Eur J Nutr (2004) [Suppl 2] 43:II/47–II/84
DOI 10.1007/s00394-004-1203-6
Joseph Rafter
Mirjam Govers
Paule Martel
Daphne Pannemans
Beatrice Pool-Zobel
Gerhard Rechkemmer
Ian Rowland
Sandra Tuijtelaars
Jan van Loo
PASSCLAIM1 – Diet-related cancer
EJN1203
Joseph Rafter
Department of Medical Nutrition
Karolinska Institute
Novum
S-14186 Huddinge, Sweden
Mirjam Govers
Numico Research
Biomedical Research Department
Bosrandweg, 20
P. O. Box 7005
6700 CA Wageningen, The Netherlands
Paule Martel
I.N.R.A. – C.R.J.
Laboratoire de Nutrition et Sécurité
Alimentaire
Domaine de Vilvert
78352 Jouy-en-Josas Cedex, France
Daphne Pannemans
Former ILSI Europe
Avenue E. Mounier 83, Box 6
1200 Brussels, Belgium
Beatrice Pool-Zobel
Friedrich-Schiller University of Jena
Department of Nutritional Toxicology
Institute of Nutrition
Dornburger str. 25
07743 Jena, Germany
Gerhard Rechkemmer
Technische Universität München
Hochfeldweg, 1
85354 Freising, Germany
Ian Rowland
University of Ulster
NI Centre for Food and Health
School of Biomedical Sciences
Cromore Road
BT52 1SA Coleraine, Northern Ireland, UK
Sandra Tuijtelaars (౧)
ILSI Europe
Avenue E. Mounier 83, Box 6
1200 Brussels, Belgium
publications@ilsieurope.be
Jan van Loo
Raffinerie Tirlemontoise – Orafti
Aandorenstraat 1
3300 Tienen, Belgium
■ Summary Background The role
of dietary factors in the aetiology
of human cancer is an area, which
has attracted intense interest in recent
years. The suggestion that approximately
one third of all cancers
may be caused by an
‘inappropriate’ balance of food
components has led to the attractive
contention that we can significantly
decrease cancer incidence
through dietary recommendations
and a change in dietary habits in
populations. Thus, a key issue
must be to establish clear criteria,
which must be met in order to be
able to make ‘cancer risk reduction’
claims for food components.
In this area, the one true marker is
the malignant human tumour,
which for practical reasons is usually
not accessible to claims. In its
absence, we must rely on alternative
markers – biomarkers/surrogate
endpoints. This paper mainly
deals with the link of these biomarkers
to the endpoint tumour
and their usefulness for making
claims. Some claims have been
made based on epidemiological
studies. Aim Can we identify targets/biomarkers
in the chain of
events from initial ‘exposure’ to
overt malignant tumour, whose
modification can be used to make
‘anticancer’ claims for food components?
Results We identified 18
targets/markers in the above chain
of events whose modification ‘have
the potential’ to be used for ‘reduction
of cancer risk’ claims for food
components. These targets/markers
fall under 5 broad headings:
tumours&preneoplastic changes;
cellular targets/markers; gut luminal
markers; angiogenesis&metastasis;
carcinogen metabolising enzymes;
genetic events. Conclusions
The strongest markers presently
available are precancerous lesions
(e.g. polyps or aberrant crypt foci)
in humans and precancerous lesions
and tumours in animal models.
The only marker that presently
can be used for a ‘reduction of disease
risk’ claim (type B) for food
components is ‘polyp recurrence’.
Type B claims cannot be made on
the basis of results in animal models.
All of the other biomarkers examined
presently lack validation
against the ‘true endpoint’, the tumour,
and thus cannot be used for
type B claims. ’Reduction of disease
risk’ claims in the area of
‘diet-related cancer’ should be
based primarily on human intervention
studies using relevant/acceptable
endpoints. An important
area for future research will be the
validation of these surrogate end-
points.
■ Key words functional foods –
diet-related cancer – colon
cancer – biomarkers – surrogate
endpoints
1 Process for the Assessment of Scientific
Support for Claims on Foods.
II/48 European Journal of Nutrition (2004) Vol. 43, Supplement 2
General introduction
Epidemiological studies indicate that the processes of
carcinogenesis and tumourigenesis are mainly induced
by environmental factors. An extensive review of the
link between nutrition and cancer on a global scale has
been recently published by the World Cancer Research
Fund (WCRF) and theAmerican Institute for Cancer Research
(AICR).Some 35% of all cases of death by cancer
are caused by high risk diet (e.g. high saturated fat/low
fibre/low consumption of fruit and vegetables), 30% is
due to tobacco smoking, together with reproductive behaviour
(7%) and alcohol abuse (3%), thus about 75%
of death by cancer is due to diet and lifestyle. Another
20% are due to an unhealthy environment (7%), infection
(9%) and pollution (4%). Finally there are about
5% of death by cancer which are caused by hereditary,
genetic factors.
Cancer incidence in Europe is highest for lung cancer
in man and breast cancer in women, followed by colorectal
cancer for both sexes and then prostate cancer in
man and lung cancer in women. Smoking is the major
cause for lung cancer and diet, but particularly a diet
high in antioxidant-rich foods (fruits and vegetables)
reduces the risk of disease even in heavy smokers.
