for the control subjects. The 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. It is possible, therefore, that
the presence of oxidative stress, which might fail to be recognized, could increase
the likelihood of detecting a protective effect.
2. Under some circumstances, the prooxidative properties of certain of the antioxidant
vitamins (vitamins C and A) may take effect. Experimental data suggest that supple-
mentation of vitamin C to iron-overload subjects may increase the oxidative DNA
damage that occurs [46]. It is worthy of note in this context that presumably healthy
men may have the hereditary disease idiopathic haemochromathosis, which leads
to iron overload, such that iron catalytic to free-radical reactions (a so-called labile iron
pool — LIP) is present in the blood plasma [47]). Interestingly, a positive correlation
has been shown between LIP and the oxidatively modified nucleoside in human
lymphocytes [48]. The absorption of non-haem iron has also been found to be affected
by ascorbic acid [49].
3. The question can be raised as to whether antioxidants in the blood and 8-oxodG in the
DNA of lymphocytes and leukocytes are representative of the situation in the target
tissue.
4. It is possible that the antioxidants themselves may allow clonal expansion to occur
and promote tumour growth by protecting initiated cells from excessive oxidant
toxicity and apoptosis that would otherwise kill them [50].
5. Paradoxically, antioxidant vitamins may have biological activities, such as those
of regulating changes in gene expression, that are separate from their direct
antioxidant effects [51].
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. On an
experimental basis, it is easy to understand the ingestion of antioxidants being associated
with lower levels of oxidative DNA damage. At the same time, many of the studies
performed are of only limited value due to methodological problems related to the study
design and the assays, and many of them lack the power of detecting 50% differences
between two separate groups. Although single studies may possess considerable power due
to specific aspects of their design such as use of repeated measurements, a major problem is
that many of these studies have too few subjects. The most likely effect ratio in healthy
subjects involves a difference of less than 10%, which means that, to obtain significant
results, hundreds of subjects would be needed, most studies encompassing far fewer
subjects than this.
At present, no firm conclusions can be reached on the basis of antioxidant intervention
studies. There is a tendency for supplementation with use of antioxidant-rich food to
decrease the urinary excretion of 8-oxodG, which is not the case with use of single
antioxidants. WBC studies provide little support for the idea that long-term antioxidant
supplementation lowers the basic level of oxidative DNA damage, although there may be
a beneficial effect in the first few hours after ingestion. This can be interpreted as
antioxidants having an overall beneficial effect on the body as a whole, yet the use of WBC
as a surrogate tissue may not be particularly well suited for detection of this effect. It should
also be borne in mind that the majority of studies involve healthy individuals, whereas the
protective effect of antioxidants may be more easily detected in subjects who are suffering
from oxidative stress. 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. 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. It should be noted, however,
that most of the studies reported have used supplements of vitamin E, which is considered
to be the least effective form of antioxidant supplementation. 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.
In the future, more attention should probably be devoted to alternative chemopreven-
tive mechanisms such as the upregulation of DNA repair systems, and to other types of
DNA damage, such as those involving bulky DNA adducts. Also the chemopreventive effect
of antioxidants on non-lymphatic tissue, which has been only sparsely investigated, should
be addressed more thoroughly before the idea of antioxidants having clearly beneficial
effects is abandoned. Hopefully, such studies will benefit from the lessons learned from
antioxidant intervention studies, particularly as regards the need of proper investigative
designs and of the validity biomarkers, which are of pivotal importance for such studies.
61
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Jakob Linseisen, Sascha Abbas
64
2.3. Measurement of serum 25-hydroxycholecalciferol

as marker of vitamin D status
Jakob Linseisen and Sascha Abbas

DKFZ, Cancer Epidemiology, Heidelberg, Germany
Vitamin D3, or cholecalciferol, is synthesized in the skin. Its precursor, 7-dehydro-

cholesterol, is converted by UV light from the sun (UVB 290–315 nm) into previtamin D3
which slowly isomerizes to vitamin D3. In addition, the human diet contains vitamin D
in the form of vitamin D3 (in food of animal origin) and vitamin D2 (ergocalciferol con-
tained in plant food, arising from the irradiation of ergosterol). In the blood vitamin D
and its metabolites are bound to the vitamin D binding protein. Vitamin D is metabolised
by hepatic 25-hydroxylase into 25-hydroxycholecalciferol (25-(OH)D), which is the major
circulating metabolite. Further hydroxylation to 1,25-dihydroxycholecalciferol (1,25-(OH)2D
or calcitriol) occurs primarily in the kidney (1α-hydroxylase). The biologically most active
vitamin D metabolite is 1,25-(OH)2D whereas 25-(OH)D has only limited biological
activity [1,2].
There is overall agreement that 25-(OH)D is the appropriate biomarker to measure
vitamin D-status in humans. Because of its serum half-life time of about three weeks [3],
this metabolite is a good indicator of the vitamin D-stores obtained both from exposure
to sunlight and ingestion of vitamin D [2]. As compared with 25-(OH)D, the half-life
time of its precursor, vitamin D (cholecalciferol), is rather short (24 h in the blood
circulation), its thus being more strongly affected by very recent sun exposure or dietary
intake of vitamin D [4]. Plasma 1,25-(OH)2D, with an even shorter serum half-life time
of 4–6 h, is also not regarded as a suitable biomarker of vitamin D status [5]; since
its conversion from 25-(OH)D is also tightly regulated, the circulating 1,25-(OH)2D level
does not provide valid information on the individual’s vitamin D status [6].
Determination of 25-(OH)D
As reflected in the recent scientific literature, the determination of circulating

25-(OH)D is not an easy task [7]. One of the major problems in measuring 25-(OH)D lies
in the molecule itself. First of all it is a very hydrophobic compound and secondly it exists
in two forms, 25-(OH)D2 and 25-(OH)D3. Because of its hydrophobic nature, 25-(OH)D
measurements are quite vulnerable to matrix effects, e.g. to lipids. Such matrix effects can
markedly diminish the validity of assays carried out [8]. Extraction from the matrix
(serum or plasma) is thus required for most analytical procedures (see Table 2.7.).
Considerable interest has developed in assays for measuring 25-(OH)D. Since the time
of first assays in 1971, strong efforts have been made to simplify them for routine use [6].
Measurement by means of HPLC is considered by most to be the gold standard here [6,8].
Although such measurement is very accurate, it has to be performed by experienced
personnel, requires expensive equipment (HPLC) and is more time-consuming than other
Vitamins and selenium: Measurement of serum 25-hydroxycholecalciferol
65
methods. Radioimmunoassays (RIA), enzyme immunoassays (EIA) and chemilumi-

nescence assays (CLPBA) are easier to handle, as well as being fast, and are thus more
frequently used. There are important differences in the performance of the various de-
tection methods, however. Table 2.7. summarizes some of the performance characteristics
of most of the assays that are available commercially.
Table 2.7. Commercially available 25-hydroxyvitamin D assays [6]
Range of Sensi- Intra- Inter-

Test principle, Sample type Assay
Extraction detection tivity assay assay Comments
Manufacturer and volume* time**
(nmol/l) (nmol/l) CV (%) CV (%)
RIA, Serum Acetonitrile ≤6 <8 <12 2 h, Calibrators and controls

DiaSorin or plasma, 10 min in the serum matrix;
50 µL no yield determination
required
RIA, Serum Two-step 4—400 ≤5 5.5 7.9 3 h, Calibrators and controls

IDS or plasma reagent 0 min in the serum matrix;
50 µL extraction no yield determination
required
ELISA, Serum None 6—360 ≤5 <6 <9 3 h, No radioactive waste;

IDS or plasma 0 min 100% cross-reactivity with
50 µL 24,25 (OH2)D
ELISA, Serum Proprietary 6.3—250 2.4 9 11 5 h, The primary antibody

Biomedica or plasma extraction 0 min is the vitamin D binding
50 µL reagent protein from serum
Chemilumi- Serum Unknown 17.5—300 18 6.6 11.2 75 min Fully automated; requires
nescence, or plasma luminometer
Nichols 20 µL
Institute
Diagnostics
HPLC, Serum Acetonitrile 15—150 4.0 5.2 8.4 20 min The laboratory must have
IDK 500 µL and C18 an HPLC unit with a silica
cartridge column
extraction
CPB, Serum, Acetonitrile 8—312 2.5 9.9 14 1 h, Uses vitamin D binding

Immundiag- plasma 10 min protein
nostics or urine
500 µL
* Represents the initial starting volume of the sample.

** Does not include extraction, counting or microplate reading times. CV — coefficient of variation; RIA — radioimmunoassay;
ELISA — enzyme-linked immunosorbent assay, HPLC — high-performance liquid chromatography, CPB — competitive protein binding;
24, 25 (OH2) D, 24,25-dihydroxyvitamin D.
66
Validity of different assays
Only few studies have compared the different commercially available assays [9–13]. Binkley
et al. [11] reported recently not only immense variation between different assays but also
variability between different laboratories in using a given assay. In their study, the serum
of 10 subjects was sent to six different laboratories. Table 2.8. lists the methods and the nor-
mal range for 25-(OH)D measurement in the laboratories that participated. The mean serum
25-(OH)D concentrations differed 2-fold between laboratories (Figure 2.4.), and the propor-
tion of subjects below an arbitrary threshold of insufficiency (32 ng/ml) varied between 17 and
90%. After spiking of the serum samples with a defined quantity of 25-(OH)D, a broad range
(17–95%) of the expected concentration was found by the different laboratories [11]
(Figure 2.5.). On the basis of their results, Binkley et al. [11] recommended the use of RIA
techniques for 25-(OH)D measurement until other methodologies have been developed further.
Table 2.8. 25-(OH)D assay methodology and the normal range employed in different laboratories [11]
Laboratory Methodology Normal range
A Acetonitrile extraction followed by in-house RIA 10—55 ng/ml

B DiaSorin (RIA) 10—40 ng/ml
C Acetonitrile extraction followed by DiaSorin (RIA) 8—38 ng/ml
D Chemiluminescent assay 20—57 ng/ml
E Acetonitrile extraction followed by DiaSorin (RIA) NA
F Chemiluminescent assay 6—54 ng/ml
G Chemiluminescent assay 10—68 ng/ml
H Ethyl acetate extraction followed by normal phase HPLC NA
NA — not applicable; RIA — radioimmunoassay; HPLC — high-performance liquid chromatography.
Fig. 2.4. Mean (SEM) results for the serum 25-(OH)D concentrations for 10 subjects as estimated by six
different laboratories (for abbreviations see Table 2.8.). The means varied widely between laboratories from 17.1
to 35.6 ng/ml (p < 0.005) [11].
67
Fig. 2.5. Mean (SEM) results for the 25-(OH)D concentrations in the serum samples of 10 subjects spiked
with the 25-(OH)D standard, as estimated by five different laboratories (for abbreviations see Table 2.8).
The means varied widely between laboratories from 27.4 to 44.6 ng/ml (p < 0.05). The increment was less than
anticipated and varied between laboratories (p < 0.005) from 7.7 to 18 ng/ml, the greatest increment being
detected by HPLC [11].
Another study compared the DiaSorin RIA assay with the Nichols chemiluminescence
assay, finding discordant serum 25-(OH)D-values. Both assays also underestimated
the 25-(OH)D-concentration as compared with the HPLC-method [10].
In accordance with these findings, several authors have called for international
standardization of the vitamin D assays that are available and that are affordable for prac-
ticing clinicians. Hollis also emphasizes the need for validation of the user’s assay system
in the laboratory in question regardless of the claims made by the manufacturers [8].
According to the latest DEQAS (Vitamin D External Quality Assessment Scheme)
report, 59% of the participating laboratories were found to have met the target [12,13].
Each of the laboratories was given five serum samples quarterly, 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). Figure 2.6. shows the latest results of this
external quality control project. These give the impression that only method 6 exceeded
the limit. However, the authors of the report state that the validity of the 25-(OH)D
results that are obtained will justifiably continue to be questioned.
There has been discussion regarding the importance of the assays detecting 25-(OH)D2
as well as 25-(OH)D3. The HPLC method enables 25-(OH)D2 and 25-(OH)D3
to be quantified whereas there are concerns regarding the detection of 25-(OH)D2 with
other assays (Nichols procedure and IDS RIA) [9]. However, a subsequent study refuted
these findings and also concluded that vitamin D2 is less potent and has a shorter
duration of action than vitamin D3 [14]. This suggests that the contribution
of 25-(OH)D2 to the overall 25-(OH)D supply is less important than had been previously
thought. Patients, however, especially those in the US, 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.
68
Fig. 2.6. Accuracy of 25-(OH)D methods used by the different DEQAS participants [13]. Each column shows
the mean deviation (% bias) and SE from the target value (ALTM) during the period of 2000–2004. The method
means of samples known to contain 25-(OH)D2 were excluded. Method 1, DiaSorin RIA; method 2, IDS RIA;
method 3, IDS EIA; method 4, competitive protein-binding assay; method 5, HPLC; method 6, Nichols automated
chemiluminescence assay.
Having an adequate definition of the normal range is essential in routine clinical

practice in order to be able to define groups that are at risk for the negative consequences
of vitamin D deficiency. In a recent publication, Hollis defined 25-(OH)D levels
below 80 nmol/L as being deficient, due to the serious health risks encountered at levels
less than this [7]. This is in contrast with most of the published studies, that set the cut-
off points at much lower levels, around 25–40 nmol/L [1,15,16]. It is important to note
that “normal” is not defined as the median population level, 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. In addition to the high degree
of uncertainty that the divergence of the results for different techniques and different
laboratories creates, epidemiological variability needs to also be taken into account,
as shown by the seasonal variation in vitamin D status that occurs [17,18].
Apart from the analytical problems, the serum 25-(OH)D concentrations in different
populations vary with latitude, season, race, age, dietary intake, and the composition
of the population being studied. There is growing evidence that there is not only a specific
seasonal decline in serum 25-(OH)D during the winter months, but there may also
be a significant proportion of the population that exhibits asymptomatic subclinical
vitamin D insufficiency. Populations at risk include nursing home residents and the
elderly, especially the home-bound elderly. In epidemiologic studies, suggested cut-offs for
insufficiency range from 10 to 30 ng/ml (25–80 nmol/l). Recent reports suggest European
and North American populations to have a higher degree of insufficiency than had
previously been thought [15,18–23]. A summary of studies on vitamin D status in the
elderly is given elsewhere [1].
69
Conclusions
In conclusion, the determination of vitamin D status is still a challenging task. Several

problems are important to bear in mind including the different forms of vitamin D
(vitamin D2, vitamin D3), the measurement variation introduced by different assays
and different laboratories, epidemiologic variation in vitamin D status and its determi-
nants, and problems in defining the appropriate threshold for vitamin D sufficiency.
Nonetheless, vitamin D metabolites have various important biological effects. This makes
strong scientific research efforts needed. Epidemiologic studies in particular can
contribute knowledge concerning the association between vitamin D insufficiency
and the risk of disease, including the risk of cancers at various sites.
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Vitamins and selenium: Selenium and cancer — selenoproteins as biomarkers
71
2.4. Selenium and cancer — selenoproteins as biomarkers

in relation to selenium supplementation
Björn Åkesson and Katharina Bruzelius

Biomedical Nutrition, Pure and Applied Biochemistry, Lund University,
and Department of Clinical Nutrition, Lund University Hospital, Lund, Sweden
Introduction
The relationship between selenium and cancer involves many different factors.
These include the forms of selenium present in the body and in the diet, their functions
and mechanisms of action, and methods employed in assessing an individual’s selenium
nutritional status — both generally, and in epidemiological studies of the risk of cancer,
as well as in connection with long-term trials for investigating the disease-preventive
potential of selenium supplementation. A variety of issues connected with this
are reviewed in two of the chapters. The present chapter concerns the various forms
of selenium and their metabolism, the occurrence of different selenoproteins used
as biomarkers of selenium status, and the use of intervention trials to study the cancer-
preventive effects of selenium supplementation. In the chapter thereafter
(Gromadziƒska et al.), the mechanisms of action involved, together with epidemiological
findings on relations between the selenium status in the body and risk of cancer are
reviewed.
Different forms of selenium
With few exceptions, nearly all of the selenium in animals, plants and microorganisms
is bound within proteins, several protein-bound forms having been identified. A major
part of the selenium in mammals is specifically incorporated into proteins of defined
biological function, so-called selenoproteins, containing the amino acid selenocysteine
(Sec; analogous to cysteine in which sulphur is replaced by selenium) (Table 2.9.).
In the diet, selenoproteins are largely found in animal foods. Another selenium-containing
amino acid is selenomethionine, which is synthesized by plants and by yeast. When
ingested by humans or other organisms, it is incorporated non-specifically into proteins
as an analogue of methionine [1]. The replacement of methionine by selenomethionine
appears to be random and to be dependent on the relative concentrations of these
amino acids [2]. Other proteins can also bind selenium as a ligand [3], but little is known
of the role it has here.
Selenite and selenate are inorganic forms of selenium used as dietary supplements.
It is uncertain whether they occur naturally in foods to any major extent. In addition to
the forms of selenium mentioned, several low-molecular-weight selenium compounds
have been shown to be present in different foods, some of these compounds being
uncharacterized [4–6].
Björn Åkesson, Katharina Bruzelius
72
Table 2.9. Some major inorganic and organic forms of selenium
Organic Inorganic
Selenocysteine (Sec) HSeCH2CH(NH2)COOH Selenite SeO32–
Selenomethionine CH3Se(CH2)2CH(NH2)COOH Selenate SeO42–
Metabolism of selenium
Different chemical forms of selenium are involved in metabolic pathways (Figure 2.7.).
Selenate and selenite are reduced by glutathione to hydrogen selenide, 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),
dimethylselenide or trimethylselenonium ions for excretion. Selenomethionine and se-
lenocysteine can also be converted to hydrogen selenide. The major selenium metabolite
excreted in urine is selenosugar, a much lesser amount being excreted as trimethyl-
selenonium ions [7]. At toxic doses, selenium is removed as dimethylselenide via exha-
Fig. 2.7. Metabolic pathways of selenium. Selenomethionine, selenocysteine and selenite can be converted into
the key metabolite hydrogen selenide (H2Se), which is turn is the precursor of selenocysteine in selenoproteins
and various excreted forms of selenium. Several compounds can be converted into methylselenol (from [8]).
73
lation. Several studies have suggested that methylated Se derivatives, such as Se-methyl-
selenocysteine and selenomethionine, are the selenium compounds most effective
in cancer prevention [8]. Several of these compounds can be converted to methylseleninic
acid or methylselenol which have anticarcinogenic effects in vitro [8].
Selenoproteins
A total of 25 selenoprotein genes were discovered in the human genome several years ago
by sequence analysis, yet the functions of many of the proteins involved are still
unknown (Table 2.10.) [1,9]. The distribution and concentrations of selenoproteins
Table 2.10. Selenoproteins in the human and their putative functions
Selenoprotein Function
15 kDa Involved in protein folding in the ER

DI1 Thyroid hormone control
cGSHPx Peroxide reduction in the cytoplasm
eGSHPx Peroxide reduction in plasma and other extracellular fluids
giGSHPx Peroxide reduction, mainly in the gastrointestinal tract
phGSHPx Reduction of phospholipid hydroperoxides
GSHPx 6 Peroxide reduction, found in embryos and in the olfactory epithelium
SelH Unknown
SelI Unknown
SelK Unkown, a membrane protein
SelM Involved in protein folding in the ER
SelN Unknown, mutations in gene associated with muscular diseases
SelO Unknown
SeP An antioxidant and selenium transport protein
SelR Reduces methionine-R-sulfoxides
SelS A membrane protein, involved in the elimination of misfolded proteins from the ER
SelT Unknown
SelV Unknown, only found in the testes
SelW Can reduce peroxides using glutathione as an electron donor
SPS2 Catalyses the formation of selenophosphate
TrxR 1 Cytoplasmic thioredoxin reductase, involved in many biological pathways
TrxR 2 Mitocondrial thioredoxin reductase, involved in many biological pathways
TrxR 3 (TGR) Thioredoxin/glutathione reductase found mainly in testes
74
in different tissues are also not well known. The cytosolic or cellular form of glutathione
peroxidase (cGSHPx) was the first selenoprotein identified [10,11]. It is found in almost
all tissues and is believed to play a part in the body’s antioxidant defence. Several other
glutathione peroxidases containing selenocysteine have been found since then. The other
two major groups of known selenoprotein enzymes are the iodothyronine deiodinases,
that regulate thyroid hormones, and the thioredoxin reductases, catalysing the reduction
of oxidised thioredoxin and other substates [1,12].
Many of the known selenoproteins catalyse redox reactions in which selenium is
at the active site. Several of the selenoproteins have an homologue protein containing
Cys instead of Sec, but the catalytic ability of these latter proteins is much lower. There
are no selenoproteins known to be present in yeast or higher plants, these organisms
tending to have homologue proteins containing Cys instead of Sec [13].
Biosynthesis of selenoproteins
Translation of the codon UGA to selenocysteine (Sec) and its insertion into proteins
is a complicated process (Figure 2.8.). In the first step, serine is bound to tRNASec and then
transformed to selenocysteine [13]. The next major step is the interpretation of UGA
as a signal to insert Sec instead of stopping the translation. This takes place when
a special stem-loop structure called the selenocysteine insertion sequence (SECIS)
is present in the mRNA. This structure is located in the 3’-untranslated region of the
mRNA in eukaryotes and immediately after the UGA codon in prokaryotes [14,15].
All known eukaryotic selenoproteins have one SECIS-element, except for SeP, which has
two [15].
Except for the SECIS element there are no features in the DNA-sequence that
selenoprotein genes are known to have in common, selenoproteins also differing
markedly in the nucleotide sequence of their SECIS elements. The SECIS consensus
sequence consists of two helices, one internal loop, one apical loop, and the SECIS core,
which is a short sequence of non Watson-Crick paired nucleotides that appear to exist
in all selenoprotein mRNA:s. Preservation of the SECIS core and of the length of the helix
between the first internal loop and the second internal loop or the apical loop are
important for the functioning of SECIS [16].
In addition to the SECIS element, additional factors are required for the insertion
of Sec in eukaryotes such as the SECIS binding protein 2 (SBP2), the Sec-specific
elongation factor (EFsec), and the recently discovered L30 protein, but the mechanisms
involved have not yet been elucidated (Figure 2.8.). In eukaryotes the Sec codon and the
SECIS element are located at some distance from each other in the mRNA. The current
conception is that the mRNA and the SBP2-EFsec loops back towards the translation
complex [13,17].
75
Fig. 2.8. Hypothesised Sec translation complex in eukaryotes. The SECIS binds to SBP2 which binds
the Sec-specific elongation factor EFSec and its Sec-tRNASec. After association with the ribosome the SBP2
and the L30 switch places, causing a conformational change which leads to the release of Sec-tRNASec,
and hydrolysis of GTP [17].
The stability of selenoprotein mRNAs is affected by the amount of selenium present

in the cell. A special feature is that transcripts of some selenoproteins are much more
stable than those of others, there being a selenoprotein hierarchy. This hierarchy is
particularly noticeable in the glutathione peroxidase family, where the order of stability
is as follows: giGSHPx ≥ phGSHPx > cGSHPx = eGSHPx, but it is not yet known how
this regulation operates [18,19]. The role of the SECIS for mRNA stability was studied by
combining the coding regions of giGSHPx, phGSHPx and cGSHPx with the 3’UTRs
(3’-untranslated regions) of each mRNA and then to study the system during selenium
deficiency. It was shown that giGSHPx and phGSHPx containing each other’s 3’UTRs
would remain stable, but not with that from cGSHPx. cGSHPx could not be stabilised
by replacing its 3’UTR with those from more stable glutathione peroxidase mRNAs.
This indicates that in the cGSHPx mRNA, at least there are factors in both the coding
and the non-coding mRNA regions that influence its stability [19].
There is also a tissue selenium hierarchy that controls the retention of selenium in the
tissues and organs under selenium-deficient conditions. In rats and mice fed a selenium-
deficient diet, the brain and the testes retain selenium whereas the selenium
concentrations in the liver and the kidneys decrease markedly [20]. The links between
selenium and cancer probably involve different selenoproteins. Accordingly an overview
of the individual selenoproteins is provided.
Individual selenoproteins
The glutathione peroxidase family