The development of breast and prostate cancer seems
to be linked to the hormonal status and both cancers are
promoted by endogenous estrogens, respectively. However,
phytooestrogens from dietary sources appear to
decrease the risk of these hormone-dependent cancers
as has been indicated by epidemiological studies.
Carcinogenesis for most cancers is a process developing
for decades (10–30 years). Several stages in the
process can be discriminated, e.g. initiation, promotion
and progression. At these various stages characteristic
molecular and cellular changes occur (see Fig.1). Many
of these different stages can be modulated by dietary
factors (food components and ingredients) either by direct
interaction with gene expression or through the
modulation of key enzyme activities involved in cell
proliferation and differentiation, respectively.
Their ability to undergo extensive proliferation is the
main characteristic of cancer cells. This finds its origin
in the typical overproduction of growth factors and/or
in the overexpression of receptors for growth factors by
these cells. In this context the action of insulin and insulin-like
growth factors appears to be especially important.
Obesity and diabetes seems to be a predisposing
condition for the development of some types of
cancers.
Cellular proliferation begins when cell-surface receptors
recognise their appropriate growth factors. Next, a
cascade of reactions mediated by cytoplasmatic protein
kinases culminates in transcriptional activation,cell-cycle
progression and cell division. This growth factor induced
mitogenic signalling can be blocked by certain
exogenic factors which prevent receptor activation or
which prevent binding of a growth factor to its receptor.
Next comes cell-cycle progression. In the cell cycle, 4
distinct phases can be distinguished. In the M-phase
there is active mitosis; the S-phase is the state of DNA
synthesis; these two phases are separated by two G or
gap phases (G1 and G2). The progression of one phase
to the other is mediated by cyclins (proteins whose concentration
rises and falls during the cell cycle) cyclin-dependent
kinases (CDKs, which are activated by specific
cyclins) and inhibitors of the assembly and activity of
cyclin-CDK complexes (CDKIs). If cells are inhibited at
any point in the cell cycle,their progression through the
cell cycle is disturbed,which in its turn prevents mitosis
and cell division.
Malignant cells are immortalised cells.This is mainly
due to the action of telomerases, which preserve the integrity
of the telomeres. These are regions at the end of
chromosomes,which are replicated by special processes,
counteracting the tendency of the chromosomes to become
shorter during a round of replication. Consequently,inhibiting
telomerase decreases the potential of
cancer cells to become immortal. It has been demonstrated
in cell systems that certain flavonoids potently
affect telomerase activity.
Besides being immortalised, tumour cells have infinite
replicative potential. DNA replication takes place in
the S-phase of the cell cycle. The process of replication
essentially duplicates the genetic material with the help
of the replication machinery (DNA polymerases, DNA
ligases, topoisomerases). Inhibiting replication ensures
that malignant cells do not progress in the cell cycle.
Healthy cells undergo apoptosis, programmed cell
death. This process is initiated by means of death sensors
and effectors. Evading apoptosis as tumours cells
do, requires mechanisms that inhibit transmission of
the death signals. Potentially any mechanisms which
T
GHOHWLRQ
S
LQDFWLYDWLRQ
1RUPDO 6PDOO DGHQRPDG\VSODVLD &DUFLQRPD
7R[LF JHQRWR[LF FRPSRXQGV LQ OXPHQ
$3& PXWDWLRQ
LQDFWLYDWLRQ
.UDV
DFWLYDWLRQ
/DUJH DGHQRPD
'1$ GDPDJH
&HOO
SUROLIHUDWLRQ
3URPRWLRQ 0HWDVWDVLV
3URWHFWLYH SK\WRFKHPLFDOV LQGXFWLRQ RI SKDVH ,,
HQ]\PHV DSRSWRVLV '1$ UHSDLU
T
GHOHWLRQ
S
LQDFWLYDWLRQ
1RUPDO 6PDOO DGHQRPDG\VSODVLD &DUFLQRPD&DUFLQRPD
7R[LF JHQRWR[LF FRPSRXQGV LQ OXPHQ
$3& PXWDWLRQ
LQDFWLYDWLRQ
.UDV
DFWLYDWLRQ
/DUJH DGHQRPD/DUJH DGHQRPD
'1$ GDPDJH
&HOO
SUROLIHUDWLRQ
3URPRWLRQ 0HWDVWDVLV
3URWHFWLYH SK\WRFKHPLFDOV LQGXFWLRQ RI SKDVH ,,
HQ]\PHV DSRSWRVLV '1$ UHSDLU
Fig.1 Molecular changes related to carcinogenesis as exemplified by the adenoma-carcinoma
sequence in colorectal carcinogenesis
J. Rafter et al. II/49
PASSCLAIM – Diet-related cancer
specifically would increase the rate of apoptosis in tumour
cells could be used in cancer therapy.
Fast growing tumours need oxygen and nutrients,
which need to be provided by new blood vessels. Malignant
cells can induce angiogenesis by secreting vascular
endothelial growth factors (VEGFs) and fibroblast
growth factors (FGF1/2). The signalling pathways leading
to angiogenesis are activated when these growth factors
bind to their receptors on the endothelial cell. The
signalling leading to angiogenesis also can occur via integrin
receptors (integrins are transmembrane proteins
promoting adhesion of cells).
When primary cancer cells come loose out of the tumour
mass,they can metastasise and invade healthy tissue
anywhere in the body,where they can further proliferate,
and destroy surrounding tissue, or hinder it in its
physiological functioning. The loosening of cells is possibly
due to altered expression of adhesion molecules,
into less ‘sticky’ ones. Another retained possibility is
through the activation of matrix metalloproteases
(MMPs).These deteriorate extracellular matrix proteins
(laminin and collagen). It was also observed that cell
surface integrin receptors, which were isolated from
metastatic cancer cells more efficiently bind to degraded
matrix proteins.
The clinical therapy of existing cancers is still primarily
based on surgery, radiation treatment and
chemotherapy. However, for example advanced colon
carcinoma are very refractive to any of these therapies.
It is put forward that cancer more efficiently can be controlled
by reducing the risk to get it, by preventing it. In
order to make a healthy cell cancerous, several errors
need to occur at the same time.Many of the external factors,
which seem to cause death by cancer can be controlled.
Nutrition entails some of the external factors
which can cause errors in a cell to occur,and as such certainly
has a potential to reduce the risk for cancer. The
enzymes involved in the biotransformation of xenobiotics,
e.g. phase I and II enzymes, are involved in toxification
of chemical carcinogens like the aflatoxins; however,
they also are involved in the metabolism of certain
phytochemicals occurring in fruits and vegetables. The
expression and activity of these enzymes has been
shown to be affected by various phytochemicals. This
may be a mechanistic link to the cancer risk reduction
observed in groups with a high consumption of fruits
and vegetables.
Nutritional studies have demonstrated effects in experimental
models. Some mechanisms of interaction
between nutrition and one or several steps described
above have been postulated, and have to some extent
been demonstrated in experimental models. Animal
models, particularly rats and mice have been used extensively
to study the processes of carcinogenesis. In
these animal studies often times carcinoma are induced
by chemical carcinogens. Recently gene knock-out mice
have been used to investigate the involvement of certain
specific genes, like APC, in the carcinogenic process.
Certainly these model systems are important to understand
the mechanisms involved in carcinogenesis; however,
a direct link to the human situation is particularly
questionable for the association between nutrition and
cancer development.
In the present paper, it will be discussed which conditions
should be met,in order to claim that food ingredients,
or foods, have a potential to reduce the risk for
cancer. The paper will focus on the numerically most
important cancers. These are colon cancer, lung cancer,
breast cancer, and prostate cancer. The following chapter
deals with sporadic tumour formation, cancers with
a strong genetic disposition will not be discussed in detail.
Colon cancer receives much attention as evidence
indicates that this is a tumour particularly amenable to
dietary influence.
Prospective, dietary intervention studies are considered
the gold standard in evidence-based nutrition for
evaluating the relation between a disease and nutritional
factors. However, especially for carcinogenesis
this is exceedingly difficult,time-consuming and expensive
to perform and also creates ethical problems if a
‘control’ group is included. For this reason, much of the
chapter deals with early/intermediate‘markers’ for cancer,
their link to the endpoint tumour and their usefulness
for making claims. It can also be mentioned that to
date some claims have been made based on epidemiological
studies.
Inflammatory bowel disease increases the risk of colorectal
carcinomas; however,this issue will be dealt with
in the ITG on Gut Health and Immunity.
Tumours and preneoplastic changes
Carcinoma incidence is the ultimate endpoint of cancer
prevention intervention studies. In rodents, using a
chemical carcinogen,this can mostly be achieved within
a year.However,human trials with carcinoma as the primary
endpoint will be looking at many years (>10 yr) to
prove a chemopreventive effect of a certain agent.Therefore,
there is a strong need for sensitive and specific intermediate
biomarkers that accurately reflect the multiple
stages of carcinogenesis. The most promising
biomarkers are discussed below.
■ Tumours and mortality in animal models
■ Definition of target. Macroscopically visible tumours
in rodents can be induced by chemical carcinogens. Examples
are 1,2-dimethylhydrazine (or its metabolite
azoxymethane) for colon cancer, dimethylbenz(a)anthracene
for breast cancer, and diethylnitrosamine for
II/50 European Journal of Nutrition (2004) Vol. 43, Supplement 2
lung cancer. In long-term studies, also the incidence of
mortality, resulting from the progression of these tumours,
can be analysed as a marker.
■ Relationship of target with cancer. The presence of
adenomas and adenocarcinomas, their size and multiplicity
are directly linked to cancer. Mortality is a hard
endpoint resulting from tumour progression.
■ Modulation of target by diet. Macroscopic tumours
can be modulated by diet in several ways: the size of tumours
can vary, as well as the number of tumours per
animal, and the number of tumour-bearing animals per
group.Also, the incidence of mortality per group can be
modulated by diet.As an example for colon cancer, several
types of dietary fibre have been frequently shown to
reduce the number of tumours in rodent models [reviewed
in 1].Antioxidants appear to have a high potential
for inhibiting lung cancer. NSAIDs are effective in
bladder cancer, and retinoids are efficacious in breast
cancer models [reviewed in 2].
■ Methodological considerations. The incidence of
macroscopic tumours has been the ‘gold standard’ endpoint
of chemoprevention studies in rodents for many
years. There are however several practical drawbacks of
these types of studies: the large groups of rodents required;
the long time scale (up to 8 months) needed for
tumours to develop; and the time-consuming histological
procedures for confirmation of the tumours. For
colon cancer, an alternative marker has been developed
and validated: aberrant crypt foci (see below, section
Precancerous lesions (aberrant crypt foci)). Therefore,
the rodent tumour model for colon cancer has gradually
lost attractiveness, due to the above-mentioned draw-
backs.