The glutathione peroxidases generally catalyse the reduction of peroxides using mainly
glutathione as the electron donor, thus contributing to the body’s defence against free
radicals. Five selenium-dependent glutathione peroxidases are known to date [9].
76
Cellular (or cytosolic) glutathione peroxidase (cGSHPx), which is found in most

tissues, catalyses the reduction of hydrogen peroxide and organic peroxides. It consists
of four identical subunits, each containing one selenium atom. Under conditions of severe
selenium deficiency, the level of cGSHPx in most tissues decreases considerably, without
any obvious damages to the host organism, which has led to speculations that this
enzyme is a form of selenium storage to some extent [21]. cGSHPx knock-out mice have
been used to explore the effects of complete loss of cGSHPx activity. 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. In model
systems with an over-expression of cGSHPx, protective adapatations against paraquat
and other challenges were shown to take place, there also being a number of negative
effects such as increased obesity and insulin resistance [22].
Gastrointestinal glutathione peroxidase (giGSHPx), which consists of four subunits,
is mainly found in the gastrointestinal tract and also in the human liver and in mammary
cells and tissue according to certain studies [23,24]. Putative functions of this enzyme are
those of protecting against ingested lipid hydroperoxides and reducing susceptibility
to colon cancer [21]. giGSHPx knock-out models show no particular changes
in phenotype to occur compared with the wild type, but knock-out of both cGSHPx
and giGSHPx leads to colitis in mice [25].
Extracellular glutathione peroxidase, eGSHPx, is a tetrameric protein produced mainly
in the kidney and then excreted into the extracellular environment [26]. Other tissues
such as liver, skeletal muscle, pancreatic, thyroid, placental and mammary gland tissue
produce this enzyme to a lesser extent. This glutathione peroxidase isoenzyme is also
found in extracellular fluids such as blood plasma, milk, amniotic fluid, lung lavage
and the aqueous humour [21,27–29]. This is the only selenoprotein that has been
identified in milk thus far. Although the glutathione concentration in the plasma is very
low eGSHPx can also use other electron donors such as the thioredoxin system [30].
The postulated roles of eGSHPx include control of peroxide transport and of the
extracellular ‘peroxide tone’ [18,21].
Phospholipid hydroperoxide glutathione peroxidase, phGSHPx, catalyses the
reduction of phospholipid hydroperoxides and is expressed in a wide range of tissues.
It differs from the three glutathione peroxidases just mentioned by being a monomer and
having a different substrate specificity. This enzyme has been implicated in inflammation
and molecular signalling. Disruption of the phGSHPx gene is lethal embryonically,
unlike disruption of cGSHPx or giGSHPx. phGSHPx occurs in mitochondrial, non-
mitochondrial and sperm-nuclei-specific forms produced from the same gene [31] and has
an important role in sperm functioning [32].
The most recently discovered member of the glutathione peroxidase family
is glutathione peroxidase 6, which has thus far only been found in olfactory epithelium
and in embryos, and its functional significance is unclear. In mouse and rat, the seleno-
cysteine in this enzyme is replaced by cysteine [9].
77
The thioredoxin reductase family

The thioredoxin reductases (TrxR) catalyse the reduction mainly of thioredoxin, but
in mammals they can also reduce other substrates, such as vitamin C. Thioredoxin
catalyses the reduction of protein disulfides and is involved in a number of vital processes,
such as DNA synthesis and the regulation of apoptosis. There are three main isoforms
of thioredoxin reductases, but a number of splice variants of TrxR 1 and 2 have also been
reported. TrxR 1 and 2 are ubiquitous being located in the cytosol and the mitochondria,
respectively [12]. The third isoform, thioredoxin/glutathione reductase (TGR), is ex-
pressed in small amounts in many tissues but is primarily found in the testis [33]. Targe-
ted disruption of the TrxR 1 or 2 genes in mouse models is embryonically lethal [22].
The iodothyronine deiodinase family

The iodothyronine deiodinase family consists of three enzymes, iodothyronine deiodinase
1, 2 and 3 (DI1, DI2, DI3), which catalyse the removal of different iodine groups from the
thyroid hormones, thus activating or deactivating them. The iodothyronine deiodinases
have high priority in the selenium hierarchy, particularly DI2 and DI3, their levels
remaining virtually unaltered in the case of selenium deficiency. In some tissues,
DI1 decreases during selenium deficiency, but not in the thyroid. DI1 is found in the liver,
kidneys and thyroid, for example, and expression of DI2 and 3 having been found in many
tissues, including bovine mammary tissue [34–36].
Selenoprotein P
Selenoprotein P (SeP), the second selenoprotein to be discovered, was designated “P” be-
cause of its being found in the blood plasma. It can contain from 1 to 17 selenocysteines,
depending on the animal species. Truncated isoforms of this protein have also been found.
SeP is expressed in most tissues, but is produced primarily in the liver and is secreted then
into the plasma. Selenoprotein P is the major form of selenium in the plasma and
is involved in selenium transport [37,38]. There are indications that it also acts as
an antioxidant in the extracellular space. It is localised in the endothelium, binding
to heparin and related carbohydrates [38]. It can reduce peroxynitrite and phospholipid
hydroperoxides [39,40], can also form complexes with mercury and cadmium [41],
and can stimulate the survival of nerve cells in culture [42]. SeP knock-out mice exhibit
low levels of selenium in the brain and testes, organs that normally are highly prioritised
during selenium deficiency. These mice die after weaning of the young, unless they
are rescued by a high-selenium diet [43].
Additional selenoproteins
Selenophosphate synthetase 2 (SPS2) catalyses the formation of selenophosphate, which
is the selenium donor for the formation of selenocysteine from serine bound in tRNASec.
SPS2 is a selenoprotein, this property indicating the existence of a feedback step in the
production of selenoproteins. Selenophosphate synthetase 1, which is not a seleno-
protein, also catalyses the formation of selenophosphate [44]. The 15 kDa selenoprotein,
78
located in the endoplasmatic reticulum (ER), is believed to be involved in protein folding

[45]. Selenoprotein M (SelM) is structurally similar to the 15 kDa protein and
is supposedly also involved in protein folding in ER [46]. Selenoprotein R (SelR) reduces
methionine-R-sulfoxides. This is an important step in the regulation of biological
processes and the management of oxidative stress in the cell. It has been identified
in prokaryotes, eukaryotes and archaea [15]. Selenoprotein N (SelN) is a 70 kDa protein
located in the ER. Although its catalytic function is still unknown, mutations in the gene
have been associated with various muscular diseases, such as rigid spine muscular
dystrophy. To date, SelN is the only selenoprotein in which a mutation of it has been
shown to cause a disease [47]. Selenoprotein S (SelS, also known as VIMP) is a membrane
protein in the ER, one that has been associated with the process of eliminating misfolded
proteins by transferring them to the cytosol [48] and also with inflammation [49].
The function of selenoprotein W (SelW) has not been elucidated fully but it has been
implicated in white muscle disease [50]. SelW occurs mainly in muscle and brain and has
been shown to act as an antioxidant, utilising glutathione to reduce peroxides [51].
A number of other selenoproteins have been discovered in man but information regarding
their function is very limited.
Indices of selenium status
The most commonly used methods for assessing the selenium status in humans involve
analysis of selenium concentrations in the blood or blood fractions. In addition,
the determination of selenium in the hair, nails and urine has been employed. Selenium
in the plasma or serum is the best known and most accessible index, usually responding
rapidly to changes in selenium status or in the dietary intake of selenium [52].
The responses to selenium intake obtained if other blood fractions are analysed can differ.
For instance, the selenium levels in whole blood or the erythrocytes appear to primarily
reflect the long-term intake of selenium [53], since the turnover of erythrocyte selenium
is slower.
Selenoproteins as biomarkers
The use of selenoproteins as markers of selenium status has only been exploited thus
far in a few large epidemiological studies. In contrast, the activity of GSHPx in plasma
has been used by a variety of laboratories in selenium supplementation studies,
and it responds rapidly to changes in selenium status, and may thus be suitable
as an indicator of short-term changes.
In general, the measurement of selenoproteins can be expected to provide information
on specific selenium functions, as compared with plasma selenium, which also includes
non-specifically bound selenium. Another important conception is that plasma
selenoproteins may not be suitable biomarkers under conditions of high selenium status,
since above a certain selenium level they tend to reach saturation. When data from
79
different cross-sectional studies was combined eGSHPx was found to approach a plateau
at a plasma selenium concentration of approximately 1 mmol/l [54]. Supplementation by
selenate resulted in glutathione peroxidase activity plateauing at a plasma selenium
concentration of 1.2 mmol/l [55].
Selenoprotein P, glutathione peroxidase and protein-bound selenomethionine are the
major selenium fractions contained in plasma [56]. Selenoprotein P accounts for at least
40% of the plasma selenium [37]. Immunoassays for measurement of this protein have
been developed [57–59]. In the following various results from their use in the authors’
laboratory are summarized (Table 2.11.).
Table 2.11. Plasma concentrations of SeP, selenium and eGSHPx in different studies (from ref. [63]).
SeP Plasma selenium eGSHPx

Study n
(a.u.) (µmol/l) (mg/l)
European countries 414 1.41 (1.39, 1.44) 1.10 (1.08, 1.12) —
Prior to LDL-apheresis 13 1.07 (0.92, 1.22) 0.73 (0.60, 0.86) 352 (306, 397)a
After LDL-apheresis 13 0.55 (0.44, 0.66) 0.41 (0.33, 0.51) 302 (259, 346)a
Finland, Trial I baseline 50 1.03 (0.98, 1.07) 0.86 (0,83, 0.88) 6.51 (6.28,6.74)b
Finland, Trial II baseline 45 1.77 (1.69, 1.85) 1.38 (1.34, 1.43) —
Cancer cases 302 1.20 (1.16, 1.24) — —
Controls 406 1.23 (1.21, 1.25) — —
Elderly subjects
(Malmö Food Study) 126—205 1.47 (1.43, 1.52) 1.14 (1.11, 1.16) 4.13 (4.0, 4.27)
Patients on HPN 38 0.69 (0.56, 0.83) 0.52 (0.41, 0.64) 1.91 (1.51, 2.31)
Latvians with differing 21 0.83 (0.54—1.15) 0.69 (0.30—1.14) 2.78 (1.20—4.32)

fish intakec 16 1.00 (0.63—1.83) 0.91 (0.46—1.47) 3.38 (2.31—4.65)
31 1.38 (0.75—2.21) 1.18 (0.66—1.56) 3.95 (2.69—5.73)
Values are means (95% CI).

a
GSHPx activity, U/l.
b
GSHPx activity, mU/mg protein.
c
Data from subgroups having (top to bottom) low, medium and high fish intake, respectively. Median values (and range) are given.
80
Selenoprotein P as a biomarker
Selenoprotein P levels were measured in healthy adults from 17 European Regions [60].
Considerable variation between different regions was found. There was a close cor-
relation between selenoprotein P and plasma selenium values, with some indication
of a plateau. In the Malmö Food Study [61] the relationship between the intake of diffe-
rent foods and selenium status was investigated. For women, significant relationships
between milk intake versus selenoprotein P and urinary selenium, and also between fish
intake versus serum and urinary selenium were found. In a study of Latvian fisherman
[62], a highly significant positive relationship between fish intake and selenium status
was obtained as well as an inverse relationship between the plasma selenium and thyroid
stimulating hormone levels. Home parenteral nutrition patients with a variety of gastro-
intestinal diseases showed much lower levels of extracellular GSHPx, total selenium
and selenoprotein P than control subjects did [64]. Concerning the association
of selenium status and exposure to toxic metals, studies of lead-exposed children
in Katowice, Poland revealed an inverse relationship between the blood lead level and the
level of selenoprotein P and of extracellular GSHPx [65]. Since the direction of causality
was uncertain, however, 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. Regarding the relationships between SeP,
eGSHPx and plasma selenium as markers of selenium status, 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, but that at normal
selenium status selenoprotein P usually correlated more highly with plasma selenium
than eGSHPx did. The levels of selenoprotein P and eGSHPx were found to vary markedly
for subjects from different regions and with different diseases.
Biomarkers of selenium status in relation to selenium supplementation
Many studies on efforts to improve selenium status through selenium supplementation

have been performed and a review of different modes of selenium supplementation has
been assembled [66]. Considerable attention has been directed at two studies conducted
in Finland regarding the use of different forms of selenium [67,68]. The scientists there
compared the responses to oral supplementation of 200 mg selenium per day in healthy
subjects on two occasions. In the first study, in which the subjects were low in selenium
status, the selenoprotein P values increased after selenium supplementation, plateauing
within two weeks [69] (Figure 2.9.). In the second study, performed after the introduction
of selenium-enriched fertilizers in Finland, the selenium status of the subjects thus being
higher, no significant increase in selenoprotein P levels was observed after selenium
supplementation and no differences were found between groups given different forms
of selenium [69]. A summary of the responses shown by the different selenium indices
in the first of these two studies is provided in Figure 2.9. In a recent study of Chinese
subjects of low selenium status given different doses of selenite and selenomethionine,
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. Full expression
of selenoprotein P was not achieved at the highest dose of either form (66 µg/d). This
suggested that selenoprotein P is a better indicator of selenium nutritional status than
glutathione peroxidase is [70].
Generally speaking, 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, 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.
Fig. 2.9. 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 [67,69]).
Biomarkers of selenium status and cancer
Experimental studies have shown that the addition of high (0.5–2 ppm) levels of selenium
to the diet has a carcinostatic effect in animals treated with carcinogenic chemicals
[71,72]. Interest in the preventive role of selenium supplementation in humans was
stimulated markedly by results from two intervention studies. Blot and coworkers [73]
found that a mixture of selenium, β-carotene and α-tocopherol reduced the total cancer
mortality and stomach cancer rate in a Chinese population. Clark and associates [74],
in turn found selenium supplementation to reduce total incidence and mortality
of cancer in an American study group (see below).
In a number of case-control studies, lower prediagnostic plasma selenium was found
for cases of cancer than for controls, particularly among men [75–81], whereas in other
studies no significant differences in this respect were found between cases and controls
[82–86]. The calculated degree of protection was found to differ between cancer sites
[81,87]. 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.).
82
Relations between the selenoprotein P level and risk of cancer
In most studies of the association between selenium status and risk of cancer, plasma
selenium has been used as a marker of selenium status. More recently the premorbid level
of SeP in the plasma of subjects who had developed cancer at different sites was studied
in a nested case-control study [88]. When cases were divided into subgroups according
to cancer site, the SeP levels for cancer of the respiratory tract were found to be
significantly lower than in matched healthy control subjects. The association between
the relative risk of getting cancer and the SeP concentration found was also estimated
from quintiles of the SeP level. For increasing quintiles, the ORs (adjusted for smoking)
were 5.2, 2.3, 2.9, 2.0 and 1.0, respectively (p for trend = 0.01). In addition, the ORs
(adjusted for smoking) in tertiles of the SeP level were calculated for the respiratory,
digestive and urinary tract and for cancer of other sites. These were 6.0, 3.4, 0.2, and 0.6
respectively, in the lowest tertile as compared with the cases in the highest tertile.
In Figure 2.10. the case-control differences of plasma selenium and SeP are compared
for major cancer sites in several different study populations. The previously reported
association of plasma selenium levels with cancer risk can very likely be explained by the
corresponding association of SeP levels. This is probably due to SeP constituting at least
40% of the total selenium in human plasma [37].
Fig. 2.10. Percentage differences in the prediagnostic selenium and SeP plasma levels between cancer cases
and controls (set at 0) for cancer at different sites. The circles represent selenium and the squares SeP. Bold
contour: p < 0.05, normal contour: p ≥ 0.05. The individual references are cited in [63].
Smoking and biomarkers of selenium status
Several factors other than selenium intake may affect biomarkers of selenium status.
Smokers were found to have significantly lower levels of selenoprotein P than non-
smokers [88]. In other studies, lower values of plasma selenium [77,85,89], whole blood
selenium [89,90], erythrocyte selenium [89], and toenail selenium [90–92] were observed
in smokers than in non-smokers. The factors contributing to the lower selenium status
in smokers are unclear. 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.
83
The finding that selenoprotein P is positively correlated with albumin level and
negatively correlated with α1-antitrypsin, both being acute-phase reactants, and that
these correlations are higher (more significant) in smokers, suggests that the
selenoprotein P levels are reduced by inflammatory activity. This agrees with findings
of Dreher and co-workers [93] indicating that the human selenoprotein P promoter
is rendered less active by cytokine treatment, which suggests a repression of selenoprotein
P expression during an acute phase reaction. Smoking may also increase oxidative stress
since cigarette smoke is a rich source of reactive nitrogen species, which together with
superoxide can produce peroxynitrite [39]. As reported by Sies and coworkers [94],
selenomethionine and glutathione peroxidase can scavenge peroxynitrite. It has also been
shown that selenoprotein P plays a role in the defence against peroxynitrite [39]. This
may also explain the slightly lower selenoprotein P concentration in smokers.
The natural presence of cadmium in tobacco smoke may also contribute to the lower
selenium status found in smokers. 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, whereas the concentration of cadmium in the blood is significantly
higher in smokers [95]. Multiple linear regression analysis of the data also suggested
a depressive effect of cadmium on the concentration of selenium in the blood, whereas
smoking alone did not serve as a true predictor of this effect. In another study, the blood
levels of selenium and cadmium and the plasma levels of selenoprotein P were measured
in children from the Katowice industrial area in Poland [65]. 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. Multiple regression analysis also indicated the blood
cadmium to increase significantly with a decrease in the selenoprotein P level, although
this association disappeared when lead was included in the model, a result that could
possibly be explained by the covariance of lead and selenium in the blood [65].
It has been reported that smokers eat less selenium than non-smokers do, which could
probably in part explain their lower selenium level [90]. In addition, in a meta-analysis
of 51 published nutritional surveys of the relationship between smoking status and
nutrient intakes, smoking was found to be significantly associated with an unhealthy
pattern of nutrient intake, which could exacerbate the risk of cancer associated with
smoking [96].
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, several intervention studies
having indicated beneficial effects [73,74]. In the Linxian trial involving supplementation
of β-carotene, α-tocopherol and selenium given for a 5.25-year period, a small but
significant reduction in total cancer mortality was obtained (RR = 0.91), in particular
mortality due to stomach cancer [73]. Patients with a history of skin carcinomas who
84
were treated with 200 mg selenium per day showed significant reductions in total cancer
mortality (RR = 0.50) and in incidence of lung (RR = 0.54), colorectal (RR = 0.42) and
prostate (RR = 0.37) cancer [74], wheras later results showed selenium supplementation
to be ineffective in preventing basal cell carcinoma and that it increased the risk
of squamous cell carcinoma and of total nonmelanoma skin cancer [97]. Experimental
findings also indicate that in some experimental systems selenium can both promote
and inhibit cancer [98]. Recent results of the SUVIMAX study showed supplementation
with vitamin C, vitamin E, β-carotene, selenium and zinc to reduce the rate of prostate
cancer in men having normal levels of prostate-specific antigen in their plasma [99].
In another Chinese investigation, selenomethionine supplementation of subjects with
mild to moderate esophageal squamous dysplasia showed there to be a nonsignificant
trend toward an increased regression and decreased progression of dysplasia as well
as a significant beneficial effect in the subgroup showing mild esophageal squamous
dysplasia [100]. A summary of results of the first generation of nutritional intervention
studies to prevent cancer have been presented [101] and future directions and criteria
for evaluating the efficacy of such interventions have been proposed [101,102].
In addition, evaluation of health claims by the FDA in the U.S. concerning the purportedly
positive effects of selenium in connection with the prevention of cancer provided certain
evidence for permitting a qualified health claim regarding selenium and cancer [103].
It is apparent from this review that selenium can play an important role in cancer
prevention, but additional studies are needed to determine whether there is also an in-
creased risk of some forms of cancer after selenium supplementation. The type of se-
lenium supplements best employed is also in need of further investigations. A better
understanding of the mechanisms by which selenium interferes with the carcinogenesis
process is a necessary focus for future research also for the evaluation of selenium related
biomarkers.
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Vitamins and selenium: Anticancerogenic activity of selenium
91
2.5. Anticancerogenic activity of selenium —

molecular mechanisms and epidemiological data
Jolanta Gromadziƒska, Edyta Reszka, Wojciech Wàsowicz

Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, ¸ódê, Poland
Introduction
There are many different aspects of the relationship between selenium and cancer,
including the forms of selenium present in the diet and in the body, their functions
and mechanisms of action, the methods used in assessing the nutritional status
of selenium — in general, in response to selenium supplementation, and in epidemio-
logical studies of risk of cancer — and the outcome of long-term trials concerned with
the disease-preventive potential of selenium supplementation. Two of the chapters deal
with these and related matters. In the present chapter the mechanisms behind the role
that selenium can play in cancer prevention, as well as epidemiological findings
regarding the relationship between selenium level and risk of cancer are reviewed. In the
previous chapter the forms and metabolism of selenium, in addition to the occurrence
of different selenoproteins, their use as biomarkers of selenium status, and intervention
trials to investigate the effect of selenium supplementation on the incidence of cancer
are summarized.
History
Historically, interest in selenium (Se) as a toxic trace element dates back to 1937 [1],
and concern for it as a carcinogenic agent dates to 1943 [2]. Nelson et al. [2] observed that
liver tumours occurred in rats fed protein-deficient fodder and grains originating from Se-
rich (5–10 ppm) regions. Of 53 animals that survived for 18 months when thus
fed, 11 were found to develop liver tumours, whereas such tumours were found in less
than 1% of the animals in the control group. Shapiro [3], however, suggested that
the pathological changes that occurred reflected regeneration of the liver rather than
a carcinogenic process. Researchers of the former Soviet Union [4] feeding Se (as selenite)
to rats at a 4.3 ppm level, observed the development of different forms of liver tumours.
Seifter et al. [5] published the alarming finding that feeding rats 0.05–0.1%
bis-(4-acetamine)-phenylselenyl hydroxide, equivalent to inclusion of 103–207 ppm Se
in the diet for a 10-day period, induced thyroid adenoma. It should be noted, nevertheless,
that since no studies have been reported of animals administered an analogous compound
that did not contain selenium, it is quite possible that the organic selenium compound
employed there had carcinogenic properties not linked with the element per se. In 1963,
however, Tscherkers et al. [6] observed the development of tumours in male rats fed
a diet containing 4.3 ppm Se, which suggests that the neoplastic changes that occurred
could be attributed to Se.
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The anticancerogenic effect of selenium in animals was first reported in 1911, when
Wassermann et al. (cf. [7]) managed to inhibit the development of placental tumours
in mice by use of Se compounds. Another early observation of such an effect was that
of Clayton and Bauman [8], who reported that in rats a diet enriched with 5 ppm Se
decreased the incidence of liver tumours brought on by 3’-methyl-1,4-dimethylamino-
benzene (3’-MeDAB). 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%, respectively, in rats to which either
of two different forms of Se were administered [9]. Supplementing a standard diet
with administration of sodium selenite was also found to reduce the number of tumours
that developed in rats that were given 2-acetyloaminofluorene (2-AAF) as a carcinogenic
agent [10–12].
Ip and Sinha [13] investigated the effect of various concentrations of Se on the
development of breast cancer induced in rats by 7,12-dimethylbenzantracene (DMBA).
Two groups of animals received a diet containing maize oil. The maize oil content in
the one case was high (25%) and in the other case low (5%). In both groups the incidence
of cancer was found to decrease with increasing level of Se in the diet. At the same time,
the Se concentration influenced neither the level of malondialdehyde (a final product
of lipid peroxidation) in the breast carcinoma cells nor the level of glutathione peroxidase
(GSH-Px) activity there [13]. The authors concluded that the protective role of selenium
was not due to its being able to inhibit lipid peroxidation or to its having any anti-
oxidative function in fat metabolism [13]. Ip [14] also studied the effect of selenium
on different stages of cancer development in rats given 5 ppm Se at different time
intervals after the administration of 10 mg DMBA. Supplying Se was found to decrease
the number of tumours induced (from 97 to 46%), particularly when it was provided at
both the initiation and the promotion stage of chemical carcinogenesis. Se supplied
during only one of these two phases had a much weaker effect [14].
Harr et al. [10] showed that adding 0.1 to 0.5 ppm Se to the diet of rats given
a carcinogenic substance led to a decrease in the occurrence of tumours from 80% (in the
non-supplemented group) to 10% (in the group receiving Se). Providing selenium at a still
higher level (2.5 ppm) reduced the incidence of cancer even more, to 3%. An interesting
study of the anticarcinogenic role of selenium was carried out by Schrauzer et al. [15],
using female mice of the C3H strain, a strain characterized by the spontaneous
development of breast adenoma in 80% or more of the cases. The animals were divided
into several groups, a control group receiving a basal diet that contained 0.15 ppm Se
and the other groups receiving, in addition to this, a supply of 0.1 to 1.0 ppm Se in their
drinking water. In the control group, the first tumours developed when the animals
were 4 months of age, whereas in the group receiving the largest amount of Se in its
drinking water (1.0 ppm) they first developed at 17 months of age. In the groups given
extra Se in their drinking water, tumours not only developed later, but they also decreased
in number with an increase in the amounts of selenium provided. These results indicated
that supplying Se in larger amounts protects animals from developing cancer or delays
93
the carcinogenic processes. The anticarcinogenic effect of Se has been found to depend
on the chemical form in which the element is administered, its dose and the agent that
induces the development of cancer [16]. 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. Cells adequately supplied with Se have been
shown to be less sensitive to effects of both endogenous and exogenous carcinogens [17].
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, viruses or transplanted
tumours [18]. 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), in the
majority of cases the reduction being quite significant [19]. Experiments in which no
effect of Se was observed were rather rare. 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. At the same time, one should
bear in mind that the results of animal studies cannot be directly extrapolated to humans.
Mechanisms responsible for the link between selenium and cancer prevention
Experimental studies have thus shown that adding high levels of selenium to the diet
of animals treated with carcinogenic chemicals has a carcinostatic effect [20]. Regarding
the mechanisms involved, it has been postulated that the chemopreventive effect is related
to the toxicity of selenium and the oxidative stress it induces, since reactive oxygen species
can promote apoptosis in vitro [21]. It has also been shown, however, that selenium
compounds can induce cell death through a mechanism distinct from oxidant toxicity
[22,23]. 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. In several studies, the activity of xenobiotic-metabolizing enzymes
in vivo has been reported to increase when selenium compounds are given, resulting
in more efficient carcinogen detoxification [24,25]. It is also possible that selenium in the
form of glutathione peroxidases, and perhaps selenoprotein P and other selenoproteins
as well, can prevent mutations by serving as free radical scavengers [26–28].
Thus a number of mechanisms for the anticarcinogenic effects of selenium and
of many selenocompounds have been proposed, including their providing protection
against oxidative damage, altering the metabolism of carcinogens, enhancing immune
responses, affecting the cell cycle, and inhibiting angiogenesis [29,30] (Tables 2.12.
and 2.13.). Some reports suggest that the action of selenium toward cells that have been
transformed earlier and toward normal cells differ [32]. The anticarcinogenic effects of Se
depend upon the chemical form of the Se-compound involved and the nature and dosage
of the carcinogen. The effects can occur at a systemic, cellular or nuclear level. The activi-
ty of many cellular targets, in which the metabolism, proliferation and differentiation
of the cells can be affected, depends on the cellular GSH/GSSG ratio. Glutathione
94
peroxidases and thioredoxin reductases are involved in ROS scavenging. This suggests
that selenoproteins participate in the regulation of intracellular signal transduction [33].
Yet non-protein selenium metabolites may also be important in the regulation of intra-
cellular communication and metabolism. Se and the selenoproteins play a regulatory role
in the following processes, for example:
— ROS-activation of protein kinases in the cytoplasm and nucleus.
— ROS-activated modification of the thiol and hydroxyl groups in the Cys and Tyr.
— Controlling changes in the cell redox potential through inducing activation of the
transcription factors and initiating de novo gene expression.
— Regulating the expression of membrane and nuclear receptors responsible for cell
maintenance, intercellular communication, and changes in cell growth.
— Affecting apoptosis, necrosis and cell survival processes [34].
Table 2.12. The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis
(ref. [16])
Form of selenium Parameter Outcome
Selenite Growth of Ehrlich ascites tumour cells in mice I

Selenite Growth of L1210 leukemic cells I
Selenodiglutathione Growth of L1210 leukemic cells I
Selenite, Selenodiglutathione Cell growth (in vitro) I
p-XSC, BSC, selenite DNA, RNA, protein synthesis I
Apoptosis E
Selenite DNA synthesis (in vitro) I
Selenite RNA and protein synthesis I
Cell death (necrosis SSB) E
Selenite Cell growth I
Selenite Cell cycle Block S/G2-M
Selenite p53 E
AP-1 I
NF-κB I
CH3SeCN, p-XSC,
Se-methylselenocysteine, DNA synthesis (in vitro) I
Cell cycle Block G1
Apoptosis E
BSC and ist glutathione ACF I
conjugates COX-2 I
p-XSC, BSC PKC, PKA I
95
Table 2.12. The effects of selenium compounds on cell growth and on the molecular targets of cancerogenesis
(ref. [16]) — cont.
Form of selenium Parameter Outcome
Selenite PKC I
p-XSC TK I
Selenite, selenium dioxide, Phospholipid/Ca+2 dependent PKC I
selenic acid
Ebselen PKC I
P-XSC, BSC JNK Dose dependent E/I
p-XSC, BSC, selenite DNA cytosine methyltransferase I
Se-methylselenocysteine PKC (in vitro) I
Se-methyselenocysteine, Cell proliferation and cell cycle biomarkers I/E/NE
triphenylselenium chloride (in vitro)
p-XSC PKC and isoprostane (in vivo) I
I — inhibition; E — enhancement, W — weak, NE — no effect; AP-1 — activator protein 1; NF-κB — nuclear factor κB; ACF — aberrant crypt foci;
COX-2 — cyclooxygenase; PKA, PKC — protein kinase A and C; TK — thymidine kinase; JNK — Jun-N-kinase;
p-XSC — 1,4-phenylenebis(methylene)selenocyanate; BSC — benzyl selenocyanate.
Table 2.13. In vitro effects of selenite and methylated selenocompounds [31]
Methylselenocyanate
Endpoints Selenite or Se-methylselenocysteine
Cell morphology Extensive cytoplasmic vacuolisation, cell detachment Normal

Membrane damage Yes No
Cell growth inhibition ++++ ++
DNA synthesis inhibition ++++ ++
Cell cycle block S/G2-M G1
DNA single strand breaks ++++ None
Cell death Necrosis Apoptosis
Gadd gene induction Late Early
Fibronectin is an extracellular component that plays an important role in intercellular

communication. Inorganic Se compounds such as selenite reduce the number of fibro-
nectin receptors at the cell surface. Since this is an immediate action, it cannot come
about through activity of the selenoproteins. Selenite activates the protein kinases
involved in the pathways of cellular response to stimulation by inflammatory agents,
whereas these processes can be inhibited by selenate [34]. Oxidation of the thiol groups
96
in the transductional proteins results in their being modified structurally, which can lead
to their becoming activated. This process in turn results in the activation of AP-1 and
of such transductional proteins as ras/rac, fos, myc and c-Jun N kinase [35,36]. In human
hepatoma cells, selenite inactivates the c-myc oncogene and activates the c-fos genes
that lead to the growth of normal cells [37]. In human colon cancer cells, Se inhibits
the expression of one of the activating zinc finger proteins that regulates the activation
of the c-myc oncogene [38].
The redox potential is an important factor in regulating the nuclear factor κB (NF-κB)
and the activation of AP-1 [39]. NF-κB is a crucial target in the pathogenesis of various
chronic inflammatory, degenerative and neoplastic diseases. It regulates the effector genes
in the promoter regions and can thus activate or repress gene expression. The up-
or downregulation of the effector genes can modulate many different cell pathways, such
as those of proliferation, growth suppression, differentiation and senescence. NF-κB
is also an important factor in regulating activation of the genes involved in the control
of cell necrosis and apoptosis, the cell cycle, the immune response, and repair processes
[39]. NF-κB activation involves changes in protein conformation and in the binding
of numerous genes, such as cytokines, for example, to the promotor regions. Reactive
oxygen species (ROS) activates the phosphorylation of one of the NF-κB subunits, I-κB,
directly. GSH-Px overexpression inhibits the translocation of another NF-κB subunit
and reduces the phosphorylation of I-κB [40–42]. This process has been found to be
inhibited in cells cultured in an Se-supplemented medium and to be intensified in the case
of Se deficiency [43]. The process the translocation of NF-κB and its binding to DNA
is multiphasic and highly complex. The phosphorylation of mitogen-activated protein
kinases (MAPK) represents one of these phases. The activation of individual kinases of the
MAPK family triggers the transcription factors involved in changes in chromatine confor-
mation and in the expression of numerous genes of proinflammatory and antioxidative
proteins, as well as the genes involved in apoptosis activation, and in cellular proliferation
or differentiation [35]. MAPK inactivation inhibits the cellular proliferation occurring
in the course of the carcinogenic process. GSH-Px and SeP are considered to have
a particular role in MAPK inactivation [44]. GSH-Px in particular can act as a suppressor
of protein kinases. The breakdown of hydroperoxides by GSH-Px and the resulting
decrease in the cellular hydroperoxide level inhibit the Se-activated kinase p38 [34].
The strength of the binding of NF-κB to DNA is influenced by Se, which modifies
the structure of Cys in the regulatory subunit of NF-κB so as to prevent oxidation of the
thiol group located within this factor [34]. The reduced thiol group in Cys is essential
for maintaining the activity of numerous transcription factors [45]. Thioredoxin (Trx)
plays a key role in this process. Oxidative stress induced by chemical or physical factors
translocates Trx to the nucleus, intensifying the binding of NF-κB and AP-1 to the DNA.
This process is made possible by intracellular reduction in the disulfide bridges (-S-S-),
mainly in regions in which DNA binding to nuclear factors occurs [46]. Through acting
as a protein disulfide reductase, TrxR thus affects the redox regulation of a variety
of enzymes and receptors, as well as such transcriptional and nuclear factors as NF-κB
97
and AP1 [47]. Trx inhibits certain kinases of the MAPK family (e.g., ASK1) directly. Se
is also known to arrest several kinase pathways important in signal transduction, such
as protein kinase A, Ca+2-dependent and -independent kinase C (PKC), diacylglycerol
kinase and thymidyl kinase [48].
It has been shown that selenium creates a dose-dependent increase in TrxR activity
and in the expression and stability of TrxR mRNA in different cancer-cell lines [39]. Trx
limits DNA synthesis, whereas Trx/TrxR activates ribonucleotide reductase, a key
enzyme in the deoxyribose formation needed for DNA synthesis. TrxR also regulates gene
expression by affecting the cellular redox status and activating numerous DNA-binding
transcriptional factors: NF-κB, AP-1, ref-1, p53, and the glucocorticoid receptors [34,49].
At the same time, not only the selenocysteine incorporated into the protein structure,
but also its metabolites, affect cellular metabolism. The various chemical forms of Se
have been shown to differ widely in their anticancer properties. Some low molecular
weight Se compounds have been shown to influence the regulation of gene expression,
reduction in the oxidative DNA damage that occurs, the bioactivation of carcinogens,
modulation of tumour angiogenesis, cellular growth, etc. [50,51]. It is important to note
that different selenocompounds are essential for the growth and differentiation of both
normal and neoplastic cells [52].
Effects of selenium on apoptosis and necrosis
In 1992 it was shown that Se(IV) compounds can induce the necrotic death of a cell
by damaging a single DNA strand [53]. It was also observed that methylated Se derivati-
ves can induce apoptosis [48]. The anticancerogenic activities of various Se compounds
are summarized in Table 2.12. [16]. It has been found that both sodium selenite and se-
lenomethionine (SeMet) suppress tumour growth in many animal models involving
a dose-dependent response [20,54,55]. Selenite has been shown to be more effective than
selenomethionine in the inhibition of cancer cell growth during chemically induced
carcinogenesis [31,54], the concentration of Se in the tissues also being found to be higher
after the administration of selenomethionine than after selenite supplementation. 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.
Effect of different selenium compounds in cancer prevention
The chemoprevention of cancer by selenium can involve the action of different

selenometabolites. Selenite, selenodiglutathione, selenomethionine and Se-methylo-
selenocysteine can be transformed into selenides. In cells with a high Se level, high
concentrations of selenides tend to also be generated. When selenides are generated
in sizeable amounts, they react with oxygen to produce superoxide and hydrogen
peroxide [56]. It is believed that the role of selenium in the inhibition of carcinogenic
processes is associated with oxidative damages caused by the redox cycle of selenides [57].
98
When Se is supplied in high concentrations, however, any surplus of it is excreted from

the body. Over 90% of the circulating Se is incorporated into the structure of the
selenoproteins, only about 5% of it being found in other metabolites [58]. It appears then
that the anticarcinogenic action of Se is due to the combined effect of selenium and
selenoproteins [33].
Regarding the selenocompounds, it is the actions of sodium selenite (Se IV),
responsible for single and double DNA strand breaks, and of selenomethionine, which is
not a DNA-damaging agent, that have been investigated most frequently [49]. 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. Experimental studies have
shown that the organic Se compounds induce apoptosis without DNA damage, even
at rather high concentrations (10–50 µM) [29]. Ip and Ganther [59,60], in connection
with studies of Se metabolism they conducted, suggested that methylated Se derivatives
are the most effective selenium compounds for cancer prevention [61]. 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, DNA strand breaks after selenite treatment being an example. The metabolic
pathways of Se-methylselenocysteine and selenobetaine involve the release of methyl-
selenol or methylseleninic acid derivatives, which have been shown in in vitro experiments
to affect apoptosis or to arrest the cell cycle (see Figure 2.7. in the previous chapter).
The results of in vitro experiments on cells treated with selenite and methylated
selenocompounds are summarized in Table 2.13. [31]. Methylselenocysteine is one of the
most important selenocompounds for chemopreventive activity [32]. It is twice as active
as selenomethionine in suppressing mammary carcinogenesis in rodents [62].
Selenium compounds can also affect activation of the p53 gene since the main dietary
selenium compound, selenomethionine, modulates p53 activity. In normal cells, p53
regulates the activity of genes involved in DNA repair processes. In human cancer cells,
p53 is often mutated and its functional activity suppressed. Selenomethionine has been
shown to act together with the p53 tumour suppressor protein in affecting the redox
status. SeMet oxidizes the thiols of the p53 molecules at 275 and 277 cysteine residues.
Seo et al. [49] demonstrated that the Se concentration is a determinant of basal p53
activity, p53-dependent DNA repair being activated within the concentration range
of 10–20 µM. Fiala et al. [63] showed that the selenium compounds as selenite,
1,4-phenylenebis-(methylene)selenocyanate and benzyl selenocyanate inhibit the activity
of DNA cytosine methyltransferase, an enzyme involved in DNA repair processes. They
suggested that this pathway may be the major mechanism involved in the chemo-
prevention that selenium compounds provide after the initiation of carcinogenesis. Two
other Se compounds, sodium selenite and methylseleninic acid, were found to cause
phosphorylation of the serines and threonines contained in the p53 molecule. This
modulation of the p53 molecule may be a specific mechanism of p53 activation.
Methylseleninic acid in a range of concentration of 0.5–1 µM was found to promote DNA
repair processes by increasing the expression of two proteins associated with the p53
99
gene, and to be involved in the pathway connected with the p53-dependent activation
of DNA repair processes [64]. There is a need of explaining how triggering of the
p53-dependent DNA repair process by two different Se compounds can be achieved when
a 10–20-fold higher Se concentration is employed. One possibility is that a small fraction
of the SeMet is metabolized to a low-molecular-weight compound [32]. Seo et al. [49]
showed that in normal human fibroblasts, selenomethionine protects the cells from DNA
damage through the induction of DNA repair. Limitations in the DNA repair capacity
appear to be a key determinant of predisposition to cancer [49,65]. 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 [49,66]. Blessing
et al. [67] described the incorporation of Se into the factors involved in DNA repair
regulation. Reducible selenium compounds (phenylseleninic acid, ebselen, selenocystine,
and 2-nitrophenylselenocyanate) were found to cause a dose-dependent decrease in the
activity of formamidopyrimidine-DNA glycosylase (Fpg), one of the enzymes involved in
DNA repair processes. These selenocompounds affect the DNA repair processes through
oxidation of the zinc finger structures and the release of zinc from DNA, as well
as damaging the integrity of the genes of the DNA repair enzymes [67,68]. They also
affect the integrity of the XPA protein (xeroderma pigmentosum group A protein), which
has an essential role in recognizing DNA lesions in the nucleotides and in the excision
of damaged nucleotides in mammalian cells [69].
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. It has been found that Se derivatives can activate the p53 gene, but that
the induction of apoptosis is not a simple result of the regulation of p53 by selenium [16].
In investigating the ability of inorganic Se compounds to induce 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. This decreased GSH
concentration in the cells induced apoptosis, which is a dominant mechanism of the cell
death these compounds bring about [70].
Selenium can also affect DNA methylation, an important epigenetic mechanism that
exerts control over gene expression [30]. The postsynthetic methylation of DNA is cata-
lyzed by the family of S-adenosylmethionine-dependent DNA methyltransferases [71].
The methylation of Se compounds to Se(CH3)2 and Se(CH3) results in a decrease in
methyl donation, this preventing DNA methylation from occurring. DNA methylation
is one of the first steps in the carcinogenesis induced by certain chemicals, such
as benzo(a)pyrene and arsenic [30].
Selenium can also act as an inductor of the enzymes participating in phase II de-
toxification [48] and it modulates expression of the enzymes involved in phase I detoxi-
fication. In vitro studies of a mammary cell line exposed to DMBA showed 1,4-phenylene-
bis(methylene)selenocyanate and its putative glutathione conjugate to inhibit the
expression of various CYP450 isoenzymes and to induce the expression of various
enzymes involved in phase II or DMBA detoxication [72].
100
Effects of selenium on immune functions

Certain anticancer properties of the selenocompounds may be associated with their
effects on cellular immunity. Selenium compounds can activate cytotoxic cells, stimulate
the expression of cytokine receptors and the proliferation of lymphocytes [73,74]. Low
Se concentrations in the tissues and in human body fluids also appear to be associated
with numerous changes in the immune system, such as suppression of the host immune
response to bacterial and viral infections, the inhibition of prostaglandin and immuno-
globulin synthesis, reduction in the activity of T lymphocytes, NK cells and macrophages,
and impairment of the body’s ability to reject implants and to destroy neoplastic tumours
[75,76]. Cellular membranes of the T lymphocytes are particularly sensitive to Se
deficiency, due to the large amounts of unsaturated fatty acids present in their structure.
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. 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. Insufficient dietary intake
of selenium results in many types of defects of the immune processes, such as in con-
nection with antibody production and specific cell immunity [77]. Selenium regulates the
immune response by stimulating natural killer cells and activating antigens of various
types to destroy the tumour cells [78]. Se can stimulate the expression of IL-2 receptors
found on activated T lymphocytes and on NK cells [79]. The anticarcinogenic effect
of selenium is also partly based on its ability to produce antitumour metabolites
(e.g. methylselenol). 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 [51,80].
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, including nutrients, micronutrients and
phytochemicals [81,82]. Research over the last decade points to a significant protective
role of Se in preventing the development of malignant neoplasms. Low plasma selenium
concentrations are thought to be associated with increased morbidity and mortality from
cancer. In a Chinese study, a statistically significant increase in morbidity from
oesophagus and stomach cancer was noted in a population with low levels of selenium,
but no such relationship was found for lung cancer [83]. An inverse association between
selenium and risk of cancer has been reported both in case-control studies and in follow-
up cohort studies. 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. Case-control studies tend to reflect the short-term Se concentrations
in the organism, such as the Se level associated with the dietary pattern at the time
of sampling in cases and controls. To date, however, the results of epidemiologic studies
101
have been rather inconsistent. Some authors have reported there to be an association
between cancer risk and Se status, whereas others have obtained null results [84,85]. The
findings for three major forms of cancer are summarized below.
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 2.14.). The significant, dose-
dependent protective activity of Se was documented in Finnish and Dutch studies [86,87]
in which Se in both serum and toenail samples was analyzed, whereas no association
between selenium level and risk of lung cancer was found in two studies of non-European
populations [88,89]. 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: 4.33, 95% CI: 0.54–34.60) after adjustments for smoking
status were made [90].
A review of epidemiologic studies of lung cancer and of the selenium concentration
found in biological material, undertaken by Zhou et al [91], indicated selenium to have
a certain protective effect, but only in populations with a low mean Se level.
In a metaanalysis of 14 epidemiologic studies, 11 of which had a prospective design,
the risk of lung cancer at high Se concentrations was found to be RR= 0.74 (95% CI:
0.57–0.97) (Table 2.14.). Interestingly, when the study population was divided
up according to Se level, the mean value obtained for groups showing a high basic
concentration of Se was RR = 0.86 (95% CI: 0.61–1.22), whereas the value for groups
with a low basic level of Se was RR = 0.72 (95% CI: 0.45–1.16). In the control group
of non-cancer patients the mean serum Se concentration was 100 ng/ml blood serum,
representing a daily Se intake of 55 mg. Of the studies considered in the meta-analysis,
only the findings for the two Finnish populations (RR = 0.41; 95% CI: 0.17–0.94
and RR = 0.20; 95% CI: 0.09–0.44) and for the Dutch population (RR = 0.50; 95% CI:
0.30–0.81) revealed a statistically significant protective effect of high Se concentrations.
In most of the reports, the protective activity of Se could be clearly discerned when
the reference group consisted of subjects showing the lowest level of this trace element.
The protective effect of Se in connection with lung cancer could also be noted in studies
examining Se concentration in the toenails. The total RR values for lung cancer
in patients showing high Se levels ranged from 0.46 (95% CI: 0.24–0.87) when toenail Se
was used as a marker of the concentration of Se in the body, to 0.80 (95% CI: 0.58–1.10)
for the serum Se level, to 1.00 (95% CI: 0.77–1.30) in studies of Se intake based on use
of a questionnaire. These finding confirm the assumption that the Se level in the toenails
can be used as a marker of long-term Se concentration [91].
102
Table 2.14. Epidemiological studies of the selenium level in the body and risk of lung cancer (according to Zhuo et al. [91])
Years of No. cases No. controls RR (95% CI) P for dose-res-