More recently,other models have been introduced for
cancer research,such as tumour-transplantation animal
models, available for breast and prostate cancer, and
mouse transgenic models. Indeed in 1990, the mutant
Min mouse was found with multiple intestinal neoplasia.
It was shown to have a mutated Apc gene, similar to
that in patients with familial adenomatous polyposis,
and in many sporadic cancers. Then other mice have
been genetically modified (with truncated Apc at different
positions or mutated Msh2 or Mlh1). They have
been used as models to evaluate the effect of diets and
chemopreventive agents in more than 70 studies [3].
Compared with the carcinogen-treated rat model, the
use of mutated mice avoids the hazard of carcinogen
handling, and leads to shorter studies. Dietary treatments
are initiated in mice by the age of 4–5 weeks,when
tumours may be already present. This timing mimics
human clinical trials,where dietary treatments are given
to adults, likely to bear minute polyps, the visible ones
having been removed before randomisation.
■ Conclusions. Chemical carcinogen-induced rodent
models are well accepted and widely used for the study
of potential chemopreventive agents. New transgenic
and implantation rodent models are interesting from a
mechanistic point of view, but should be interpreted
with great care. For instance, in the APC Min mouse,
adenomas develop mainly in the small intestine and in
the first weeks of life, which is very much unlike the human
situation.
■ Precancerous lesions (aberrant crypt foci)
■ Definition of target. Aberrant crypt foci (ACF), first
described in 1987 [4], are precancerous lesions of the
colonic epithelium, which consist of a number of crypts
that are larger than normal crypts, slightly elevated
above the mucosal surface,with a thick epithelial lining.
■ Relationship of target with cancer. Several lines of evidence
strongly suggest that ACF with certain morphological,
histological, cell kinetic and genetic features are
precursor lesions of colon cancer in both rodents and
humans [reviewed by 5]:
ț ACF are induced by all colon carcinogens in a dosedependent
manner [6];
ț In ACF, a shift of proliferation towards the luminal
surface of the epithelium occurs, like in adenomas
[7].Apoptosis is far less defined in experimental ACF.
ț The number of ACF, as well as the number of crypts
per ACF (so called crypt multiplicity),are affected by
chemopreventive agents, and predict tumour incidence.
A recent review by Corpet [1] reported a significant
correlation between the potencies against
ACF and tumours for 57 agents studied for both biomarkers.
Using two different protocols, the interference
of chemopreventive agents with the initiating
phase and the post-initiative phase of carcinogenesis
can be discriminated [8].
ț ACF correlate with colon cancer risk and adenoma
size and number in humans.For instance,the number
of ACF was higher in patients with Familial Adenomatous
Polyposis, and dysplasia was shown in most
cases[9].Also,thenumberofACFinresectedsections
of thenormalmucosaof cancerpatientswashigherin
regions with higher colon cancer mortality [10].
■ Modulation of target by diet. The following parameters
can be modified by diet: number of ACF in colon;
number of crypts per ACF; proliferation rate in ACF.For
instance, lycopene [8, 11] and fish oil [12–14] have been
shown to reduce the number of ACF in rats. See also the
review by Corpet [1].
■ Methodological considerations. ACF can be easily visualised
with methylene blue staining and scored with a
J. Rafter et al. II/51
PASSCLAIM – Diet-related cancer
low-magnitude microscope in resected colon samples of
rodents and humans, without tissue sectioning. Several
studies have described ACF visualisation in vivo during
endoscopy in cancer patients, with variable results
[15–17]. This technique is technically difficult, but
promising, and has to be further developed and vali-
dated.
Although most data support the use of ACF as a surrogate
end point for the screening of agents for chemoprevention,
there is still some discussion whether ACF
are true preneoplastic lesions. The azoxymethane dose
that yields one cancer per rat, yield 100–200ACF per
colon, indicating that most ACF regress and never become
cancers. In some ACF, hyperplastic or carcinoma
tissue has been observed [18].Also,crypt multiplicity in
ACF was a predictor of tumour incidence in several
studies [e.g.19].Most probably only larger foci,with altered
morphology, dysplastic, with altered cell kinetics,
and with mutations in some genes involved in carcinogenesis,
will progress toward cancer.
Since, a few agents have shown opposite effects on
ACF and on tumours (for example,dietary cholic acid,a
known colon tumour promoter decreases ACF) other
types of micro-lesions have been studied (that are enhanced
by cholic acid).
β-Catenin-Accumulated Crypts (BCAC), were first
described by Yamada etal. (2003) [20]. As they are only
identified in the histological sections of en face preparations,
BCAC are not yet ready to be used for routine
chemoprevention studies.
Mucin Depleted Foci (MDF), first described by
Caderni etal. (2003) [21]. Fifteen weeks after injections
of azoxymethane to rats,six to ten MDF could be scored
on the unsectioned colon stained with High-Iron Diamine
Alcian Blue (HID-AB). Thus, in contrast with
BCAC, MDF might be used for chemoprevention stud-
ies.
It is possible that MDF and BCAC are two views of the
same lesion.Indeed,mucin production is usually absent
in BCAC,and it is likely that MDF cells accumulate beta-
catenin.