Author Study Se index Gender Population follow up (mean Se level) (mean Se level) (high vs. low Se) ponse trend
Comstock et al. [92] NCCa Serum All USA 15—18 258 (0.108 ppm) 515 (0.110 ppm) 0.65 (0.41–1.02) 0.08
Goodman et al. [89] NCC Serum All USA 5—14 356 (119.1 (19.6) µg/l) 356 (117.7 (18.5) µg/l) 1.20 (0.77–1.88) 0.49
Kabuto et al. [93] NCC Serum All Japan 11—13 77 (113.0 µg/l) 120 (119.1 (2.0) µg/l 0.56 (0.20–1.43) NA
M; 112.2 (2.3) µg/l F)
Knekt et al. [94] NCC Serum Male Finland 8—12 153 (57.0 (16.7) µg/l)b 153 (61.0 (13.5) µg/l)b 0.66 (0.37–1.19) 0.001
Knekt et al. [86] NCC Serum All Finland 16—19 77 (53.2 (24.3) µg/l)c 145 (57.8 (16.9) µg/l)d 0.41 (0.17–0.94) 0.46
Ratnasinghe et al. [88] NCC Serum Male China 4—5 108 (46.5 µg/l) 216 (45 µg/l) 1.20 (0.60–2.40) 0.52
Garland et al. [90] NCC Toenail Female USA 3.5 47 (0.811 (0.166) µg/g) 47 (0.897 (0.308) µg/g) 4.33 (0.54–34.60) 0.17
Hartman et al. [95] NCC Toenail Male Finland 5—8 250 (0.537 (0.134) mg/kg) 250 (0.550 (0.129) mg/kg) 0.20 (0.09–0.44)e NA
f
0.61 (0.27–1.41)
Van den Brandt et al. [87] cohort Toenail All The Netherlands 3.3 317 (0.529 (0.206) µg/g male; 2459 (0.547 (0.126) µg/g male; 0.50 (0.30–0.81) 0.006
0.537 (0.080) µg/g female)g 0.575 (0.109) µg/g female)
Hu et al. [96] CC Diet All China — 227 (33.12) µg/day 227 (33.07 µg/day) 1.30 (0.70–2.20) 0.48
Kromhout et al. [97] cohort Diet Male The Netherlands 25 63 (NA) 870 (64.6 (15.2) µg/day) 0.98 (0.41–2.36) >0.01
Zhou et al. [98] CC Diet Female China — 290 (36.10 (16.0) µg/day) 290 (39.80 (33.0) µg/day) 0.76 (0.47–1.15) 0.11
a Nested case-control; b data from 189 sets; c data from 91 individuals; d data from 177 individuals; e male subjects randomized in the earliest in the trial; f male subjects randomized in the 5th year; g data from 370 individuals.
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 2.15.).
Several studies have shown that selenium can be of specific help in combatting prostate
cancer [99]. In the Health Professionals Follow-Up Study, in which the Se concentration
in the toenails served as a measure of long-term Se intake, the odds ratio (OR) for
the development of cancer that was associated with a high level of Se intake
was 0.49 (95% CI: 0.25–0.96) [100]. In the Netherlands Cohort Study, which involved
a 6.3 years’ follow-up period, high Se concentration in the toenails was associated
with an appreciably lower risk of prostate cancer [101]. In two large case-control studies
of male populations in Great Britain and Canada, however, the Se level in the toenails was
not found to be associated to any marked degree with the level of risk of prostate cancer
[102,103]. 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, although a protective effect of a high Se
level (fifth quintile) was found (OR = 0.38, 95%CI: 0.17–0.85). In addition, the protective
effect of high Se levels and high α-tocopherol concentrations in the plasma was only
particularly strong when the γ-tocopherol level was high [104]. 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 = 0.5; 95% CI: 0.3–0.9) [105]. A high prediagnostic Se level was found,
in the Physicians’ Health Study, to be associated (at a 13-year follow up) with a sig-
nificantly reduced risk of prostate cancer, especially men who had a PSA concentration
equal to or higher than 4 ng/ml [106]. The lack of a protective effect of Se in connection
with risk of prostate cancer risk was observed in the men participating in the Carotene
and Retinol Efficacy Trial (CARET) [89]. 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. The findings of this review
showed that the pooled RR of prostate cancer for a particular Se intake, defined
as the average of the 1st and 4th quintiles or the 1st and 3rd quartiles, was 0.72 (95% CI:
0.61-0.84) in cohort studies and 0.74 (95% CI: 0.61–1.39) in case-control studies,
indicating that the intake of selenium was able to reduce the risk of prostate cancer [107].
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 2.16. In a US case-control study
of the relation between the risk of colonic malignant or benign tumour and the serum Se
level, no protective effect of higher Se levels was found in either of the groups investigated
[108]. A lower Se concentration in the serum was found to be associated with a stronger
tendency to be afflicted with a colorectal tumour [109]. Also, colorectal cancer patients
with low Se concentrations were found to have a significantly lower survival time
104
Table 2.15. Epidemiological studies of the selenium level in the body and risk of prostate cancer
Years of No. cases No. controls RR (95% CI) P for dose-res-

Author Study Se indicator Population
follow up (mean Se level) (mean Se level) (high vs. low Se) ponse trend
Knekt et al. [94] NCCa Serum Finland 8—12 46 (59.6 (19.4) µg/l)b 46 (58.3 (14.8) µg/l)b 1.00 (0.42—2.40) 0.707
Goodman et al. [89] NCC Serum USA 5—14 235 (114.8 (19.6) µg/l) 456 (114.3 (20.4) µg/l) 1.02 (0.65—1.60) 0.69
Yoshizawa et al. [100] NCC Toenail USA 5 181 (0.82 µg/g) 181 (0.96 µg/g) 0.39 (0.18—0.84) 0.05
Helzlsouer et al. [104] NCC Toenail USA 1—6 117 (0.77 µg/g) 233 (0.79 µg/g) 0.38 (0.17—0.85) 0.12
(2000)
Van den Brandt et al. [101] Cohort Toenail The Netherlands 6.3 540 (0.530 (0.090) µg/g) 1211 (0.547 (0.126) µg/g) 0.69 (0.48—0.99) 0.008
Nomura et al. [105] Case-control Serum USA (Japanese- — 249 (128.0 µg/l) 249 (131.6 µg/l) 0.5 (0.3—0.9) 0.02
Americans)
Li et al. [106] NCC Plasma USA 13 586 (0.106 (0.018) µg/g) 577 (0.108 (0.018) µg/g) 0.78 (0.54—1.13) 0.16
Allen et al. [103] Case-control Nails UK — 300 (0.622 µg/g) 300 (0.611 µg/g) 1.24 (0.73—2.10) 0.581
a Nested case-control; b data from 51 sets.
Table 2.16. Epidemiological studies of the selenium level in the body and risk of and colorectal cancer
Se indi- Years of No. cases No. controls RR (95% CI) P for dose-res-
Author Study cator Gender Population follow up (mean Se level) (mean Se level) (high vs. low Se) ponse trend
Knekt et al. [94] NCCa Serum Male Finland 8—12 29 (63.3 (18.9) µg/l)b 29 (64.0 (18.2) µg/l)b 1.01 (0.18—5.65) 0.643
Knekt et al. [94] NCC Serum Female Finland 8—12 48 (64.0 (18.7) µg/l)c 48 (65.3 (15.3) µg/l)c 1.10 (0.42—2.92) 0.724
Wallace et al. [112] NCC Serum All USA 1—4 276 (131.5 (19.7) µg/l) 276 (130.3 (17.8) µg/l) 0.76 (0.44—1.30) 0.50
Nelson et al. [108] Case Serum All USA — 25 (138 µg/l) 138 (134 µg/l) 1.7 (0.5—5.9) NA
-control
Garland et al. [90] NCC Toenail Female USA 3.5 89 (0.863 (0.146) µg/g) 47 (0.843 (0.186) µg/g) 2.04 (0.88—4.75) 0.12
Van den Brandt Cohort Toenail Male The Netherlands 3.3 Colon 121 1209 0.82 (0.43—1.58) 0.326
et al. [111] (0.535 (0.092) µg/g) (0.547 (0.123) µg/g) 0.91 (0.41—2.00)
Rectal 76 0.131
(0.593 (0.411) µg/g)
Van den Brandt Cohort Toenail Female The Netherlands 3.3 Colon 112 1246 0.77 (0.41—1.45) 0.733
et al. [111] (0.560 (0.106)) (0.575 (0.108))
Rectal 36 1.58 (0.59—4.22) 0.273
(0.578 (0.091))
a Nested case-control; b data from 32 sets; c data from 59 sets.
105
106
and a lower cumulative cancer-related survival rate than such patients with higher Se
concentrations did [109]. However, 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, selenoprotein P (SeP) concentration or glutathione peroxidase
activity in the plasma [110]. Colorectal cancer risk was also analyzed in a 41-month
follow-up of the Nurses’ Health Study [90], no significant association being found
between risk of cancer and toenail Se status. A similar result was obtained in a 3.3-year
follow-up study of a Dutch cohort in which toenail Se level was used as an indicator
[111]. In a Canadian case-control study, however, a significant inverse association was
obtained between toenail Se level and risk of colon cancer (OR = 0.42; 95%CI: 0.19–0.93)
[102]. 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 [94,112].
To gain further insight into the anti-carcinogenic potential of selenium, a pooled
analysis of data from three clinical trials of colorectal adenoma was conducted:
the Wheat Bran Fiber Trial, the Polyp Prevention Trial, and the Polyp Prevention Study.
The selenium level was measured in blood specimens of 1763 trial participants.
After adjustment for age, gender, smoking status and study site, 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,
although this result was only statistically significant in the case of the Polyp Prevention
Study (OR: 0.57, 95% CI: 0.34–0.95) [113].
To sum up, a number of epidemiologic studies show a low Se level, especially in males,
to be associated with an elevated risk of lung and prostate cancer. No inverse association
of Se level and cancer risk was found for lung cancer in females, possibly due in part
to the rather low proportion of women in the study population [94]. In contrast
to prostate cancer, breast cancer was not found to be influenced by the selenium level
[114–116]. The majority of studies on relations between selenium level and occurrence
of colorectal cancer have yielded null results for both males and females. However,
there are some reports of a significant inverse relationship between blood Se levels and
the prevalence of adenomatous polyps [117,118]. The potential role of dietary Se in the
early prevention of colorectal neoplasms would need to be confirmed, and the preventive
role of Se regarding cancer of this type, as found in the Polyp Prevention Study [113],
would need further verification as well.
Summary
Results of epidemiologic and laboratory studies have indicated selenium to have

a protective role in counteracting or preventing the development of cancer. A low level
of selenium concentration in the body was shown to be associated with a higher risk
of lung, prostate or colorectal cancer. However, a variety of confounding factors such
as geographical location, gender, age, environmental exposure, genetic susceptibility,
and the like need to be taken into account. There is a need of clarifying the role of Se
107
in the etiology of certain types of cancer through further epidemiologic investigation.

We now have evidence from laboratory studies of selenium compounds affecting cell
growth, the cell cycle, DNA repair, gene expression, and signal transduction. In the
experiments involved, the effects of both organic forms of Se (SeMet and methylated
selenocompounds) and inorganic forms of it (selenites and selenates) were evaluated.
The metabolic pathways of the action of the two forms differ and depend on the basic
levels of the compounds in question. Various hypotheses endeavoring to explain the
connection between the metabolic pathways in which Se is involved and the effects these
can have on chemically induced cancerogenesis, such as in connection with the regulation
of cell signaling and of the redox status, the modulation of transcriptional factors, and the
activation of DNA repair. Results of animal and in vitro studies have shown that the form
of the selenium compound (methylated selenocompound) in question may be of critical
importance to the chemoprotective actions it can perform.
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3. Bioactive components in foods
3.1. Anticarcinogenic effects of flavonoids
Margaret M. Manson
Cancer Biomarkers and Prevention Group, University of Leicester, Leicester, UK
Introduction
Many thousands of different flavonoids are found in plant species, with major dietary
sources including fruit, vegetables, tea, chocolate and soy. Total daily intake can range
from 50–800 mg. Those flavonoids which have been studied in most detail exhibit many
properties which could be protective against heart disease, ageing and cancer. These
polyphenolic compounds are classified, according to structure, as flavonols (quercetin,
kaempferol), flavones (luteolin, apigenin), flavanones (myricetin, narginin, hesperetin),
isoflavones (genistein, daidzein), anthocyanins (cyanidin, pelargonidin, petunidin),
catechins (epicatechin, epicatechin-3-gallate) and chalones (xanthohumol). Amongst
their health-promoting properties are antioxidant, antiviral, anti-allergic, anti-inflam-
matory, and anticancer activities. Such chemopreventive agents can be effective at differ-
ent stages of the carcinogenic process, both by blocking initiation and by suppressing
the later stages involving promotion, progression, angiogenesis, invasion and metastasis.
Several recent reviews have summarised the potential chemopreventive mechanisms
for a number of flavonoids [1–4]. Some recent data for the well-studied flavonoids
apigenin, epigallocatechin gallate (EGCG), genistein, resveratrol, quercetin, the chalone,
xanthohumol and the novel flavonol, tricin, are summarised here.
Blocking mechanisms
Possible ways in which initiation of carcinogenesis can be blocked include prevention

of reactive oxygen species attack on DNA, altered metabolism of procarcinogens in favour
of conjugation and excretion of reactive metabolites, inhibition of carcinogen uptake into
cells and enhanced DNA repair.
Many flavonoids possess antioxidant or free radical scavenging potential, which varies
depending on the hydroxylation status of the benzene rings. Examples include quercetin
(a flavonol in vegetables, apples and onions), xanthohumol (a chalone in hops and beer)
and genistein (an isoflavone in soy). An early study by Duthie et al [5] reported
that quercetin protected human lymphocytes from hydrogen peroxide-induced DNA
damage. Similar findings were reported by Wilms et al [6], who also found that quercetin
protected human lymphocyte DNA from bulky adduct formation following treatment
with benzo[a]pyrene. Also in this study, volunteers consumed quercetin-rich blue-
berry/apple juice for 4 weeks, which led to a significant increase in antioxidant capacity
of plasma.
Margaret M. Manson
116
Flavonoids can interact with the aryl hydrocarbon receptor (AhR) as agonists or anta-
gonists, depending on structure and cell context. Such interactions influence the
expression of drug metabolising enzymes such as cytochromes P450 [7]. They have also
been shown to influence the multi-drug resistance phenotype acquired by many tumour
cells. Quercetin and silymarin were found to inhibit MRP1/4/5-mediated drug transport
from intact erythrocytes with high affinity, in a manner which suggested that they
interact at the substrate-binding sites. Such interactions might influence bioavailability
of anti-cancer drugs in vivo and could be considered for combination therapies [8].
In another recent study [9], the flavonols, quercetin and kaempferol, reduced P-glyco-
protein expression and function in multi-drug resistant human cervical carcinoma KB-IV
cells, while the isoflavones, genistein and daidzein, modulated intracellular drug levels
by inhibiting function, without affecting expression.
Xanthohumol possesses several useful properties to block carcinogenesis including
modulation of enzymes involved in carcinogen metabolism and detoxification (inhibition
of Cyp1A, induction of quinone reductase activity), scavenging of ROS, including hydro-
xyl and peroxyl radicals, along with inhibition of superoxide anion radical formation
and nitric oxide production [10].
Suppressing mechanisms
Mechanisms which result in suppression, or even better, elimination of tumour cells,

include growth inhibition by induction of cell cycle arrest or apoptosis. A significant
number of flavonoids, alone and in combination, have been shown to induce G2/M arrest
in SW480 and CaCo2 human colon carcinoma cells [11]. Tricin, a novel flavonol in rice
bran, was shown to inhibit the growth of breast tumour cells, causing G2/M arrest, but not
apoptosis [12]. In a subsequent study by the same group [13], tricin decreased the number
of intestinal adenomas in Apcmin mice by 33%, with inhibition of COX-1 and COX-2
activity. The latter led to a 34% reduction of PGE2 levels in small intestinal mucosa
and blood. Xanthohumol was also found to inhibit COX-1 and COX-2 activities, and
to be antiestrogenic [10]. The inhibitory effect of other flavonoids on COX-2 expression
and activity has been reviewed by O’Leary et al. [14]. During later stages of carcinogenesis
additional useful mechanisms include inhibition of angiogenesis, invasion and metastasis.
A range of tumour suppressing activities is shown for apigenin (Table 3.1.) and quer-
cetin (Table 3.2.). Resveratrol, genistein and EGCG (reviewed in [2]) have a number
of effects in common with those detailed here for apigenin and quercetin, namely
inhibition of signalling through the EGFR family, NF-κB, and pAkt, induction of cell cycle
arrest involving a decrease in cyclin D1 and phosphorylation of Rb, accompanied
by upregulation of p21 and p27, and induction of apoptosis involving release of cyto-
chrome c from mitochondria, activation of caspases 3 and 9 and downregulation of Bcl
family members. However, depending on cell type and experimental conditions,
flavonoids can both up- and down-regulate key molecules, including JNK, AP-1, p21, p27,
cdc2, cyclin D1, p53, and PI3K.
Bioactive components in foods: Anticarcinogenic effects of flavonoids
117
Table 3.1. Chemopreventive suppressing effects of apigenin
Tissue/cell type Mechanism Effect Reference
Jurkat T cells ↓chymotrypsin-like activity of 20S and 26S Apoptosis [15]

proteosomes; ↑Bax; ↑IκBα
PWR-1E, LNCaP, PC-3, DU145 ↑ROS; ↓Bcl2; ↑cleavage of caspases 3,7,8,9 Apoptosis [16]
prostate tumour cells and cIAP-2
[17]
Breast and prostate cancer ↓fatty acid synthase activity Growth inhibition
cells and apoptosis
[18]
NUB-7, LAN-5, SK-N-BE ↑p53; ↑p21; ↑Bax; ↑cleavage of caspase 3 Apoptosis (p53-dependent)
neuroblastoma cells
[19]
Breast cancer cells ↓Her2; ↓Akt; ↑cleavage of caspase 3; Apoptosis; ↓ colony
↑DFF-45 cleavage; ↓cyclin D1/D3 & cdk4; formation
↑p27
[20]
PC-3 prostate cancer cells ↓p50; ↓p65; ↓NF-κB-DNA-binding; ↓IκBα ↓Bcl2, cyclin D1, COX-2,
degradation and phosphorylation; ↓IKKα MMP9, NOS-2, VEGF
actvity; ↓TNFα activation of NF-κB Apoptosis
[21]
DU145 prostate cancer cells ↓cyclin D1/2 & E; ↓CDK2/4/6; ↑p21, p27, Growth inhibition, G1 arrest,
p16, p18; altered Bax:Bcl2 ratio; apoptosis
↑cyt c release; ↑APAF-1; ↑IκBα ; ↓NF-κB
p50 & p65
HER2-overexpressing breast ↓PI3K & Akt activity; ↓PI3K-HER2 docking; Apoptosis [22]
tumor cells ↓HER2/neu phosphorylation; ↑HER2
degradation
A549 lung cancer cells in vitro ↓Akt; ↓p70S6K1;↓HIF-1α; ↓VEGF Growth and angiogenesis [23]
and in nude mice inhibition
OVCAR-3 and A2780/CP70 ↓Akt; ↓p70S6K1;↓HIF-1α; ↓VEGF; ↑p53; Angiogenesis inhibition [24]
ovarian cancer cells ↓HDM2
HCT 116 colon carcinoma cells ↑ERK & p38 phosphorylation and activity ↑phosphorylation [25]
of Elk & ATF2
HCT 116, HT-29 ↓CK2; ↓TNF a-induced NF-κB activation Apoptosis; [26]
colon cancer cells
Margaret M. Manson
118
Table 3.2. Chemopreventive suppressing effects of quercetin
Tissue/cell type Mechanism Effect Reference
HL60 human myeloid ↑Bax; ↑phosphoBcl2; ↓Pgp Apoptosis [27]

leukeamia cells
Jurkat T cells Inhibiting chymotrypsin-like activity of 20S Apoptosis [15]

and 26S proteosomes; ↑Bax; ↑IκBα
Breast and prostate cancer ↓fatty acid synthase activity Growth inhibition [17]
cells and apoptosis
Colonic aberrant crypt foci ↑Bax; ↓Bcl2; ↑cleavage of caspase 9 Suppression by 4-fold; [28]
apoptosis ↑3-fold
MiaPaCa pancreatic ↓phosphoFAK Decreased invasion [29]

tumour cells
MCF7 breast tumour cells ↑PTEN; ↑p27; ↓Akt Growth inhibition [30]
and apoptosis
LNCaP, PC3 prostate ↓Sp1 interaction with AR; ↑c-jun Inhibition of androgen [31]
tumour cells receptor activity
HT29, SW480 colon ↓ErbB2/3; ↓Bcl2; ↓phosphoAkt Growth inhibition [32]