■ Conclusions. From the above it can be concluded that
ACF are a valuable biomarker in rodents, providing a
quantitative assessment of the development of colon
cancer. To judge the relevance in humans of ACF and a
fortiori of MDF,not enough data are available at the mo-
ment.
■ Adenomatous polyps
■ Definitionoftarget. Adenomatous polyps are well demarcated,
circumscribed lumps of epithelial dysplasia.
Adenomas are present in all segments of the large bowel,
but their distribution tends to parallel that of colorectal
malignancies, so that about 70% of adenomas are localised
in the left colon and rectum. Endoscopic examination
for the detection of adenomatous polyps is a
common diagnostic procedure, in which single or multiple
polyps can be visualised and removed surgically.
Common clinical practice is that all polyps detected
during endoscopy are resected.
■ Relationship of target with cancer. Adenomatous
polyps in the colon and rectum are generally considered
to represent the most likely precursor lesions for colorectal
cancer in humans. They can vary in the number,
size, histological type and degree of dysplasia [22]; all
are important determinants of potential malignancy.
Adenoma size and number have been shown to be associated
with adenoma recurrence [23], and removal of
polyps decreases the risk of colon cancer [24]. Furthermore,
it was shown that invasive carcinomas developed
at sites where large polyps (>1cm) were not excised
[25].FAP patients,who have hundreds of polyps in their
large bowel, will almost all develop cancer if left untreated
[26]. In addition, adenomatous polyps and cancer
share the same aetiological factors as well as molecular
genetic alterations.
■ Modulation of target by diet. The following parameters
can be modified by diet: size, number, growth and
recurrence of polyps; histological features.As an example,calcium
has been shown to reduce the recurrence of
colorectal polyps in a few studies [27, 28]. On the other
hand, a beneficial effect of fibre supplementation on
polyp recurrence could repeatedly not be demonstrated
[28–30].
■ Methodological considerations. The major drawback
for using polyp recurrence as a biomarker in human
chemoprevention trials is the long time scale required to
allow recurrence of the polyps: at least 3–5 years is
needed,which makes these studies complex and very expensive.
In addition, the cohort of patients studied
should be quite large.An alternative is found in studying
the growth (or regression) of non-resected polyps over
time. This allows results to be obtained earlier. However
the awareness that detected polyps are deliberately not
being removed, sometimes encounters ethical objections,as
it will increase the risk of developing malignant
lesions.
A further limitation of the use of polyp recurrence as
a biomarker of colon cancer risk is the fact that polyps
occur only in part of the general population [31].Therefore,
these persons might already reflect a certain susceptibility
to colon cancer and might not be representative
of the general public.
Finally, the majority of adenomatous polyps do not
progress into invasive cancer. Therefore, although many
studies have indicated that polyp recurrence may be
II/52 European Journal of Nutrition (2004) Vol. 43, Supplement 2
modulated by chemopreventive agents, it remains to be
determined whether dietary or pharmacological modulation
of polyp recurrence leads to a reduction in the incidence
of colon cancer.
■ Conclusions. However, even taking into account the
above-mentioned reservations,the recurrence of adenomatous
polyps is still probably the most reliable surrogate
end-point in human chemoprevention studies,
since it measures the occurrence of a lesion in the pathway
leading from normal mucosa to cancer.In addition,
polyp size and their histological features (tubular/villous)
are important biomarkers too.
However, caution should be taken in interpreting the
results of studies which show no or even a negative effect,
such as the large fibre intervention trials, mentioned
above. Because of the overwhelming amount of
available scientific evidence that dietary fibre is protective
against colon cancer, it becomes clear that also
polyp recurrence is a biomarker that might be hindered
by methodological issues.
■ Tumour markers
Tumour markers are molecules that indicate either the
presence or the progress of malignancy. Tumour markers
are directly secreted or exfoliated by tumour tissue.
They are useful for diagnosis,progression of disease and
efficacy of cancer therapy, rather than for screening of
healthy subjects.
Widely used tumour markers are PSA (prostate-specific
antigen) for prostate cancer, and oestrogen and
progesterone receptors for breast cancer [32]. A change
in concentration can easily be detected in a blood test.
As an example of dietary modulation, PSA levels in
blood can be decreased by supplementation with vitamin
D [33].
For colon cancer,no such marker is widely used.Several
biochemical markers have been identified as risk indicators
of colonic neoplasia, such as insulin and insulin-like
growth factor [34], and plasminogen
activators and inhibitors [35]. Epidermal growth factor
receptor has been detected in lung and colon cancer patients
[36].Very promising are recently developed molecular
markers. These detectable mutations include 8p,
18q, DCC, p1, p53 and K-ras [reviewed by McLeod, 37].
In general, the currently available tumour markers
lack sensitivity for early cancer diagnosis, and lack
specificity for scoring malignancy. However, improvements
in molecular and genetic techniques have permitted
the detection of more sensitive and more specific
markers, such as tumour derived DNA and RNA circulating
in plasma, so that genetic mutations that occur
during tumour progression can be detected in a blood
sample. For colon cancer, although a large number of
studies directed at molecular markers have been reported
to provide prognostic or predictive indicators,
none of the potential markers has yet come to a widespread
clinical application [37]. For developing health
claims, only markers that have diagnostic value on the
level of dietary intervention, such as PSA for prostate
cancer, are important.
Cellular targets/markers
This section presents the main targets, at the cellular
level, which have consequences for carcinogenesis during
the post-initiation phase and can be modulated by
food components.