cancer cells and apoptosis
SW480 colon cancer cells ↓β-catenin/Tcf transcriptional activity ↓c-myc [33]
A549, H1299 human lung ↑cyclin B1; ↑phospho cdc2; Growth inhibition; [34]
carcinoma cells ↑survivin; ↑p53; ↑p21 G2/M arrest
PC3 prostate cancer cells ↓HSP70 Apoptosis [35]
One recent report by Fenton and Hord [36] has suggested a novel chemopreventive
mechanism for flavonoids. In normal colon, epithelial cells migrate to the apex of the
crypt, a process involving the APC gene, which is often mutated in colon cancer. These
authors reported that apigenin, epicatechin, 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.
During the carcinogenic process, both hypermethylation of the promoter regions
of tumour suppressor genes and hypomethylation of oncogenes can occur, resulting
in under- or over-expression. Both EGCG [37] and genistein [38] have been shown
to reactivate a number of key genes, such as the cell cycle inhibitor p16 and the retinoic
119
acid receptor (RARβ), in several different cancer cell types. The mechanism proposed was
through inhibition of DNA methyltransferase, which, in the case of EGCG, involved
direct interaction with the enzyme.
Combined effects
There is now accumulating evidence for the additive, synergistic or antagonistic effects
of combinations of more than one chemopreventive agent. For example Mertens-Talcott
et al. [39], using MOLT-4 human leukaemia cells, showed that quercetin and ellagic acid
acted synergistically in inducing apoptosis. Ellagic acid potentiated the inducing effect
of quercetin on levels of p21 and phosphorylation of p53 at serine 15. Phosphorylation
of JNK1/2 and p38 was increased by the combination, while quercetin alone only induced
p38 phosphorylation. Neither the generation of ROS, nor quercetin stability were
affected by ellagic acid. Combinations of flavonoids were found to have an inhibitory
effect on the breast cancer resistance protein (ABCG2), suggesting the potential use
of ‘flavonoid cocktails’ to reverse multi-drug resistance in treatment of this cancer [40].
Conclusions
Flavonoids, like other types of dietary chemopreventive agents, exhibit a wide range of po-
tentially useful activities for cancer prevention. Their blocking activities include antioxidant
effects, modulation of drug metabolising enzymes and multidrug resistant genes. Sup-
pressing activities include inhibition of signalling pathways responsible for cell proliferation
and survival, and induction of apoptosis, mainly through intrinsic pathways involving Bcl
family members, mitochondrial membrane depolarisation, cytochrome c release and acti-
vation of caspases. They can also induce cell cycle arrest by modulating key components
of cell cycle regulation, including cyclins, cyclin dependent kinases and inhibitors.
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Bioactive components in foods: Biomarkers of dietary polyphenol intake for studying diet-cancer relationships
123
3.2. Biomarkers of dietary polyphenol intake

for studying diet-cancer relationships
Jakob Linseisen and Sabine Rohrmann

Division of Clinical Epidemiology, German Cancer Research Centre, Heidelberg, Germany
Introduction
The primary aim of analytical cancer epidemiology is to detect associations between

exposure variables and disease endpoints, associations that are accompanied and sup-
ported by basic research findings enabling one to identify and understand insofar as
possible the steps contained in the causal chain of disease development. Nutritional
epidemiology deals with dietary factors that are difficult to assess. Long-term dietary
habits are usually explored in epidemiological studies by use of food frequency question-
naires (FFQ). Dietary measurements tend to suffer, however, from imprecision, parti-
cularly concerning dietary components provided by only certain kinds of foods,
and components for which bioavailability is low, such as various secondary plant
products. The use of biomarkers for such compounds should overcome some of the me-
thodological problems in nutritional epidemiology just referred to. The analytical data
obtained are objective and more precise, measurement error being independent of that
contained in the corresponding questionnaire data. The validity and reproducibility
of nutritional biomarker measurements, however, needs to be demonstrated in advance
of their use in epidemiological and etiological studies [1].
Polyphenols
Polyphenols are provided by plant-derived foods, including beverages. The basic flavonoid
structure and various examples of them are given in Figure 3.1. Some of the polyphenols
are found in a rather wide variety of foods (kaempferol contained in many vegetables,
for example), whereas others are limited to only a few kinds of food (such as apigenin
found in parsley and celery). Flavonoids are classified in several different sub-groups,
these including the flavonols, flavones, flavanols, flavanones, isoflavones, lignans,
anthocyanins and proanthocyanins; phenolic acids consist of two main subgroups,
the hydroxybenzoic and the hydroxycinnamic acids.
Estimates of dietary intake estimations are hampered by incomplete or missing data
in food composition tables. Even more problematic, is the fact that the polyphenols
markedly differ from one another in their bioavailability and intestinal metabolism.
Current evidence from bioavailability studies suggests that the bioavailability varies
from about 0.3 to 43% (based on urinary excretion) of the dose administered, reaching
plasma concentrations of 0–4 µmol/l at an intake of 50 mg aglycone equivalents [2].
Bioavailability is determined by different factors, the sugar moiety of the compound
in question and its further metabolism by the gut microflora, for example. Isoflavones
and gallic acid are polyphenols that are absorbed to the highest extent, followed
Jakob Linseisen, Sabine Rohrmann
124
by the catechins, flavanones and quercetin glucosides, although the kinetics involved
differ considerable. The proanthocyanidins, the galloylated tea catechins and the antho-
cyanins are much less available. Thus far, data on the phenolic acids are very limited.
Flavonoids undergo extensive first-pass phase II metabolism in the intestinal epithelial
cells and the liver, their being substrates for methylation, sulfation, and glucuronidation.
For many of the polyphenols, their half-life time in the plasma is short, concentrations
of them reaching baseline levels within 24 hours [3]. The steady-state levels of the plasma
polyphenols should only be achievable through regular intake of the foods in question.
The plasma concentrations present in free-living (fasting-status) subjects are even lower
than the abovementioned range obtained after intervention. Measurement of the
compounds found in urine samples, ideally 24-hour samples, is a promising approach,
due mainly due to the higher concentrations of polyphenols — at least after extraction
and enrichment — as compared with plasma samples. The polyphenol concentrations
contained in biological specimens are estimated in most studies after hydrolysis
of the glycosides involved, the aglycones being quantified.
Fig. 3.1. The basic structure of flavonoids and the chemical structure of selected polyphenols.
125
Laboratory techniques
Two recent reviews provide an overview of the analytical methods used to determine
the structure and content of the flavonoids and phenolic acids contained in foods
and in food-based matrices [4,5]. Although in principle these methods can be applied
to human specimens, the clean-up procedures for this type of samples may be more
complicated than required for many food systems.
Hydrolysis
In foods, the flavonoids are usually glycosylated and the phenolic acids are ester-bound.
Hydrolysis (acidic or enzymatic) is frequently used to simplify the analytical procedure,
the respective aglycones and acids being subsequently detected and quantified. In biologi-
cal specimens, flavonoids mainly undergo modification by means of phase II enzymes
leading to methylated, sulfated and glucuronidated compounds. Accordingly, frequent
use is made of enzymic hydrolysis by means of sulfatase and glucuronidase unless a study
aims at determining the exact metabolites. Phenolic acids undergo further metabolism
and degradation, although a part of them is excreted unmodified.
Clean-up procedures
For human specimens, such as plasma, serum, or urine samples, solid phase extraction
(SPE)-columns provide the most convenient solution for removing from a sample any
matrix compounds that would disturb the analysis. Some techniques such as liquid
chromatography-mass spectrometry (LC-MS), do require only minor sample-preparation
steps.
Separation and detection systems

The two major separation techniques for the quantification of polyphenolics are HPLC
and GC, combined with different detection systems (Table 3.3.), although the use
of LC-MS is becoming increasingly common. For the flavonoids, HPLC has become the
method of choice, and the phenolic acids are genarally quantified by means of GC
after derivatisation. Identification of compounds by means of mass fragmentation is used
as a gold standard. However, a single mass-selective detector often fails to fulfil
the requirements for sensitivity. Thus, new developments include the creation
of HPLC-ESI-MS-MS systems and similarly coupled devices. For the structural
characterization of compounds, mass spectrometric techniques need to be used.
The availability of antibodies for isoflavones and lignans has enabled the antibody-
based assays with a high degree of sensitivity to be developed [6]. For the purpose
of quantification, the fluorescence emitted is recorded by means of a plate reader, with
the option of time-resolved measurement. Thus far, the scientific literature contains
no report on use of the metabonomics techniques (NMR, LC-MS) to characterise clusters
of polyphenolic compounds that can potentially be used as biomarkers.
126
Table 3.3. Principal techniques and detection systems used for the identification and quantification
of flavonoids and phenolic acids in human specimens (metabonomics/NMR excluded)
Separation Detection system Substances class technique
HPLC UV/VIS spectroscopy (Diode array detection) Flavonoids, Phenolic acids
Mass spectrometry Flavonoids, Phenolic acids
Electrochemical Flavonoids, Phenolic acids
Fluorometric Flavonoids, Phenolic acids
GC Mass spectrometry Phenolic acids
LC Mass spectrometry Flavonoids, Phenolic acids
Immunoassays Time-resolved fluorescence Isoflavones, lignans
HPLC — high-performance liquid chromatography; GC — gas chromatography; LC — liquid chromatography.
Bioavailability studies and other short-term interventional studies
A report was published vey recently summarizing all scientific studies (n = 97) that has
been conducted thus far on the bioavailability of polyphenols [7]. Most of the studies
concerned only one or some few compounds within a given subclass of polyphenols.
Kinetic data from the experiments in question are summarized in Table 3.4. (according
to [7]). Relatively low plasma concentrations were obtained in each case, even after
the administration of polyphenol preparations or polyphenol-rich food corresponding
to 50 mg aglycone equivalents. Differences between the various compounds and of classes
of polyphenol are striking nevertheless.
Table 3.4. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1
(according to Manach et al. [7])
Tmax (h) Cmax (µmol/l) Urinary excretion2 Elimination half-life (h)

(% of intake)
Mean (SEM) Range Mean(SEM) Range Mean(SEM) Range Mean (SEM) Range
Daidzin 6.3 (0.6) 4.0–9.0 1.92 (0.25) 0.36–3.14 42.3 (3.0) 21.4–62.0 5.3 (0.8) 3.4–8.0
Daidzein 4.9 (1.0) 3.0–6.6 1.57 (0.52) 0.76–3.00 27.5 8.5 (0.8) 7.7–9.3
Genistin 6.5 (0.6) 4.4–9.3 1.84 (0.27) 0.46–4.04 15.6 (1.8) 6.8–29.7 7.8 (0.7) 5.7–10.1
Genistein 4.1 (0.6) 3.0–5.2 2.56 (1.00) 1.26–4.50 8.6 7.1 (0.3) 6.8–7.5
Glycitin 5.0 1.88 (0.38) 1.50–2.26 42.9 (12.0) 19.0–55.3 8.9
Hesperidin 5.5 (0.1) 5.4–5.8 0.46 (0.21) 0.21–0.87 8.6 (4.0) 3–24.4 2.2
127
Table 3.4. Pharmacokinetic data from 97 studies concerning the bioavailability of polyphenols1
(according to Manach et al. [7]) — cont.
Tmax (h) Cmax (µmol/l) Urinary excretion2 Elimination half-life (h)

(% of intake)
Mean (SEM) Range Mean(SEM) Range Mean(SEM) Range Mean (SEM) Range
Naringin 5.0 (0.1) 4.6–5.5 0.50 (0.33) 0.13–1.50 8.8 (3.17) 1.1–30.2 2.1 (0.4) 1.3–2.7
Quercetingluc. 1.1 (0.3) 0.5–2.9 1.46 (0.45) 0.51–3.80 2.5 (1.2) 0.31–6.4 17.9 (2.2) 10.9–28.0
Rutin 6.5 (0.7) 4.3–9.3 0.20 (0.06) 0.09–0.52 0.7 (0.3) 0.07–1.0 19.9 (8.1) 11.8–28.1
(Epi)catechin 1.8 (0.1) 0.5–2.5 0.40 (0.09) 0.09–1.10 18.5 (5.7) 2.1–55.0 2.5 (0.4) 1.1–4.1
EGC 1.4 (0.1) 0.5–2.0 1.10 (0.40) 0.30–2.70 11.1 (3.5) 4.2–15.6 2.3 (0.2) 1.7–2.8
EGCG 2.3 (0.2) 1.6–3.2 0.12 (0.03) 0.03–0.38 0.06 (0.03) 0.0–0.1 3.5 (0.3) 2.5–5.1
Gallic acid 1.6 (0.2) 1.3–1.5 4.00 (0.57) 2.57–4.70 37.7 (1.0) 36.4–39.6 1.3 (0.1) 1.1–1.5
Chlorogenic acid 1.0 0.26 0.3
Caffeic acid 1.4 (0.6) 0.7–2.0 0.96 (0.26) 0.45–1.35 10.7
Ferulic acid 2.0 0.03 27.6 (17.6) 3.1–61.7
Anthocyanins 1.5 (0.4) 0.7–4.0 0.03 (0.02) 0.001–0.20 0.4 (0.3) 0.004–5.1
Proanthocyanidins 2.0 0.02 (0.01) 0.008–0.03
1
All the data were converted so as to correspond to a supply of 50 mg aglycone equivalent.
2
Usually represent 24-h urine samples.
Tmax — time to reach Cmax; AUC — area under the plasma concentration-time curve; EGC — epigallocatechin; EGCG — epigallocatechin gallate.
A review of short-term intervention studies (n = 93) in which polyphenols were

administered (either as isolated compounds or in the form of foods or food extracts)
to human subjects was published recently [8]. The authors examined evidence relating
to the effects of these polyphenols had on biomarkers used to test various patho-
physiologically relevant hypotheses in vivo.
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, flavanones or isoflavones) as biomarkers
of polyphenol intake [9–16]. 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, although one study in particular failed
to support this conclusion [12]. The magnitude of the variation in the plasma polyphenol
concentrations found between free-living subjects following their usual diet habits was
128
found to be rather high (Table 3.5.), the intra-individual variation also being high [14].
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 0.75, 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, and that the correlations are much lower when intake calculations are
based on data obtained either three or seven days prior to blood sampling. Due to
the short half-life time of most polyphenols, close to of steady-state plasma con-
centrations of them can only be achieved if the substance in question is consumed
regularly, a precondition most likely to be fulfilled by compounds such as kaempferol that
are widely distributed in plant foods, and if the bioavailability of the compound is not
particularly low.
Table 3.5. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men
on their usual diet, participating in a cross-sectional study (according to Bolarinwa and Linseisen [16].
Mean (SD) Min. Max.

Compound Median
(nmol/l)
Gallocatechin 68.6 91.2 (17.1) 0.0a 561.9
Protocatechuic acid 413.0 520.8 (77.2) 48.0 2641.9
Epigallocatechin 742.8 1031.3 (153.4) 0.0a 4528.3
Catechin 82.1 107.8 (15.6) 0.0a 388.1
Gentisinic acid 1357.8 2160.6 (275.9) 0.0a 6849.7
Epigallocatechingallate 158.9 267.7 (48.6) 0.0a 1348.7
Caffeic acid 372.4 477.3 (67.2) 0.0a 2290.8
Vanillic acid 159.5 260.3 (42.7) 0.0a 1121.6
Epicatechin 114.0 277.7 (78.5) 0.0a 2542.9
Syringic acid 478.3 908.1 (203.5) 0.0a 7103.0
p-Coumaric acid 644.6 1304.6 (254.3) 0.0a 6103.4
Ferulic acid 460.6 652.1 (94.6) 58.8 2904.4
Salicylic acid 339.1 6990.0 (3077.9) 26.2 104468.3
Ellagic acid 53.8 140.1 (41.4) 0.0a 1253.4
Daidzein 0.0a 10.1 (2.8) 0.0a 59.9
Quercetin 78.5 108.7 (15.9) 19.7 563.8

129
Table 3.5. Concentrations (nmol/l) of selected flavonoids and phenolic acids in plasma samples of 41 men
on their usual diet, participating in a cross-sectional study (according to Bolarinwa and Linseisen [16] — cont.
Mean (SD) Min. Max.

Compound Median
(nmol/l)
Naringenin 79.7 122.5 (21.7) 0.0a 533.1
Luteolin 388.0 545.8 (80.8) 0.0a 2555.2
Genistein 108.1 157.1 (24.1) 0.0a 639.7
Hesperetin 27.6 37.2 (4.9) 0.0a 151.7
Kaempferol 36.8 126.1 (40.4) 0.0a 1356.8
Apigenin 5.3 9.3 (1.9) 0.0a 52.5
Isorhamnetin 14.2 31.0 (7.3) 0.0a 204.8

a
Below the detection limit.
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,
the correlation coefficients for selected flavonols and flavanones being between 0.28
and 0.38 [13]. The urinary polyphenol excretion rates show a high degree of variability,
just as was indicated already for the plasma concentrations. To give an example
of the polyphenol concentrations found in 24-h urine samples of subjects on a habitual
diet, Nielsen and coworkers [13] reported average (SD) concentrations of quercetin,
kaempferol, naringenin, phloretin, and total flavonoids as being 25(23), 50(32), 701(659),
76(110), 1638(1316) µg/24 h, respectively.
Plasma and urinary polyphenol concentrations are not expected to reflect long-term
or habitual dietary intake, although this has not been investigated tested extensively.
One study reported correlation coefficients of between 0.24 and 0.74 for plasma
isoflavone concentrations, the dietary intake estimates being based on FFQ data [11].
In large-scale epidemiologic (etiological) studies of disease-related effects of dietary
polyphenol levels, little use has been made of biomarker measurements. Hertog and
colleagues were the first to analyze commonly consumed foods in terms of their flavonol
and flavone content by means of HPLC. Their work provided the basis for the estimation
of dietary flavonol and flavone intake. In the following, several studies on associations
with the risk of cardiovascular diseases and with cancer at different sites, some of which
appear very promising, are taken up. Only few studies concerning the use of biomarkers
of polyphenol intake were found to be available (Table 3.6), except as regards the intake
of phytoestrogens (isoflavones and lignans).
130
Table 3.6. Epidemiologic (observational) studies of the risk of cancer in which biomarkers of dietary polyphenol
intake (other than that of isoflavones and lignans) were employed
N, cases/ Specimen;
Author Type Cancer site Result
controls Flavonoid
Dai et al. 2002 [17] CCS 250/250 Urine; Breast NS

citrus flavonoids
Zheng et al. 1999 [18] CCS 60/60 Urine; Breast NS

total phenols
Sun et al. 2002 [19] Cohort 232/772 Urine; Gastric Significant

tea polyphenols and esophageal inverse association
CCS — case-control study; NS — not significant.
One of the major reasons for the frequent use of biomarkers of phytoestrogen intake
in epidemiological studies (see Table 3.7.) may be the ready availability of appropriate
immunoassays suitable for measurements on large numbers of samples, enabling the
attainment of the requirement of sufficient statistical power.
Table 3.7. Epidemiological studies of the risk of breast cancer risk in which biomarkers of dietary isoflavone
and mammalian lignans intake were employed.
N, cases/ Specimen;
Author Type Result
controls Phytoestrogen
Ingram et al. [21] CCS 144/144 Urine; equol, enterolactone Significant inverse association
Zheng et al. [18] CCS 60/60 Urine; isoflavones NS
Murkies et al. [22] CCS 18/20 Urine; isoflavones Significant inverse association
Dai et al. [17] CCS 250/250 Urine; isoflavones, lignans Significant inverse association
Piller et al. [23] CCS 220/237 Plasma; enterolactone Significant inverse association
Pietinen et al. [24] CCS 194/208 Serum; enterolactone Significant inverse association
den Tonkelaar Cohort: 88/268 Urine; genistein, enterolactone NS

et al. [25] nested CCS
Hulten et al. [26] Cohort: 248/492 Plasma; enterolactone Significant positive association
nested CCS for highest and lowest categories
Grace et al. [27] Cohort: 97/187 Serum, Urine; Higher risk when isoflavone
nested CCS 114/219 isoflavones, lignans estimates are higher
CCS — case-control study; NS — not significant.

131
In view of the usually rather low sample volumes available in epidemiologic studies,
the analysis of polyphenol content is restricted in most cases to only a few com-
pounds or classes of compounds. Accounts of analytical procedures that permit
a wide variety of polyphenols to be determined in a single run were published
recently [16,20].
Specificity and sensitivity
In applying mass-selective detection methods at least to standard mixtures or biological

specimens spiked with the compound in question, the concentrations were found
to be high enough to enable the characteristic mass fragments to be identified.
This differs from other methods, such as HPLC-UV/VIS, ECD (electron capture
detection), or use of fluorescence detectors, in which peak identity cannot be con-
firmed with sufficient certainty. Antibody-based assays are also subject to failures
in specificity due to cross-reactions with other matrix compounds. For all these
methods, confirmation of their results by use of MS-based techniques is necessary.
MS-based systems also have their problems in connection with concentrations close
to the detection limit, however, where the characteristic fragments may be absent.
Sensitivity is a major issue with regard to analytical methods for determining
polyphenols in biological specimens. In epidemiological studies in particular,
in which the sample volumes available are usually very small, the degree of sensitivity
a method provides can be decisive for whether it can be used or not (along with other
factors, such as analytical time and costs). Detection limits very close to 1 nmol/l
of polyphenols have been found for techniques involving HPLC-ECD, HPLC-MS-MS,
and TR-FIA (immunoassay). The sensitivity achievable with HPLC-MS, LC-MS,
GC-MS and HPLC with use of fluorometric detection is slightly lower.
Reproducibility, validity and reliability
For each well-developed method, satisfactory figures concerning the analytical precision
and accuracy are available. The higher values here are those for the MS-based
techniques (coefficient of variation < 5–7%, recovery 90–105%), the immuno-assays
being located at the other end of the scale, having coefficients of variation close
to or above 10% and recovery rates frequently at < 90%. However, working at
the detection limit of a method is always a challenge, and reports on the quality
of the method in question are usually obtained clearly above the detection limit.
As already mentioned, MS-based systems also have their problems when the con-
centrations involved are very low. To the best of our knowledge, except for some few
small studies [14], no systematic findings on repeated within-subject samples or on los-
ses during sample storage are available.
132
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9. de Vries JH, Hollman PC, Meyboom S, Buysman MN, Zock PL, van Staveren WA, et al. Plasma
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11. Verkasalo PK, Appleby PN, Allen NE, Davey G, Adlercreutz H, Key TJ. Soya intake and plasma
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Br J Nutr 2001;86:415–21.
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15. Ritchie MR, Morton MS, Deighton N, Blake A, Cummings JH. Plasma and urinary phyto-oestrogens
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Theodore G. Sotiroudis, Soterios A. Kyrtopoulos
134
3.3. Anticarcinogenic compounds of olive oil

and related biomarkers
Theodore G. Sotiroudis and Soterios A. Kyrtopoulos

Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, Athens, Greece
Olive oil is an important ingredient of the Mediterranean diet. Epidemiological studies

demonstrate rather conclusively that populations within Europe consuming this diet have
a particularly low incidence of a number of common cancers [1,2]. A plethora of minor
constituents in olive oil have been identified as being effective agents mitigating against the
initiation, promotion and progression of multistage carcinogenesis. These include toco-
pherol and carotenoid antioxidants, that have been thoroughly studied, a number of simple
and bound phenolics (tyrosol, hydroxytyrosol, secoiridoids and lignans), the triterpene
hydrocarbon squalene and the phytosterol β-sitosterol [1–3]. The occurrence of these
constituents also calls for the development of specific nutritional biomarkers that reflect
the nutritional status of these dietary constituents with respect to their intake
or metabolism and that can provide information useful for nutritional epidemiology
regarding the effects of disease processes that can occur [4]. A brief overview is presented
of recent findings concerning the bioavailability of certain minor but important olive oil
minor components (polyphenols, lignans, squalene and β-sitosterol), considered as putative
nutritional biomarkers in relation to cancer the incidence of cancer.
Phenolic compounds
HPLC chromatography of the methanol extract of virgin olive oil reveals seven major
polyphenol peaks corresponding to hydroxytyrosol, tyrosol, oleuropein, the aglycone
of ligstroside, two secoiridoids (dialdehydes related to oleuropein and ligstroside
but lacking the carboxymethyl group at C4), and a peak containing the lignans
(+)-1-acetoxypinoresinol and (+)-pinoresinol [1–3] (Table 3.8., Figure 3.2.). Oleuropein
and its metabolites tyrosol and hydroxytyrosol, which represent major antioxidants
in olive oil, are dose-dependently absorbed in humans after the ingestion of realistic doses
of virgin olive oil. When olive oil samples containing increasing amounts of a phenolic
extract of olive oil were administered to human volunteers, a dose-dependent decrease
in the urinary excretion of the F2-isoprostane 8-iso-PGF2α, a biomarker of in vivo lipid
peroxidation processes, was observed. This indicates olive oil phenolics to maintain their
antioxidant activities in vivo. It has also been shown that olive oil phenolics are excreted
in the urine as glucuronide conjugates and that the urinary free tyrosol concentration
is responsive to the dietary intake of virgin olive oil. In addition, a statistically significant
negative correlation has been found between homovanillyl alcohol (HValc, a major
metabolite of hydroxytyrosol, together with homovanillic acid — HVA) and isoprostane
excretion, the excretion of both HValc and HVA also being significantly correlated
with the dose of administered hydroxytyrosol. Thus, HValc in urine reflects the in vivo
Bioactive components in foods: Anticarcinogenic compounds of olive oil and related biomarkers
135
concentration of hydroxytyrosol [5–11]. After the ingestion of olive oil of low phenolic
content plasma glutathione peroxidase activity was found to decrease postprandially,
but this was not observed after the intake of olive oils of moderate to high phenolic
content [12]. An HPLC method for the simultaneous determination of oleuropein and
of its metabolites hydroxytyrosol and tyrosol in human plasma has been developed [13,14].
Table 3.8. Concentrations of the major phenolic compounds found in virgin olive oil
Compounds mg/kg
Tyrosol 27.45 [2], 2.65–4.75 [3]
Hydroxytyrosol 14.42 [2], 1.83–4.71 [3]
Oleuropein aglycone 103–205 [3]
Total secoiridoids 27.72 [2]
Lignans 41.53 [2], 38–65 [3]
References [2] and [3].
Fig. 3.2. Structures of certain phenolic compounds detected in olive oil. Hydroxytyrosol (A), oleuropein
aglycone (B), (+)-pinoresinol (C).
Recent findings suggest that olive oil may also affect the bioavailability of other food
bioactive components with a chemopreventive potential. It was observed in this respect,
that the concentration in human plasma of lycopene, a biomarker of the intake
of tomato-rich food and hypothesized to be responsible for reducing the risk of various
136
cancers, increased dramatically after the consumption of tomatoes cooked in olive oil,
as compared to the consumption of tomatoes cooked without olive oil [15]. The con-
sumption of tomato products prepared together with olive oil, but not with sunflower
oil, was found to improve the antioxidant activity of plasma [16].
Lignans are plant compounds metabolized in the gut to produce the phytoestrogens
enterolactone and enterodiol. Phytoestrogens have an anticarcinogenic potential through
the anti-estrogenic, anti-angiogenic, proapoptotic and anti-oxidant mechanisms
established for them [17,18]. Recent findings suggest that enterolactone is more rapidly
metabolized in human colon epithelial cells and/or excreted by them than enterodiol is,
that the phase II metabolism of enterolactone and enterodiol already may take place
during their uptake in the colon, and that the epithelial cells in the colon may be
responsible for this metabolism [19]. 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, their reaching a steady state. Plasma enterolignan
concentrations can thus be considered to be good biomarkers of dietary lignan exposure
and be used to evaluate the effects of lignans [20]. A number of in vitro and animal studies
support a role for lignan-rich foods and of purified lignans in the modulation of cancer
events in the breast, the prostate and the colon, whereas the findings of epidemiological
studies are controversial [18]. Nevertheless, a tendency for a lower risk of breast cancer
to be associated with higher plasma concentrations of enterolactone, restricted almost
entirely to estrogen-receptor alpha negative breast cancer has been found, suggesting
that dietary lignans may be important in the etiology of breast cancer, particularly
in premenopausal women [21].
Squalene
It has been suggested that the lower risk of cancers of various types associated with high
olive oil consumption (as compared with other human foods) may be due to the presence
of squalene (reviewed in [1]). This triterpene hydrocarbon is found mainly in nonedible
shark liver oil, while virgin olive oil is a major source of phytosqualene, its content ranging
from 800 to 12,000 mg/kg. If virgin olive oil were the sole source of dietary fat, the squalene
intake would be more than 200 mg/d [22]. Nevertheless, very little is known concerning
the postprandial metabolism of squalene. It has been observed that postprandial squalene
metabolism is age dependent [23], and that the content of squalene in the whole plasma
and in the lipoprotein fractions (where its ratio to cholesterol is highest in the VLDL
and the intermediate density lipoproteins [24]) varies directly with the triglyceride content
and is increased in hypertriglyceridemia, which expands the plasma pool of this
metabolically active hydrocarbon [25]. Experiments in vitro and animal models suggest
squalene to play a tumour-inhibiting role, which is most probably based on its strong
inhibitory action on the catalytic activity of beta-hydroxy-beta-methylglutaryl-CoA
reductase, leading to a reduced farnesyl pyrophosphate availability for prenylation of the ras
oncogene [26]. Although animal studies have enhanced our understanding of the possible
137
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 long-
term effects of greater consumption of squalene are unknown. Several factors must
be taken into account when examining the evidence for squalene’s inhibition of carcino-
genesis factors, such as the effective dose used and exposure time [27]. At present, therefore,
its use as a nutritional biomarker is hardly to be considered.
Phytosterols
Phytosterols are plant sterols that are structurally similar to cholesterol and that possess
anticarcinogenic properties [27]. Together with squalene, they represent markers of chole-
sterol synthesis and absorption and are transported together with cholesterol in serum
lipoproteins [24]. β-Sitosterol, one of the most common phytosterols and the main olive
oil sterol [1], together with campesterol are the two predominant phytosterols in the
blood. It has been suggested that the high reproducibility and high reliability over time
(consistency of the plasma phytosterol level over time) of the plasma measurements
of these sterols makes them suitable for clinical and population-based studies of cancer
prevention [28]. In recent years, functional foods high in phytosterol-ester content
for lowering the cholesterol level have been developed. Although phytosterols
act as immune modulators and anticancer agents in vitro [29], the protection (if any)
that high concentrations of phytosterol provide against the development of cancer
in humans has not been adequately examined, further study of this being needed.
Conclusions
Since the phenolic content of the olive oil consumed may account for the postprandial
antioxidant activity in vivo after the ingestion olive oils of moderate to high phenolic
content, we suggest that these biomolecules, or certain polyphenol metabolites in human
plasma and urine, can serve as practical biomarkers for olive oil consumption and as
an alternative biomarker for future epidemiological studies in dietary cancer prevention
and health promotion.
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in cancer prevention. Eur J Cancer Prev 2004;13:319–26.
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droxytyrosol and tyrosol in human urine after olive oil intake. Anal Biochem 2001;294:63–72.
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13. Tsarbopoulos A, Gikas E, Papadopoulos N, Aligiannis N, Kafatos A. Simultaneous determination
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15. Fielding JM, Rowley KG, Kooper P, O’Dea K. Increases in plasma lycopene concentration
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2005;135:795–801.
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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.
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John D. Hayes, Michael O. Kelleher, Ian M. Eggleston
140
3.4. Anticarcinogenic effects

of glucosinolate breakdown products
John D. Hayes1, Michael O. Kelleher1 and Ian M. Eggleston2

1
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY
2
Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom
Glucosinolates and their association with cancer chemoprevention
Epidemiological studies have revealed that regular consumption of cruciferous vegetables,

such as broccoli, Brussels sprouts, cabbage, cauliflower, kale, swede and turnip, is associa-
ted with a reduced incidence of cancer [1,2]. Furthermore, greater health benefit may
be obtained from raw as opposed to cooked vegetables [3]. The types of neoplastic disease
in man that these vegetables appear to protect against include colorectal cancer [4],
lung cancer [5], and possibly prostate cancer [6]. Feeding experiments in animals have
also suggested broccoli can protect against liver cancer [7]. Cruciferous vegetables
uniquely contain glucosinolates at approximately 20 µmol/g dry mass of vegetable [8,9],
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 [10,11].
Glucosinolates are substituted β-thioglucoside N-hydroxysulfates, formed by the plant
from any one of eight amino acids, namely, alanine, valine, leucine, isoleucine,
phenylalanine, methionine, tyrosine and tryptophan [2]. Over 115 naturally occurring
glucosinolates have been identified. Each cruciferous vegetable contains a mixture
of glucosinolates that varies according to the strain of the plant [8,12–15]. The
glucosinolate content is primarily under genetic control, though it can be influenced by
environmental factors [16,17]. Much of the diversity amongst glucosinolates arises from
the addition of different sized alkyl groups to the side chain of those amino acids,
principally valine, phenylalanine and methionine, used in their biosynthesis; this variable
elongation of amino acid side chains entails repetitive additions of methyl groups through
a series of transamination, condensation, isomerisation and decarboxylation reactions
[18]. As shown in Figure 3.3., the synthesis of glucosinolates proceeds through the
conversion of elongated amino acids to their oxime derivatives, catalysed by members
of the cytochrome P450 (CYP) 79 family [19]. Subsequently, the oxime is metabolised
to a thiohydroximate, which is in turn conjugated with glucuronic acid to form
a desulfoglucosinolate before finally being sulfated to yield the glucosinolate [2].
The task of establishing a link between the ingestion of particular glucosinolates and
their possible health benefits is not straightforward. This endeavour is simplified to some
extent by the fact that relatively few glucosinolates are present in the human diet.
The most common of these are the methylsulfinylalkyl glucosinolates glucoiberin
and glucoraphanin, the olefinic glucosinolates sinigrin, gluconapin, glucobrassicanapin
and progoitrin, and the aromatic glucosinolate gluconasturtiin (Table 3.9.) [9,20].
Glucoraphanin has been reported to be abundant in broccoli [9], though certain broccoli
Bioactive components in foods: Anticarcinogenic effects of glucosinolate breakdown products
141
strains also contain substantial amounts of glucoiberin [21]. Sinigrin has been reported to
be the predominant glucosinolate in Brussels sprouts, cabbage, cauliflower and kale [9];
gluconapin is also found in high levels in Brussels sprouts [9]. Substantial amounts
of progoitrin are present in many cruciferous vegetables [9]. The aromatic glucosinolate
gluconasturtiin is present in watercress. The indolyl glucosinolate glucobrassicin is pre-
sent in Savoy cabbage, Brussels sprouts and cauliflower [9,22], and whilst not abundant
it can elicit distinct pharmacological effects.
Table 3.9. Trivial names of some glucosinolates with the corresponding side-chain (R) composition
Name R side-chain
Sinigrin 2-Propenyl
Gluconapin 3-Butenyl
Glucobrassicin 3-Indolylmethyl
Glucobrassicanapin 4-Pentenyl
Progoitrin 2-Hydroxy-3-butenyl
Glucoiberin 3-Methylsulfinylpropyl
Gluconapoleiferin 2-Hydroxy-4-pentenyl
Glucocheirolin 3-Methylsulfonylpropyl
Glucoerucin 4-Methylthiobutyl
Glucoberteroin 5-Methylthiopentyl
Fig. 3.3. Synthesis of glucosinolates. The R group is derived from the original amino acid and is highly variable.
142
Production of isothiocyanates, thiocyanates, nitriles, cyano-epithioalkanes

and oxazolidine-2-thiones from glucosinolates
Evidence suggests that inhibition of carcinogenesis by glucosinolates is not primarily
attributable to this class of compound, but rather it appears to be due to certain of their
breakdown products. Hydrolysis of these phytochemicals is catalysed by myrosinase
(β-thioglucoside glucohydrolase, EC 3.2.3.147), an enzyme that is physically segregated
from glucosinolates within the intact plant by virtue of the fact that it is sequestered
in specialised “myrosin” cells [23]. Upon wounding of the vegetable, for example during
harvesting, during freeze-thawing, during food preparation, or during chewing
whilst eating, myrosinase is released from the “myrosin” cells and is able to hydrolyse
glucosinolates within the damaged plant. In addition, myrosinase activity may be
present in human colonic microflora, suggesting that glucosinolates can be hydrolysed
in the gastrointestinal tract during digestion of food [24,25]. 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. The outcome of the reaction with myrosinase depends on the nature
of the aglycone, as well as the reaction temperature, the pH and the presence of ferrous
ions (Figure 3.4A. and 3.4B.).
Fig. 3.4A. Hydrolysis of glucosinolates. At high or neutral pH the formation of isothiocyanates is favoured
while at low pH the formation of nitriles is favoured. 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, resulting in the formation of an epithioalkane.
143
ESP — epithiospecifier protein.
Fig. 3.4B. Hydrolysis of sinigrin. Following damage to the plant tissue the glucosinolate sinigrin is hydrolysed
by myrosinase resulting in the formation of four distinct compounds. On the right-hand side of the figure,
an arrow shows that allyl thiocyanate, formed from sinigrin, can convert spontaneously to form allyl
isothiocyanate [26].
The thiohydroximate-O-sulfates formed from methylsulfinylalkyl, olefinic and aromatic

glucosinolates undergo a Lossen rearrangement, with the elimination of sulfate, to form
their respective isothiocyanates (ITCs), thiocyanates or nitriles [10,23]. Certain
thiocyanates that are formed during a Lossen rearrangement, such as allyl-ITC, are unstable
and can undergo a relatively slow spontaneous conversion to their respective isothiocyanate
[26]. Elemental sulfur is also formed in certain circumstances. At neutral pH, hydrolysis
of glucosinolates with aliphatic or aromatic side chains gives rise primarily to isothiocya-
nates (ITCs). The glucosinolates glucoiberin, gluconapin, glucoraphanin, glucobrassica-
napin and sinigrin yield 3-methylsulfinylpropyl-ITC, 3-butenyl-ITC, 4-methylsulfinyl-
butyl-ITC (sulforaphane), 4-pentenyl-ITC and 2-propenyl-ITC (allyl-ITC), respectively.
At low pH, the thiohydroximate-O-sulfates formed by myrosinase from gluco-
sinolates with a side chain containing a double bond (e.g. sinigrin, gluconapin and
glucobrassicanapin) may, in the presence of an epithiospecifier protein (ESP) and ferrous
ions, give rise to a cyano-epithioalkane [27]. In this case, ESP interacts with myrosinase
to promote sulfur transfer from the S-glycosyl unit to the alkenyl chain derived from the
amino acid part of the aglycone [28]. Thus, at pH 4 and in the presence of Fe2+ ions,
144
myrosinase and ESP convert sinigrin to 1-cyano-2,3-epithiopropane [29]. Gluconapin

can similarly be converted by the combined actions of myrosinase and ESP to 1-cyano-
-3,4-epithiobutane [30,31]. Likewise, glucobrassicanapin can be hydrolysed to 1-cyano-
-4,5-epithiopentane [31]. Progoitrin, a (2R)-hydroxy-3-butenyl glucosinolate, is also
converted in the presence of myrosinase, ESP and Fe2+ ions to an epithionitrile [32] and in
the case of epi-progoitrin ((2S)-hydroxy-3-butenyl glucosinolate), it can be hydrolysed by
myrosinase to crambene (1-cyano-2-hydroxy-3-butene) [33,34]. Two cDNAs for ESP have
recently been cloned from Arabidopsis and broccoli and the purified proteins characterized
following their heterologous expression in E. coli [35,36].
If the aglycone generated by myrosinase is from a glucosinolate with a side chain
lacking a double bond, the sulfur atom may be lost and a nitrile formed [37–39].
This reaction may involve ESP, and is diminished by heating [40]. A few glucosinolates
produce thiocyanates though the mechanism involved is unclear [23]. Upon hydrolysis
by myrosinase, those aglycones from glucosinolates that contain β-hydroxylated side-
chains form oxazolidine-2-thiones, as a consequence of spontaneous cyclization.
Examples of these include progoitrin, glucoconringiin and gluconapoleiferin [2].
Production of indoles from glucosinolates
The indolyl glucosinolates glucobrassicin and neoglucobrassicin are synthesised by the

plant from tryptophan. The best studied of these is glucobrassicin. At neutral pH,
hydrolysis of glucobrassicin by myrosinase does not generate an ITC, but rather gives rise
to indole-3-carbinol and a thiocyanate ion (Figure 3.5.); this reaction probably proceeds
Fig. 3.5. Production of indoles from glucosinolates. 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.
145
through a Lossen rearrangement generating an unstable ITC intermediate [22]. At acidic

pH, hydrolysis of glucobrassicin yields indole-3-acetonitrile, hydrogen sulfide and ele-
mental sulfur [22]. In the acidic environment of the stomach, indole-3-carbinol condenses
to form various compounds including indolo[3,2-b]carbazole and 3,3∂-diindolylmethane,
both of which have potent pharmacological effects [41]. It can also combine with ascorbic
acid to form ascorbigen [42] (Figure 3.6.).
Fig. 3.6. Structures of indoles produced from glucosinolates — 3,3’-Diindolylmethane (DIM), indolo [3,2-b]
carbazole (ICZ) and ascorbigen (ASG).
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, 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. These include induction of antioxidant and detoxi-
fying genes, inhibition of CYP enzyme activity, cell cycle arrest, and stimulation of apop-
tosis. There is a dose-dependency in these responses: generally, induction of cyto-
protective genes and inhibition of CYP activity occurs at relatively low concentrations
of phytochemical, whereas activation of cell cycle arrest and apoptosis occurs at higher
levels of phytochemical [43,44]. A major problem exists in interpreting experiments
utilizing vegetable extracts because of uncertainty about attributing biological effects
to specific phytochemicals. 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, there is a relative abundance of information about isothio-
cyanates and indoles, when compared with the data about thiocyanates, nitriles, cyano-
epithioalkanes, and oxazolidine-2-thiones.
Induction of gene expression mediated by Nrf2
Isothiocyanates increase the expression of antioxidant and detoxication proteins, such

as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1),
both in vivo and in vitro [45,46]. Agents such as ITC that increase GST and NQO1
146
enzymes without increasing arylhydrocarbon hydroxylase activity, catalysed by the

phase I drug-metabolising enzyme cytochrome P450 (CYP) 1A1, are sometimes called
mono-functional inducers [47]. Sulforaphane, 3-methylsulfinylpropyl-ITC, allyl-ITC,
7-methylsulfinylheptyl-ITC, 8-methylsulfinyloctyl-ITC, benzyl-ITC and phenethyl-ITC,
induce NQO1 in the mouse Hepa-1c1c7 hepatoma cell line [48,49] (Figure 3.7.). Many
of these compounds induce GST P1-1 in the rat liver RL-34 cells [50]. The mouse nqo1 gene
contains a functional antioxidant response element (ARE) in its 5∂-upstream region [51],
as does the rat GSTP1 gene (called GPEI) [52]. The induction of these two genes by ITCs
is mediated by the Nrf2 (Nuclear Factor-Erythroid 2 p45-related factor 2) bZIP (basic-
region leucine zipper) transcription factor that is recruited to the ARE as a heterodimer
with small Maf protein [51]. Indeed, as far as is known, all genes that are induced by ITCs
contain an ARE in their promoters and are regulated by Nrf2.
Fig. 3.7. Structures of isothiocyanates which induce NQO1: 1) Allyl-ITC; 2) Phenethyl-ITC; 3) Benzyl-ITC;
4) 3-methylsulfinylpropyl-ITC; 5) 7-methylsulfinylheptyl-ITC; 6) 8-methylsulfinyloctyl-ITC.
Examination of nrf2–/– and wild-type mice, suggests AREs are present in the promoters
of at least 100 genes [53–55]. The battery of genes regulated by Nrf2 includes those
encoding aldo-keto reductase (AKR), carboxyl esterase, ferritin, glutamate cysteine ligase
catalytic (GCLC) and modifier (GCLM) subunits, GST, heme oxygenase 1, NQO1,
metallothionein, microsomal epoxide hydrolase, multidrug resistance-associated protein,
thioredoxin, thioredoxin reductase, UDP-glucuronosyl transferase. Many of these
genes are induced by sulforaphane in vivo in an Nrf2-dependent fashion in the stomach,
small intestine and liver of rodents [53,54,55–58]. Importantly, feeding broccoli seed
to mice increased the levels of GCLC, GST and NQO1 in the gastrointestinal tract
in an Nrf2-dependent fashion [21]. It is thought that ITCs possess the ability to induce
ARE-driven gene expression because they are thiol-active [59]. Through this characte-
ristic, ITCs modify cysteine residues in the Cullin 3:Rbx1 E3 ubiquitin ligase substrate
adaptor Keap1, leading to its inhibition and inability to serve as a substrate adaptor
147
required for the ubiquitination of Nrf2 under homeostatic conditions [60–62]. Consistent
with this view, exposure of RL-34 cells or HepG2 cells to sulforaphane causes stabilisation
and rapid accumulation of Nrf2 [63,64]. Surprisingly, allyl-ITC does not increase Nrf2
stability [64] and there may therefore be other factors involved in enzyme induction
by these phytochemicals. Treatment of cells with benzyl-ITC causes a rapid increase
in the level of reactive oxygen species, and this may also contribute to gene induction [46].
It has been found that administration of cranbene to Fischer 344 rats causes
an elevation in hepatic GST and NQO1 enzyme activities, but not CYP1A1, suggesting
it is a mono-functional inducer [65]. By comparison with sulforaphane, cranbene was
found to be an approximately equally potent inducer in the rat [66]. However, 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. The identity of this metabolite is not known.
The indole-containing glucobrassicin breakdown products can activate gene
expression by several mechanisms. Indole-3-carbinol is a modest inducer of Nrf2-depen-
dent ARE-driven gene expression in the liver and small intestine of mice [52,67]. Although
indole-3-acetonitrile has not been studied in the Nrf2 knockout mouse, it is a good
inducer of GST enzyme activity in mouse liver and small intestine [68], a fact that implies
it works through Nrf2.
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[3,2-b]carbazole and 3,3∂-diindolylmethane.
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.
Examination of the dose of indole required to double XRE-driven reporter gene expression
shows that indolo[3,2-b]carbazole is a much more potent inducing agent than 3,3∂-di-
indolylmethane, indole-3-carbinol or ascorbigen [43,69]. Amongst genes for drug-
metabolising enzymes, mouse, rat and human CYP1A1 are prototypic XRE-regulated
genes. This cytochrome has O-deethylase activity towards ethoxyresorufin, and there
is abundant evidence from enzyme assays, western and northern blotting that CYP1A1
is inducible by indolo[3,2-b]carbazole and 3,3∂-diindolylmethane [2]. The promoters
of other genes including rat and human NQO1, rat ALDH-3, rat GSTA2, rat UGT1A1,
and rat UGT1A6 contain an XRE, as does the mouse BAX gene, and it is therefore antici-
pated that activation of AhR by glucobrassicin-derived indoles will influence significantly
the metabolism of xenobiotics (for a review, see [70]). Interestingly, the mouse nrf2 gene
promoter also contains an XRE [71], indicating that AhR ligands could lead to an
increased production of Nrf2 mRNA; this may not necessarily lead to an increase in Nrf2
protein levels. Whether indolo[3,2-b]carbazole and 3,3∂-diindolylmethane can increase
the stability of Nrf2 protein is not known, but unless they are metabolised by CYPs,
and thereby generate reactive oxygen species, this seems unlikely [72].
148
In addition to induction of CYP1A1 by indoles, other cytochromes are inducible, such