These targets are known to have common regulatory
pathways. Indeed, various endogenous or exogenous
agents,including dietary compounds,may affect several
of these targets simultaneously.
Cellular targets have two main limitations:
ț Very often, these targets are studied using cellular
models (spontaneously transformed rodent cell
lines, or human tumour cell lines) that do not completely
reflect the in vivo situation in a normal tissue.
ț The choice of the cell type is also critical, since cell
specificity of regulatory mechanisms could influence
the outcome of the study.
So, for cellular targets, results that are mostly obtained
from in vitro and animal studies are not sufficient to
base a claim on but they could be used as supportive ev-
idence.
■ Cell proliferation
■ Definition of target. In adults, most cell types (especially
epithelia) undergo a number of cell divisions, before
differentiating and dying. In each tissue, cell proliferation
is induced and finely regulated by the binding of
endogenous hormones and growth factors to their specific
receptors expressed in the tissue.
Cell division itself is regulated by a complex interplay
of many genes that control the cell cycle, with DNA
replication and mitosis as major checkpoints. The cell
cycle is tightly regulated to minimise transmission of genetic
changes to subsequent cell generations. Progression
through the cell cycle is under the control of different
types of proteins: cyclins (A, B, D, E),
cyclin-dependent kinases (CDKs: cdk2, cdk3, cdk4,
cdk6, cdc2...) and inhibitors of cyclin-CDK complexes
of theWAF1/CIP1 family (p21),the KIP family (p27,p57)
and the INK4 family (p16, p15, p18). Tens of molecules
have been identified as components of the signalling
cascades which couple regulation of the cell cycle and
detection of DNA damage. The product of the tumour
J. Rafter et al. II/53
PASSCLAIM – Diet-related cancer
suppressor gene p53 is one of these key regulatory proteins
[38].
■ Relationship of target with cancer. Cell proliferation
plays a crucial role at different stages of multistage carcinogenesis
[39]:
ț Throughout the successive cell cycles, errors in DNA
replication due to genetic instability,which are not or
incorrectly repaired, can result in DNA sequence
changes (gene mutations). Also, DNA replication
converts DNA alterations (DNA adducts) due to
stress (irradiation, exposure to carcinogens) to DNA
sequence changes. Thus, in proliferating tissues, various
genetic changes can progressively accumulate.
ț Clonal proliferation of the genetically damaged cells
leads to the formation of foci,nodules and then to tumours
which can escape from the control of neighbouring
cells.
Perturbation of cell proliferation is observed in all malignant
tissues. For example, in colon, the mucosal cell
proliferation, which is normally restricted to the stem
cell zone,extends to the entire crypt during carcinogenesis
and leads to the formation of aberrant crypt foci,
polyps, adenomas or carcinomas. Patients with a high
risk of colorectal cancer (e.g. FAP) have a corresponding
high mucosal proliferation [40].Refer also to section
on‘short chain fatty acids’ under Metabolites below.
■ Modulation of target by diet. Cell proliferation is inhibited
by many chemopreventive agents, including
retinoids, vitamin D, polyphenols, phytooestrogens,
fatty acids,sodium butyrate,indole-3-carbinol.They act
in different ways on the regulation of cell proliferation:
at the level of growth hormones and growth factors or
their receptors or on the genes and proteins regulating
the cell cycle [for review, see 41, 42].
■ Methodological consideration. To assess cell proliferation,
different methods are available:
ț Cell growth curves are done with cell cultures in
vitro.
ț DNA synthesis, determined by incorporation of precursors:
tritiated thymidine or bromodeoxyuridine
(BrDU) into cells or tumour fragments in vitro.
ț Distribution of cells in different cell cycle phases, using
flow cytometry.
ț Measurement of proliferation biomarkers, detected
using immunohistochemistry. Among them, Ki-67
and PCNA (proliferating cell nuclear antigen) are nuclear
proteins whose level varies as a function of the
cell cycle. Ki-67, whose function is unknown, is expressed
throughout the cell cycle except G0 [43].
PCNA, which is an auxilliary protein to polymerase
delta, is expressed during the G1 phase of the cell cycle
[44].Recently a monoclonal antibody to recombinant
parts of Ki-67 antigen, named Mib-1, has been
developed.It appears as a more reliable tool for measuring
proliferation activity in human tissues [45].
In clinical trials,cell proliferation is frequently evaluated
by the BrdU index or PCNA content. These methods
have both advantages and disadvantages, which have
been recently discussed by Renehan etal. [46].
■ Conclusion. Inhibition of cell proliferation could be a
valuable surrogate marker. The possibility to use it as a
basis for a claim may depend on the methodological approach
(dynamic or static assessment, measurement in
cellular or animal models, preferably in humans).
A nutritional inhibition of cell proliferation could be
beneficial in the case of tumour cells (therapy),whereas
it remains questionable in the case of normal cells (pre-
vention).
■ Apoptosis
■ Definition of target. Apoptosis is a cell suicide mechanism
that enables multi-cellular organisms to regulate
cell number in tissues and to eliminate unneeded or ageing
cells. Apoptosis is a programmed cell death. In can
be distinguished both morphologically and functionally
from necrosis, which is a pathological cell death, resulting
from gross insults such as prolonged ischaemia that
affects many adjacent cells simultaneously. In contrast,
apoptosis typically occurs in single cells. Normally, it is
initiated by endogenous stimuli, such as the absence of
vital growth factors or hormones and the action of cytokines,
like tumour necrosis factor α or Fas ligand [47,
48].