as CYP1B1 and CYP19 [73], as are the drug metabolising enzymes AKR, GST T1-1,
sulfotransferase and UGT1 [74]. Furthermore, 3,3∂-diindolylmethane can induce expres-
sion of the transcription factors ATF3, c-Jun and NF-IL6 as well as genes involved
in cell growth, such as growth arrest and DNA damage (GADD) GADD34, GADD45 and
GADD153 [75,76]. Also, p21 is induced by indole-3-carbinole [77]. It is not however clear
whether the genes for ATF3, c-Jun, NF-IL6 and the GADD proteins are regulated through
XREs and whether the process is mediated by AhR.
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. This viewpoint is probably an oversimplification and does not
take into account the multiple changes in gene expression that indoles affect.
Bonnesen et al [43] have reported that treatment of human colon LS-174 cells with
indolo[3,2-b]carbazole before exposure to benzo[a]pyrene provides a small measure
of protection against DNA damage as measured by the comet assay. Most importantly,
prior treatment of the LS-174 cells with both indolo[3,2-b]carbazole and sulforaphane
before exposure to benzo[a]pyrene was found to confer substantial protection against
genotoxicity, and this protection was greater than was achieved by either phytochemical
alone [43].
Inhibition of carcinogen activation by glucosinolate breakdown products
Certain isothiocyanates can block the activation of several carcinogens to their ultimate
carcinogenic forms. 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-carcino-
gens by CYP isoenzymes [78,79]. Inhibition of CYP can also be achieved by sulforaphane
[80,81]. In the case of the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone,
the ITCs with highest lipophilicity and low reactivity of their NCS group had the greatest
ability to inhibit lung tumorigenesis [82].
Inhibition of histone deacetylase by isothiocyanates
The isothiocyanate sulforaphane has been shown to inhibit histone deacetylase (HDAC)
[83,84]. This function is likely to alter gene expression substantially. It may also have
profound implications for cell fate as a change in the balance between histone acetyl
transferase (HAT) and HDAC could alter tumourigenesis. Indeed, recognition of this
possibility has lead to considerable recent interest in the ability of HDAC inhibitors to act
as both chemopreventive and chemotherapeutic agents.
Within the cell, DNA is tightly coiled around an octamer of histone proteins
in a structure known as a nucleosome, the basic structural unit of chromatin. Each
of the histone proteins contains an evolutionary conserved amino tail protruding from
149
the nucleosome, which can determine the accessibility of the DNA to transcription
factors. The tail is also subject to many post-translational modifications including
acetylation. 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, allowing transcription
factors access to the regulatory regions of genes. Conversely, removal of acetyl groups
causes the tail to wrap tightly around the DNA thereby preventing interaction with
the transcription machinery. The addition and removal of the acetyl groups is carried
out by HAT and HDAC, respectively. In pre-cancerous and cancerous cells, tumour
suppressor genes are associated with deacetylated histones resulting in the inactivation
of these genes. Inhibition of HDAC may prevent the removal of acetyl moieties from
histones thus allowing transcription of the tumour suppressor genes.
Sulforaphane has been shown to diminish HDAC activity with a concomitant rise
in histone acetylation in prostate cancer cells [84], human embryonic kidney cells [83]
and human colorectal cancer cells [83]. The link between inhibition of HDAC activity and
the resultant increase in transcription of tumour suppressor genes has been reported
for p21 [84,85], p53 [86] and Bax [84,85]. Equally, mammalian HDAC is capable of down-
regulating p53 function, by deacetylation of histones associated with the p53 gene,
resulting in a reduction in its transcriptional activity. In addition, sulforaphane has been
found to cause a G2/M phase delay with an increase in apoptotic cell fraction in a time-
and dose- dependent fashion [87].
Thus the ability of sulforaphane, along with other dietary HDAC inhibitors such
as diallyl disulfide [88], to affect chemoprevention via its ability to alter chromatin
structure is of acute importance to the welfare of the cell.
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, apoptosis and
differentiation [89]. However, the majority of the studies into mechanisms by which this
class of chemical inhibit cell growth have focussed on sulforaphane and phenethyl-ITC.
For example, 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; this was associated with a higher level of apoptosis
and lower proliferation in animals on the ITC containing diet [90].
In human PC-3 prostate cancer cells, 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 [91,92]. The loss
of Cdc25C was reported to be due to proteasomal activity, and was accompanied
by its translocation from the nucleus to the cytoplasm [92]. Relocation of Cdc25C was
found to be controlled by its phosphorylation at Ser-216, mediated through activation
of checkpoint kinase 2 (Chk2) [84]. Cell proliferation by ITCs may also be achieved
by distrupting cytoskeletal structure and tubulin polymerisation [93,94].
150
Administration of ITCs to cells at growth suppressive concentrations results

in the rapid generation of reactive oxygen species (ROS), within 1 h of exposure, which
appears to be necessary for cell death [92,95]. The generation of ROS by ITCs
is accompanied by depletion of intracellular GSH and is achieved through the rapid export
of ITC-glutathione and ITC-cysteinylglycine conjugates via MRP1 and Pgp-1 efflux
pumps [96]. Consistent with the view that production of ROS is necessary for apoptosis,
overexpression of catalase suppresses ITC-initiated programmed cell death, as does
pre-treatment with N-acetylcysteine [92]. Furthermore, addition of GSH subsequent
to ITC treatment can block apoptosis [44,97]. Treatment with ITCs leads to a loss
of mitochondrial membrane potential and release of cytochrome c from mitochondria
[98]. There is evidence that ITCs can activate both the intrinsic and extrinsic caspase
cascades, though this may be cell specific. For example, in PC-3 cells sulforaphane can in-
crease Fas protein levels and activate caspase-8 whilst simultaneously targeting mito-
chondria and activating caspase-9 [92]. In human bladder cancer UM-UC-3 cells, benzyl-
ITC and phenethyl-ITC are more effective at activating caspase-9 than caspase-8 [99].
By contrast, in human leukaemia HL60 cells, caspase-8 plays a major role in apoptosis
stimulated by phenethyl-ITC [100]. The levels of pro-apoptotic proteins Bak and Bax,
which neutralize the antiapoptotic effects of Bcl-2, are increased by phenethyl-ITC
and sulforaphane in PC-3 prostate cancer cells, and this may lead to induction of Apaf-1
[91,92,101]. Furthermore, the pro-apoptotic proteins Bok and Bim EL are also induced
by ITCs, and this is thought to amplify the effects of Bak and Bax [92]. Besides increasing
the levels of these pro-apoptotic proteins, ITCs down-regulate the anti-apoptotic proteins
Mcl-1 and Bcl-xL , though the effect is cell-specific [101]. Various ITCs have been shown
to activate c-Jun N-terminal kinase (JNK) [102,103], and this is mediated by extracellular
signal-regulated kinases, ERK1/2 [104]. The use of inhibitors indicated that JNK
is essential for phenethyl-ITC to cause cytochrome c release and caspase-3 activation
in human HT-29 colon adenocarcinoma cells [105]. However, the mechanism by which
JNK activates caspases remains unclear.
Stimulation of cell cycle arrest and apoptosis by indoles
Treatment of human MCF-7 breast cancer cells with 100 µM indole-3-carbinol

inhibits proliferation through affecting a G1 cell cycle arrest [106]. This may in part
be due to 3,3∂-diindolylmethane rather than indole-3-carbinol as significant quantities
of the indole spontaneously condense to the dimer in culture conditions [107]. Cell cycle
arrest at G1 occurs as a consequence of indole-3-carbinol inhibiting both cyclin-dependent
kinase (CDK) 2 and CDK6. In the case of CDK6, expression of the gene is reduced because
indole-3-carbinol attenuates recruitment of the Sp1 transcription factor to the CDK6
promoter [108]. Furthermore, in HaCaT keratinocytes, treatment with 400 µM indole-3-
carbinol induces the CDK4/6 inhibitor p15INK4b mRNA and protein causing hypophospho-
rylation of Rb protein [109]. It therefore appears that cyclin D-CDK6 activity can
be inhibited by dual mechanisms. In the case of CDK2, indole-3-carbinol has been
151
reported to decrease the kinase activity in MCF-7 cells and inhibit phosphorylation
of Rb protein [77]. The reduction in CDK2 activity is attributed to a selective alteration
in the size of the complex in which it is contained, from an active form within
a 90 kDa complex to a lower activity form within a 200 kDa complex [110]; the 90 kDa
and 200 kDa complexes include forms of cyclin E that differ in size, and the larger
complex also contains an additional 75 kDa cyclin E immunoreactive protein. Further-
more, the reduction in CDK2 activity is accompanied by redistribution of the kinase
in the 200 kDa complex from the nucleus to the cytoplasm, suggesting that
indole-3-carbinol can influence the nucleocytoplasmic shuttling of the kinase [110].
In cells that contain wild-type p53, such as MCF-10A, treatment with 300 µM
indole-3-carbinol or 30 µM 3,3∂-diindolylmethane has been found to result in activation
of the ATM signalling pathway, an increase in p53 protein levels, induction of p21 [111].
These changes result in prevention of the CDK2-mediated G1/S transition [111].
Indole-3-carbinol, but not 3,3∂-diindolylmethane, was found to inhibit the expression
of the androgen receptor in human lymph node carcinoma of prostate (LNCaP) cells
as well as the probable downstream target gene prostate specific antigen [112]. It is
possible that down-regulation of this receptor represents an antiproliferative mechanism
in prostate cells.
Indoles can affect apoptosis in breast and prostate cancer cells. Treatment of PC-3
prostate cancer cells with 60 µM indole-3-carbinol inhibits the EGF-induced
autophosphorylation of PI3K and Akt [113]. Thus, the Akt/PI3K cell survival pathway
appears to be targeted by indole-3-carbinol. Also, nuclear translocation of NF-κB
is inhibited by 3,3∂-diindolylmethane through a reduction in phosphorylation
of IκBα [114,115].
In HCT-116 human colon cells, indole-3-carbinol can induce nonsteroidal anti-
inflammatory drug-activated gene-1 (NAG-1), a TGF-β family member associated with
pro-apoptotic activities [116]. This may also mediate the anti-tumour effects of indoles.
Concluding comments
It is becoming clear that glucosinolate breakdown products can influence the initiation
and progression of carcinogenesis. They also appear to influence apoptotic responses
to chemotherapeutic agents, such as tamoxifen [77]. 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. Specifically, there are
few data about chemopreventative activities of thiocyanates, nitriles, cyano-
epithioalkanes and oxazolidine-2-thiones. It is unclear whether formation of thiocyana-
tes, nitriles, cyano-epithioalkanes and oxazolidine-2-thiones from glucosinolates, at the
expense of forming isothiocyanates, is undesirable from a cancer chemoprevention
perspective. It is unclear whether the activity of ESP, which reduces the formation
of isothiocyanates from glucosinolates, is undesirable. If so, ESP should possibly
152
be eliminated by genetic means from commercial crops. Furthermore, relatively little

is known about the pharmacokinetic properties of glucosinolate breakdown products
in the human, and without this information it is difficult to relate responses of cells
in culture to certain concentrations of phytochemical to what happens in the in vivo
situation. These are areas that warrant further examination.
Mammalian cells display marked dose responsiveness to phytochemicals: at low doses
of phytochemical, cytoprotective adaptive responses are activated, whereas at higher
doses cell cycle arrest and apoptosis occurs. It is presently unclear how these different
types of response are co-ordinated by the cell and how decisions about whether adap-
tation, growth arrest or apoptosis is chosen as the appropriate response are determined.
Identification of mechanisms that control such outcomes will be useful.
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Theo M. de Kok, Simone G. van Breda
160
3.5. Combined action of different dietary compounds

preventive of cancer
Theo M. de Kok and Simone G. van Breda

Department of Health Risk Analysis and Toxicology, University of Maastricht, Maastricht, The Netherlands
Introduction
The relatively consistent epidemiological finding that the consumption of whole foods
of different types such as fruits, 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. In the
research conducted, numerous bioactive compounds have been isolated and identified,
and their potential health-promoting effects evaluated extensively, both in vitro and in vivo.
One of the key problems of research in this field, 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. 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, vegetables, whole grains, and the like.
Although relatively high doses of single bioactive agents may show potent anticarcinogenic
effects, the cancer-preventive effects that certain whole foods and diets are shown to have
can perhaps better be explained in terms of the chemopreventive properties that interactions
between the different dietary ingredients involved create. In this chapter, evidence that
bioactive compounds act synergistically is reviewed.
Evaluating the effects of phytochemicals that act synergistically
Carcinogenesis is an extremely complex multistep process in which numerous molecular

mechanisms play an important role. Cancer-preventive dietary compounds may interfere
at a variety of different levels with these processes. Table 3.10. summarizes various mecha-
nisms by which phytochemicals can modulate the risk of cancer [1,2]. The combinations
of phytochemicals that natural foods contain can reduce the risk of cancer by affecting
different overlapping and complementary mechanisms. Isolated and purified compounds,
in contrast, may lose their biological activity or fail to behave in the same way as in the com-
plex matrix that the original item of food represents. 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, which are believed to have cancer-preventive effects. The intended positive
effects of an increased intake of β-carotene or vitamin C as dietary supplements, in contrast,
are far from certain. Some studies show no reduced incidence of cancer as a result of taking
supplements of vitamin C [3] or β-carotene [4], and there are even reports of increased
occurrence of lung cancer in smokers receiving dietary β-carotene supplements [5,6].
In addition to characterizing the chemopreventive effects of individual compounds,
therefore, evaluation of the effects of synergistically acting phytochemicals is needed.
Bioactive components in foods: Combined action of different dietary compounds preventive of cancer
161
Table 3.10. 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. [2].
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 3.11. presents a selection of relevant
studies concerning such synergistic effects. Suganuma et al. [7] reported that the effects
of tritium-labelled epigallocatechin gallate (EGCG) being incorporated into human
lung cancer cells were enhanced by epicatechin (EC), another green tea polyphenol,
one without a galloyl moiety. Epicatechin was found to promote the occurrence
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. These effects, induced by a tea polyphenol
having a galloyl moiety, were also enhanced in a dose-dependent way by EC. 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.
Table 3.11. Selection of studies of the synergistic effects of mixtures of polyphenols and of combinations
of these with other types of phytochemicals
Combination
Synergistic effect Mechanisms involved Reference
of compounds
Polyphenol mixtures
EGCG and EC, Inhibition of growth of human Enhanced cellular uptake of EGCG, Suganuma
sulindac or tamoxifen lung cancer cells enhanced apoptosis, reduced et al. [7]
release of TNF-α
EGCG and EC Inhibition of cell growth Increased production Horie et al. [8]
and induction of apoptosis of caspases-3, -8 and -9;
in gastric carcinoma cells Extracellular production
of oxygen species
EGCG, EC, EGC and ECG Modulation of CYP1A1 Antagonism of TCDD-induced Williams
expression in human transcription of human CYP1A1 et al. [10,11]
hepatocytes via interaction with the Ah-receptor
Polyphenols and other phytochemicals
Green/black tea and soy Inhibition of prostate tumours, Reduction in serum levels of Zhou et al. [12]
(SPC)* tumour weight and metastasis testosteron and DHT
Green/black tea and soy Inhibition of breast tumour Inhibition of tumour angiogenesis, Zhou et al. [13]
(SPC) cell growth reduced levels of estrogen receptor-α
protein and of serum IGF-I.
Green tea infusions and grape Reduced tumour cell growth Inhibition of tNOX, induction of Morré and Morré
or grape skin extracts apoptosis, [16]
Polyphenols, vitamin E, A Reduced oxidative stress Reduced formation of lipid Gorelik et al. [18]
and ß-carotene hydroperoxides and malondialdehydes;
reduced co-oxidation of vitamins E, C
and ß-carotene
EGCG, EC, EGC and ECG Reduced oxidative stress in Reduction in α-tocopheryl radicals, Zhou et al. [19]
or gallic acid micelles and human LDL trap-ping of lipid peroxyl radicals and Zhou et al. [21]
and α-tocopherol regene-ration of vitamin E Liu et al. [22]
163
Table 3.11. Selection of studies of the synergistic effects of mixtures of polyphenols and of combinations
of these with other types of phytochemicals — cont.
Combination
Synergistic effect Mechanisms involved Reference
of compounds
12 red wine polyphenols Increased antioxidant potential Regeneration of phenoxy radicals De Beer et al. [25]
by phenolic compounds Jørgensen
et al. [27]
Different types of vegetables Up- and down-regulation No indication of specific Van Breda
(cauliflower, carrots, of genes involved in synergetic mechanisms et al. [23,24]
peas and unions) carcinogenesis, interpreted
mainly in preventive terms
* SPC — Soy Phytochemical Concentrate.
Synergistic effects of green tea catechins, both on the inhibition of cell growth and the
induction of apoptosis, were also found in gastric carcinoma cells [8]. Various gastric cell lines
were shown to differ in their susceptibility to EGCG treatment. EC alone had virtually
no effect on carcinomic cell growth or induction of apoptosis, but it had a significant
synergistic effect on the induction of apoptosis when combined with other catechins. After
this combined treatment, the activity levels of caspases 3, 8 and 9 became elevated, indicating
them to be involved in catechin-induced apoptosis. Interestingly, catalase was found to block
the synergistic effects of EC and EGCG, suggesting that the reactive oxidative species
and production of hydrogen peroxide play a part in the synergistic mechanisms here [8].
Since the cytochrome P450 (CYP) enzymes are responsible for the metabolism of many
environmental carcinogens, the modulation of their expression and activity by phyto-
chemicals is a potential mechanism by which the risk of cancer can be reduced. Some
of the cytochrome P450 genes are expressed constitutively, whereas others are inducible
by xenobiotic compounds or by phytochemicals. Enzyme induction usually enhances detoxi-
fication, but under some circumstances substrates may be activated to become mutagens,
carcinogens or cytotoxic substances [9]. The induction of CYP1A enzymes by PAH
and by dioxins such as TCDD occurs at the transcription level and is mediated by the cytosolic
aryl hydrocarbon receptor (AhR) [9]. Williams et al. [10] demonstrated that complex green tea
extracts exert mixed agonist/antagonist activity on the Ah-receptors, whereas EGCG acts
as a strict AhR antagonist. 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,
being due instead to the effects of the complex mixture involved. 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).
The optimal degree of synergy was achieved by a combination of the four major tea cate-
chins — EC, EGCG, epigallocatechin (EGC), and epicatechin gallate (ECG) — and it was not
found to be improved further by the addition of other compounds [11].
164
Synergistic effects of polyphenols and other phytochemicals

In two different studies, Zhou et al. 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 [12,13]. A phytochemical soy concentrate (SPC) and green and black tea in-
fusions were employed (Table 3.11.). In further investigations it was shown that bioactive
compounds in tea (particularly EGCG [14]) and soy (the soy isoflavone genistein
as well as SPC) inhibit growth of prostate cancer and tumour metastasis in vivo [15].
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, the latter
a biologically more active metabolite of testosterone and a prerequisite for the
development of benign prostatic hyperplasia and prostate cancer [12]. In an immune-
deficient mouse model in which MCF-7 human breast cancer cells were implanted, SPC
in combination with green tea showed a synergistic inhibition of tumour cell growth.
This was accompanied by inhibition of tumour angiogenesis, reduced expression
of estrogen-receptor (ER) alpha and reduced serum levels of the insulin-like growth factor
(IGF)-I, all of these being factors in breast cancer development. The modulation of these
two different mechanisms may be an explanation of the synergistic effects of the com-
bined phytochemicals [13].
A tumour-specific growth protein possessing NADH oxidase activity (tNOX) has
emerged as a potential target of the anticancer action of plant polyphenols and flavonoids
[16]. NOX proteins are located at the cell surface and are responsible for the increase
in cell size that occurs following cell division [17]. Cells in which NOX activity is blocked,
such as by phytochemicals, are unable to enlarge. They cease to divide and eventually
undergo apoptosis. Recently, Morré and Morré [16] showed there to be an exceptionally
strong (10-fold) synergy between grape polyphenols and tea catechins in the inhibition
of tNOX. The strongest synergistic activity was found for ethanol extracts of grape skins,
whereas no activity was detected for extracts of grape seeds, indicating that the effects
were not caused by resveratrol, which is found predominantly in the seeds. These results
suggest that more effective cancer prevention and cancer therapy could be achieved
by use of combinations of different phytochemicals.
Polyphenols and dietary antioxidant vitamins can also have synergistic inhibitory
effects on lipid peroxidation and on the co-oxidation of dietary antioxidants. 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, though to a lesser
extent) [18]. In the gastric fluid, vitamin C can enhance the activity of polyphenols
through a synergistic antioxidant effect. In line with this, the authors suggested that the
antioxidant network in the stomach can decrease the level of hydroperoxides and other
cytotoxic compounds, increasing at the same time the amounts of vitamin antioxidants
reaching the blood system. The authors indicated that the resulting synergistic increase
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, 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 com-
binations of polyphenols and other antioxidants [18]. By studying the kinetics of the
reaction of α-tocopherolyl radicals with green tea polyphenols by means of stopped-flow
electron paramagnetic resonance, Zhou et al. [19] demonstrated clearly that several green
tea polyphenols (EC, EGCG, EGC, ECG and gallic acid) can effectively reduce α-toco-
pheroxy radicals so as to allow α-tocopherol to be regenerated. These green tea polyphe-
nols were also found to trap the initiating radicals (ROO•) as well as the propagating lipid
peroxyl radicals (LOO•). It is particularly the elimination of the pro-oxidant effect of vita-
min E (or the so-called tocopherol-mediated peroxidation) that can occur in the absence
of other oxidants [20]. This, combined with an α-tocopherol regenerating reaction invol-
ving coexisting antioxidants, plays a major role in enhancing the antioxidant efficiency
of vitamin E. 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 [21,22].
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, the synergistic effects of taking whole foods or complex mixtures
of compounds have been reported. In two recent animal studies on the effects
of vegetable consumption on the modulation of gene expression, it was found that
most of the genes that were differentially expressed before and after vegetables were
consumed, the changes in gene expression could be interpreted as a cancer preventive
effect [23,24]. 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. 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. On the other hand,
the individual vegetables were able to modulate genes which were not significantly
modulated by the mixture, indicating that combinations of different foods containing
different complex mixtures of phytochemicals can also have an antagonistic effect
on gene expression.
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. In one study,
the Trolox equivalent antioxidant capacity (TEAC) value and phenolic composition were
determined for a large number of pinotage wines [25]. When the contributions of the
separate phenolic compounds were calculated, 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. Taking account of the different mixtures found of the 12 phenolic compounds
that were present, and assuming their concentrations to be those typical for red wines,
166
indicated 16 to 23% of the antioxidant activity to be synergistic. This implies that,

in addition to the synergistic effects between the different phenolic compounds,
synergistic effects between these and the other wine constituents may have contributed
to the TEAC values. The author excludes a potential role of sulphur dioxide in the
regeneration of phenolic compounds from their phenoxyl radicals as it does not contribute
to the total antioxidant potential at the concentrations normally present in red wines [26].
Jørgensen et al. [27] demonstrated, however, the regeneration of quercetin from
its phenoxyl radical by the presence of (+)-catechin. This suggests that the regeneration
of phenolic compounds from phenoxy radicals is a mechanism that can contribute
to the synergistic effects observed.
Other synergistic effects

In addition to investigating the synergistic effects of various phytochemicals, 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 [1,28]. Administering multiple agents can increase the efficacy and potency
of the chemopreventive measures taken and reduce toxic side-effects. Various combi-
nations of drugs have been proposed for further clinical development based on their
synergistic activity as shown in vitro or in animal studies. Use of retinoids in combination
with such SERMs (selective estrogen receptor modulators) as tamoxifen or raloxifene
[29,30] are examples of this. Also, the effects reported by Suganuma et al. [7] of using
EGCG to induce apoptosis in human lung cells in vitro have been synergistically enhanced
by use of cancer preventive agents such as sulindac and tamoxifen. This effect has also
been evaluated in an animal study in which treatment of rats with a combination
of EGCG and sulindac was found to reduce aberrant crypt formation after their being
treated with azoxymethane for colon carcinoma [31]. In that study, EGCG and sulindac
were also found to synergistically enhance apoptosis. 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, its also reducing side
effects, particularly those of sulindac, without a loss in treatment potency. A review
of the effects of various combinations of chemotherapeutic and chemopreventive
approaches in dealing with cancer has appeared recently [28].
Conclusions
An increasing number of in vitro and in vivo studies indicate that combinations of dietary
chemopreventive agents can result in a significant level of activity being achieved
at concentrations at which any single agent is virtually inactive. The fact that many
of these phytochemicals act synergistically may why some food items or diets show
cancer preventive effects that cannot be explained on the basis of their separate bioactive
ingredients alone. Although our understanding of the molecular mechanism behind the
observed combinatorial effects of these chemopreventive agents is still limited, it appears
167
that many different combinations of complementary modes of action may be involved.

There is a clear need of further study of the synergistic effects of dietary phyto-
chemicals. This may contribute to the improvement of cancer treatment and cancer
prevention. Especially the development of new dietary supplement regiments, cancer
therapies and nutraceuticals can benefit from improved insight into the mechanisms
behind the synergistic effects that both natural and synthetic chemopreventive
compounds show.
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3.6. Probiotics and prebiotics — potential

anticarcinogenic food components
Joseph Rafter
Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden
Introduction
An example of a functional food, 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’ [1,2]. Probiotics usually
refer to highly selected lactic acid bacteria, e.g. Lactobacillus spp. Bifidobacterium spp.
and Streptococcus spp., with defined gut survival properties and associated biological
activities and which can be ingested in fermented milk products or as a supplement.
The list of healthful effects attributed to probiotic bacteria is extensive [3] and includes:
alleviation of lactose intolerance symptoms; serum cholesterol reduction; anticancer
effects; alleviating constipation; relieving vaginitis to name but a few. The vast majority
of studies on the anticancer effects deal with colorectal cancer (CRC), although there
are some on breast [4] and bladder cancer [5].
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. Diet makes
an important contribution to CRC risk [6] implying that risks of CRC are potentially
reducible. Evidence from a wide range of sources supports the view that the colonic
microflora is involved in the etiology of CRC. This has led to intense interest in factors,
such as probiotics, 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 have the potential to improve host health’) [3] and syn-
biotics (combinations of pro- and prebiotics), that can modulate gut microflora and its
metabolism. Evidence for protective effects of pro- and prebiotics on cancer is derived
from in vitro studies, animal models, epidemiology and human intervention studies.
Overall, 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- or prebiotics alone. The evidence from
animal studies provides strongest support for anticancer effects and data from human
studies (epidemiology and experimental) are limited.
Evaluation of anticarcinogenic dietary effects
Evidence from human studies

Consumption of lactobacilli by healthy volunteers has been shown to reduce the muta-
genicity of urine and faeces associated with the ingestion of carcinogens in cooked meat.
Administration of L. acidophilus to eleven volunteers on a fried meat diet known to in-
crease faecal mutagenicity, resulted in a lower faecal mutagenic activity after 3 days
Bioactive components in foods: Probiotics and prebiotics — potential anticarcinogenic food components
171
compared to faecal mutagenic activity after 3 days consumption of ordinary fermented

milk (although not significant) [7]. High levels of mutagenicity also appeared in urine
on days 2 and 3 of the fried meat and ordinary fermented milk dietary regimen. During
L. acidophilus administration, the urinary mutagenic activity on days 2 and 3 was
significantly lower compared to the ordinary fermented milk period. In most cases,
an increase in the number of faecal lactobacilli corresponded to a lower mutagen
excretion, particularly in urine. Hayatsu and Hayatsu [8] also demonstrated a marked
suppressing effect of orally administered L. casei on the urinary mutagenicity arising
from ingestion of fried ground beef in the human.
As yet, there are few epidemiological studies addressing the association between
fermented dairy products and colorectal cancer. Consumption of large quantities of dairy
products such as yoghurt and fermented milk containing Lactobacillus or Bifidobacterium
may be related to a lower incidence of colon cancer [9]. An epidemiological study
performed in Finland demonstrated that, despite the high fat intake, colon cancer
incidence was lower than in other countries because of the high consumption of milk,
yoghurt, and other dairy products [10,11]. In two population-based case-control studies
of colon cancer, an inverse association was observed for yoghurt [12] and cultured milk
consumption [13], adjusted for potential confounding variables. In another case control
study, an inverse relationship for yoghurt consumption with risk of large colon adenomas
in men and women was reported [14]. 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 [4,15]. On the
other hand, two companion American prospective studies, the 1980–1988 follow-up
of the Nurses’ Health Study and the 1986–1990 Health Professionals follow-up study,
did not provide evidence that intake of dairy products is associated with a decreased risk
of colon cancer [16]. In a cohort study in the Netherlands, 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,
although a weak non-significant inverse association with colon cancer was observed [17].
In summary, it would appear that the case control studies indicate protective effects
while the prospective studies do not.
In conclusion, data from human intervention studies are of paramount importance
in providing evidence that probiotics, prebiotics or fermented milk consumption are
causally related to reduction in cancer risk. Thus, this is an area of high priority for future
studies. 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. Once such markers are available, it will become possible
to perform studies in healthy volunteers, at-risk groups and patients. It will be important
to define dose and time relationships and it would appear at present, from animal studies,
the most profitable approach would be to use combinations of pro- and prebiotics.
There will also be, in the near future, the opportunity to exploit genomics and proteomics
in investigations of effects of pro/prebiotics on gene expression and post-transcription
Joseph Rafter
172
events in colonic biopsies and to identify human groups responsive to pro/prebiotic

intervention. It will also be particularly important to use data on mechanisms of action
to develop hypothesis based intervention studies in humans.
Of relevance here is a clinical trial which is presently ongoing i.e. to examine the effect
of a synbiotic preparation on colon cancer risk biomarkers in humans (SYNCAN project,
funded by EU, and involving 8 research centres in Europe; http://www.syncan.be).
It involves a twelve-week randomised, double blind, placebo controlled trial of a food
supplement containing Lactobacillus GG, Bifidobacterium Bb-12 and Raftilose Synergyl
in adenoma patients. In this study, all of the ”state of the art” colon cancer risk
biomarkers, including colonic mucosal markers, faecal water markers and immunological
markers, are being measured. 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.
Evidence from laboratory animal studies

There are several good animal models for colon cancer which have proved useful
for identifying dietary factors which may protect us against the development of this
tumour. End points used are the tumours themselves or early lesions, such as aberrant
crypt foci (ACF). ACF are putative preneoplastic lesions from which adenomas
and carcinomas may develop. In recent years, there have been many studies, using these
models, which clearly demonstrate a protective effect of dietary supplements of lactic
acid bacteria against colon tumour development.
Oral administration of lactic acid bacteria has been shown to effectively reduce DNA
damage, induced by chemical carcinogens, in gastric and colonic mucosa in rats.
Pool-Zobel et al. [18] reported, using the comet assay, that Lactobacillus acidophilus,
L. gasseri, L. confusus, Streptococcus thermophilus, Bifidobacterium breve and B. longum were
antigenotoxic toward N’-nitro-N-nitrosoguanidine (MNNG). These bacteria were also
protective toward 1,2-dimethylhydrazine (DMH)-induced genotoxicity. Metabolically
active L. acidophilus cells, as well as an acetone extract of the culture, prevented
MNNG-induced DNA damage, while heat-treated L. acidophilus was not antigenotoxic.
Among different cell fractions from L. acidophilus, the peptidoglycan fraction and whole
freeze-dried cells were antigenotoxic.
Certain strains of lactic acid bacteria have also been found to prevent putative
preneoplastic lesions or tumours induced by carcinogens. Goldin et al. [19] showed that
a specific strain of L. casei subsp. 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. Overnight cultures
of L. acidophilus also inhibited the formation of ACF, induced by azoxymethane (AOM)
[20]. Although B. adolescentis culture and its supernatant did not show an inhibitory
effect in this study [20], feeding of bifidobacteria suppressed the ACF formation induced
by AOM [21,22] or DMH [23,24]. Consumption of B. longum or inulin was associated
with a decrease in AOM-induced colonic small ACF in rats and combined administration
173
significantly decreased the incidence of large ACF [25]. In addition, 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, and that
the additional colonisation of B. breve reduced the number of ACF with four or more
crypts/focus and crypt multiplicity which are reliable predictors of malignancy [26].
Reddy and Rivenson [27] reported that lyophilised cultures of B. longum administered
in the diet to rats inhibited liver, colon and mammary tumours, induced by the food
mutagen 2-amino-3-methyl-3H-imidazo(4,5-f)quinoline (IQ). Goldin and Gorbach [28]
showed that dietary supplements of L. acidophilus not only suppressed the incidence
of DMH-induced colon carcinogenesis but also increased the latency period in rats.
Feeding of fermented milk increased the survival rate of rats with chemically induced
colon cancer [29]. Dietary administration of a lyophilised culture 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 [30]. Ingestion of B. longum also
significantly inhibited AOM-induced cell proliferation, ornithine decarboxylase activity
and expression of the ras-p21 oncoprotein. Recently, there was a report on the anti-
tumourigenic activity of the prebiotic inulin, enriched with oligofructose, in combination
with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis in the AOM-induced
colon carcinogenesis rat model [31]. The authors concluded that, while a possible
protective effect of probiotics was observed, the results indicated that the prebiotic
decreased AOM-induced carcinogenesis. The mechanisms by which they act are less clear,
but the data presented suggested that they may act through a combination of mecha-
nisms involving an increase in short-chain fatty acid (SCFA) production, lower prolife-
rative activity and a variation in the expression of some enzymes involved in the patho-
genesis of colon cancer.
There is additional direct evidence for anti-tumour activities of lactic acid bacteria
obtained in studies using pre-implanted tumour cells in animal models. It has been
demonstrated that feeding of fermented milk or cultures containing lactic acid bacteria
inhibited the growth of tumour cells injected into mice [32,33]. Sekine et al. [34] using
whole peptidoglycan isolated from B. infantis strain ATCC15697, reported that a single
subcutaneous injection significantly suppressed tumour growth. In addition, five
intralesional injections resulted in 70% tumour regression in the mice.
More recently, mindful of the fact that the composition and metabolic activities
of the intestinal flora of experimental animals are significantly different from those
of humans [35], we exploited human flora associated (HFA) mice to test the effects
of a probiotic mixture on a parameter of relevance for colon carcinogenesis i.e. DNA
adduct formation [36]. Indeed, the results from a previous report from our laboratory,
demonstrated that human intestinal microflora had different effects than mouse
microflora concerning DNA adduct formation after exposure to mutagens [37].
The probiotic mixture, Biothree®, used in this study contained Streptococcus faecalis T-110,
Clostridium butyricum TO-A and Bacillus mesentericus TO-A, which are acid resistant
in contrast to most bacteria, which do not survive contact with gastric acid. It has been
Joseph Rafter
174
reported that S. faecalis T-110 and C. butyricum TO-A showed strong symbiosis with each
other and the growth of enteropathogens (enterotoxigenic Escherichia coli, Salmonella
typhimurium, Vibrio parahaemolyticus, C. difficile and C. botulinum) was inhibited in mixed
cultures of S. faecalis T-110 and C. butyricum TO-A [38]. It has also been reported that
B. mesentericus TO-A stimulated the growth of Bifidobacterium by producing
3,3-dihydroxyazetidine [39,40]. Biothree® is used as a clinical therapy in Japan. It is effec-
tive for the improvement of symptoms caused by abnormal intestinal flora, i.e. diarrhoea
and constipation. Interestingly, the results of this study demonstrated that the above
probiotic mixture had an effect to significantly decrease the DNA adduct formation
in the colonic epithelium induced by the food mutagen 2-amino-9H-pyrido[2,3-b]indole
(2-amino-alpha-carboline; AAC), given by gavage. Two possible mechanisms may be in-
volved: reduction of direct exposure to AAC and/or induction of DNA repair of the DNA
adducts in the colonic epithelium.
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. However, such mechanisms might include: alteration of the meta-
bolic activities of intestinal microflora; alteration of physicochemical conditions in the
colon; binding and degrading potential carcinogens; quantitative and/or qualitative
alterations in the intestinal microflora incriminated in producing putative carcinogen(s)
and promoters (e.g. bile acid-metabolizing bacteria); production of antitumourigenic
or antimutagenic compounds; enhancing the host's immune response; effects on physio-
logy of the host.
Conclusion
Many healthful effects are attributed to the probiotic bacteria and some of these effects
have more scientific support than the anticancer effect. The strongest evidence for anti-
cancer effects of probiotics comes from animal studies and evidence from human studies
(epidemiology and experimental) is still limited. An important goal for the future should
be carefully designed human clinical trials to corroborate the wealth of experimental
studies. Also, as mentioned above, there are several possible mechanisms which might
explain how lactic acid bacteria might protect against tumour development in the colon.
It is possible that different strains target different mechanisms. All of the mechanisms
have various degrees of support, mainly originating from in vitro and animal experiments
and some of them even have some support from human clinical studies. Thus, more work
needs to be done to identify the specific strains and strain characteristics responsible
for specific antitumour effects and the mechanisms by which these effects are mediated.
However, even with the above reservations in mind and mindful of the limited number
of human studies available, the use of lactic cultures for human cancer suppression
is interesting, holds promise and certainly deserves more scrutiny.
175
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Annex ECNIS participants
Nofer Institute of Occupational Medicine (NIOM)

Poland, Âw. Teresy 8, 91-348 ¸ódê, coordinator, ecnis@ecnis.org
Deutsches Krebsforsforschungszentrum (DKFZ)

Germany, Neuenheimer Feld 280, 69-120 Heidelberg, J.Nair@dkfz.de
University of Copenhagen (UC)

Denmark, Norregade 10, DK-1017 Copenhagen, steffen.loft@farmakol.ku.dk
Karolinska Institutet (KI)

Sweden, SE-171 77 Stockholm, dan.segerback@biosci.ki.se
Fondazione ISI (ISI)

Italy, Viale Settimo Severo 65, 10133 Turin, giuseppe.matullo@unito.it
The National Hellenic Research Foundation (NHRF)

Greece, Vassileos Constantinou Avenue 48, 11635 Athens, skyrt@eie.gr
University of Leicester (ULEIC)

United Kingdom, University Road, LEI 7 RH Leicester, pbf1@leicester.ac.uk
National Institute of Environmental Health (NIEH)

Hungary, Gyáli út 2-6, H-1097 Budapest, schoketb@okk.antsz.hu
Nicolaus Copernicus University,

Collegium Medicum in Bydgoszcz
Poland, Jagielloƒska 13, 85-067 Bydgoszcz, ryszardo@cm.umk.pl
Genetics Research Institute & Ospedale Policlinico (GRI)

Italy, Strada Della Carita 10, 20135 Milan, taioli@policlinico.mi.it
Johannes Gutenberg-Universität Mainz (JOGU)

Germany, Saarstrasse 21, 55099 Mainz, oesch@uni-mainz.de
Finnish Institute of Occupational Health (FIOH)

Finland, Topeliuksenkatu 41 a A, 00250 Helsinki, Ari.Hirvonen@ttl.fi
Vrije Universiteit Brussel (VUB)

Belgium, Pleinlaan 2, 1050 Brussels, mkirschv@vub.ac.be
Lund University (ULUND)

Sweden, Paradisgatan 5C, SE-22100 Lund, bjorn.akesson@tbiokem.lth.se
Institute of Cancer Research (ICR)

United Kingdom, Old Brompton Road 123, SW7 3RP London, david.phillips@icr.ac.uk
180
Katholieke Universiteit Leuven

Belgium, Oude Markt 13, 3000 Leuven,
K. U. Leuven R&D Department, Groot Begijnhof 58-59, B-3000 Leuven,
ludwine.casteleyn@lin.vlaanderen.be
Maastricht University (UM)

The Netherlands, Universiteitssingel 50, 6200 MD Maastricht, f.vanschooten@grat.unimaas.nl
Biochemical Institute for Environmental Carcinogens Prof. Dr Gernot Grimmer Foundation (BIU)
Germany, Lurup 4, 22927 Grosshansdorf, albrecht.seidel@biu-grimmer.de
Institut Catala d'Oncologia (ICO)

Spain, Gran Via S/N Km27, 08907 L'Hospitalet de Llobregat, Barcelona, cagonzalez@ico.scs.es
Utrecht University, Institute of Risk Assessment Science (IRAS-UU)

The Netherlands, Heidelberglaan 8, 3508 TC Utrecht, H.Kromhout@iras.uu.nl
University of Dundee (UNIVDUN)

United Kingdom, Nethergate, DD1 4HN Dundee roland.wolf@cancer.org.uk
International Agency for Research on Cancer (IARC)

France, Cours Albert Thomas 150, 69372 Lyon, boffetta@iarc.fr
NETIX Skrzypczyƒski, Krzysztofowicz Sp. J. (NETIX)

Poland, Stawki 2, 00-193 Warszawa, mask@netix.pl
Leocordia AB (Leocordia AB)

Sweden, Selagarden, Stallarholmen, lennart.moller@csb.ki.se
Imperial College London (ICL)

United Kingdom, Exhibition Road, London, p.vineis@imperial.ac.uk

Dietary Vitamins, Polyphenols Selenium and Pro Bio Tics

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Dietary Vitamins, Polyphenols Selenium and Pro Bio Tics

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ECNIS is a Network of Excellence within the European Union’s Sixth Framework Programme,

Compiled and edited by Björn Åkesson and Per Mercke

Technical editor: Katarzyna Rogowska

Published by Nofer Institute of Occupational Medicine

1.1. Introductory overview and future prospects

Appendix 1.2. Overview of possible anticarcinogenic food components

2. Vitamins and selenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.1. Biomarkers of exposure to and effects of vitamins A, C and E

2.2. Antioxidant vitamins and cancer risk: is oxidative DNA damage

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

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

Theodore G. Sotiroudis, Soterios A. Kyrtopoulos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

3.4. Anticarcinogenic effects of glucosinolate breakdown products

John D. Hayes, Michael O. Kelleher, Ian M. Eggleston . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

3.5. Combined action of different dietary compounds preventive of cancer

Theo M. de Kok, Simone G. van Breda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

3.6. Probiotics and prebiotics — potential anticarcinogenic food components

Joseph Rafter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

Annex ECNIS participants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Vitamins and selenium

Bioactive compounds in foods

Dietary assessment methodology and the development and validation of biomarkers

Table 1.1. Some major issues regarding nutritional biomarkers

Markers of dietary intake vs. markers of nutritional status

Dietary components as biomarkers

Functional biomarkers of nutrients

Use of nutritional biomarkers

Nutrient biomarkers in relation to genetic polymorphism

It is generally assumed that genetic polymorphism influences the response of biomarkers

Biomarkers and response to dietary supplements

Biomarkers for the assessment of dietary effects on carcinogenesis

Biomarkers used as a surrogate endpoint in studies of the effect that dietary

Application of omics technologies

Appendix 1.2. Overview of possible anticarcinogenic

There is a vast range of dietary compounds proclaimed to have anti-carcinogenic proper-

PubMed search No. of refs

Table 1.3. An overview of putative anticarcinogenic food components as assembled

Class of compound Example of bioactive

Table 1.3. An overview of putative anticarcinogenic food components as assembled

Class of compound Example of bioactive

Table 1.3. An overview of putative anticarcinogenic food components as assembled

Class of compound Example of bioactive

Methylxanthines Caffeine Tea, coffee, Manson [18]

References (selected from the PubMed search described above)

2.1. Biomarkers of exposure to and effects of vitamins A, C and E

Biomarkers for vitamin A in body fluids

Reversed-phase HPLC 4 20 25 [7]

Biomarkers of vitamin A related effects in the eye

Table 2.2. Biological indicators of subclinical VAD*

Prevalence cutoffs for defining a public health problem and assessing

Biomarkers of oxidative stress after supplementation with vitamin A

Biomarkers of vitamin C in body fluids

However, since treatment with dithiotreitol is known to reduce dehydroascorbate to