Apoptosis is characterised by membrane blebbing,
cell shrinkage, chromatin condensation and DNA fragmentation
followed by rapid engulfment by neighbouring
cells or macrophages [49].
It involves a series of death sensors and effectors.Cellular
death receptors (e.g.tumour necrosis factor receptor
TNFR,death receptor CD95 Fas) are sensors that initiate
the death pathway. Death signals typically are
focused on the mitochondria where the release of cytochrome
C (haeme-containing protein that transfers
protons during cellular respiration) catalyses apoptosis.
Caspases (death effectors) finally transmit the death signal:
these proteases specifically cleave vital proteins of
the nuclear lamina, such as poly (ADP-ribose) polymerase,
and the cytoskeleton (actin, fodrin, lamin),
which results in cell disassembly.
Endogenous molecules, which have other activities
in the cell, regulate apoptosis. Some regulatory transmembrane
proteins of the Bcl-2 family,such as Bax,Bak
and Bad, are pro-apoptotic effectors. The tumour suppressor
gene p53 (of which the corresponding p53 pro-
II/54 European Journal of Nutrition (2004) Vol. 43, Supplement 2
tein functions in the checkpoint control that arrests human
cells with damaged DNA in G1) can induce expression
of Bax, as well as death receptors.Antiapoptotic effectors
in the Bcl-2 family include Bcl-2 and Bcl-XL.
These are central in inhibiting signals addressed to the
mitochondria, which leads to cell survival. Other genes
preventing programmed cell death are for instance NFκB,
which expresses cell survival genes, which induce
IAPs, which bind to and inhibit certain caspases.
■ Relationship of target with cancer. Apoptosis provides
a protective mechanism against neoplasia by removing
genetically damaged stem cells from the epithelium before
they can undergo clonal expansion [50]. Loss of
apoptosis may result in disordered cell growth. It contributes
to tumour development but also to tumour initiation,progression
and metastasis.Defects in apoptosis
allow neoplastic cells to survive beyond senescence,
thereby providing protection from hypoxia and oxidative
stress as the tumour mass expands.
Evading apoptosis as tumour cells do,requires mechanisms
that inhibit transmission of the death signals.
Genetic alterations of regulators of apoptosis in tumours
have been described: for example, mutations of
p53 gene in more than 50% of tumours, overexpression
of Bcl-2 due to chromosomal translocation in lymphomas
[for review, see 51].
■ Modulation of target by diet. Many chemopreventive
agents have been found to induce cell death. They include
retinoic acid, perillyl alcohol, curcumin, EGCG,
apigenin, quercetin, chrysin, resveratrol and genistein
[41].
In the case of the colorectal mucosa, different kinds
of dietary components have been shown to modulate
apoptosis both in vitro and in vivo: they include polyunsaturated
fatty acids (PUFA), the short-chain fatty acid
butyrate and some flavonoids and glucosinolate breakdown
products [for review see: 50].
■ Methodological consideration. Various complementary
techniques have been developed to monitor apoptosis.
Since the common methods have their own set of
limitations, it is recommended to confirm the results by
using a combination of the following techniques [52]:
ț Morphological analysis of apoptotic cells (electron
microscopy) and counting of apoptotic bodies
(membrane-bound structures containing nuclear
fragments and condensed cell contents).
ț Detection of DNA fragmentation in the nuclei of
apoptotic cells in tissue sections, by the TUNEL
(TdT-mediated dUTP nick end labelling) method
[53].One important weakness of the TUNEL method
is that it may detect necrotic cells containing damaged
DNA.
ț Annexin V binding: annexin V binds to membranebound
phosphatidyl serine, a constitutive anionic
membrane phospholipid that is normally restricted
to the inner leaflet of the plasma membrane lipid bilayer
but is selectively exposed on the surfaces of cells
as they undergo apoptosis.In vivo uptake of 99mTc-radiolabeled
annexinV,assessed by SPECT imaging,allows
non invasive monitoring of cell death dynamics
[54, 55].
ț Immunodetection of caspase activity,using antibodies
directed against caspase-cleaved substrates, has
been recently developed. Such a method would be
very useful in future clinical trials [56].
■ Conclusion. Induction of apoptosis could be a valuable
surrogate marker. The possibility to use it as a basis for
a claim will depend mainly on the methodological approach
(use of a combination of complementary assays
to overcome artefacts and difficulties related to measuring
apoptosis in humans and interpretation).
As previously mentioned for cell proliferation, a nutritional
induction of apoptosis could be beneficial in
the case of tumour cells (therapy), whereas it remains
questionable in the case of normal cells (prevention). It
must be kept in mind that it is the balance between proliferation
and apoptosis that is important.
■ Differentiation
■ Definition of target. Normal cells do not live indefinitely
due to senescence.Senescent cells cannot be stimulated
to divide further, become resistant to programmed
cell death and acquire differentiated
functions. Differentiation is obviously associated with
slowing of the cell cycle.
Differentiation can be described as the process by
which, during development or tissue maturation, cells
acquire functions that are specific of the tissue or cell
type. It results from the induction of a differential expression
of genes, some becoming activated and others
repressed.
■ Relationship of target with cancer. It is generally
agreed that the malignant neoplastic cell is less differentiated
than the normal adult cell in the organ from which
the cancer originates.However,for a long time,there has
been a controversy on the link between transformation
and de-differentiation. Nevertheless, malignant neoplastic
transformation occurs only in cells that are capable
of dividing. In general, it appears that the commitment
to a given pathway of cellular differentiation is
irreversible, but the steps of terminal differentiation
may be reversible.
■ Modulation of target by diet. There are a number of
examples of animal malignant tumours or human can-
J. Rafter et al. II/55
PASSCLAIM – Diet-related cancer
cer cells in culture that can be induced to lose their malignant
phenotype by treatment with certain differentiation-inducing
agents: differentiation of murine embryonal
carcinoma cells by exposure to retinoic acid or
sodium butyrate; differentiation of human acute
promyelocytic (HL-60) cells in culture by sodium butyrate,
vitamin D3 and retinoic acid analogues. Among
dietary compounds,curcumin and phytic acid have also
been shown to induce differentiation of various tumour
cell lines [57, 58].
The best examples of cancer treatment through induction
of cellular differentiation is the treatment of
acute promyelocytic leukaemia and oral leukoplakia
with retinoids [59].Since the 1980s,vitamin D3 has been
recognised as a potent antiproliferative and prodifferentiation
agent (more recently, it has also been shown to
induce apoptosis and to inhibit tumour invasion and angiogenesis).Several
epidemiological studies support the
hypothesis that vitamin D3 is an important endogenous
cancer protective agent. Trials of systemic 1,25 (OH)2D3
and vitamin D3 analogues are now under way in patients
with various malignant diseases [for review, see 60].
■ Methodologicalconsideration. Various morphological
signs of differentiation are studied. Some of them are
specific to the cell type taken into consideration:
ț Increase of the nuclear/cytoplasmic ratio (for mouse
embryonal carcinoma PCC4 cells, for example).
ț Morphological differentiation of human teratocarcinoma
NT2 cells in neurons, of epidermal precursors
in keratinocytes, of myelopoietic progenitors/stem
cells in monocytes-macrophages, and mononuclear
precursors in osteoclasts [60, 61].
ț Induction of tissue-specific molecular markers: for
example in breast cancer cells, expression of milk
components (lipid droplets, β-casein mRNA) and
down-regulation of the Her2/neu membrane receptor
[62].
ț Expression of components of the cytoskeleton, like
lamininA,actin,cytokeratin,which are considered as
differentiation-specific markers [57].
■ Conclusion. Induction of cell differentiation could be
a valuable surrogate marker. The possibility to use it as
a basis for a claim may depend on the use of a set of reliable
and complementary markers. The science base
would be more solid if the results from different methods
are consistent.
■ Cyclo-oxygenase 2 (COX-2)
■ Definitionoftarget. Cyclo-oxygenase (COX) is an important
modulator protein in the eicosanoid pathway. It
catalyses the conversion of arachidonic acid to
prostaglandins.It is a bifunctional enzyme that has both
cyclo-oxygenase and peroxidase activities. There are
two COX isoforms that differ both in their tissue distribution
and their regulation.COX-1 is a constitutively expressed
“housekeeping” gene involved in processes like
gastric acid secretion, vascular homeostasis and water
reabsorption in the kidney. COX-2 is inducible and
thought to be involved in different processes such as inflammation
and ovulation [63].
■ Relationship of target with cancer. COX-2 is overexpressed
in many cancers including oesophagus, stomach,
colon, lung, pancreas, head and neck, and prostate
[for review, see 64]. Recent studies using animal models
provide evidence that COX-2 is mechanistically linked
to cancer development: 1) over-expression of COX-2 in
transgenic mice induces mammary gland tumours [65];
2) knocking out of the COX-2 gene in the Apc∆716 mouse
reduces the number and size of intestinal polyps and of
skin papillomas [66, 67].
Both COX-2 and its substrates, the prostaglandins,
play an active role in carcinogenesis, since they affect
xenobiotic metabolism,angiogenesis,apoptosis,inflammation,
immuno-suppression and invasiveness [for review,
see 64]. Several population-based studies have
shown that regular use of non-steroidal anti-inflamma-
torydrugs(NSAIDs),whichareCOXinhibitors,decrease
therelativeriskof colorectalandprostatecancer[68,69].
■ Modulation of target by diet. Inhibitors of the enzyme
both reduce the formation of tumours in many tissues
and suppress the growth of established tumours [64].
Many studies are currently performed with celecoxib,
aspirin, curcumin, and genistein, for example.
It has been demonstrated that lipid components of
the human faecal fraction in intimate contact with the
epithelium (faecal water) and specific bile acids can alter
the transcription of COX-2.Available data support an
influence of diet on the levels of the faecal water components.In
view of the knowledge that the amount of COX-
2 is important, since there is a correlation between its
level of expression and the size of the colorectal tumours
and their propensity to invade underlying tissue, even
small effects over time on the transcriptional regulation
of this enzyme by dietary components may be important
for tumour development in the colon.
■ Methodological consideration. COX-2 expression in
cells or frozen sections of animal or human tissues is
generally studied by immunohistochemistry or immunoblotting
[for a detailed description of the techniques,
see ref 70].
■ Conclusion. Inhibition of COX-2 activity is a valuable
surrogate marker,since convincing data obtained in humans
with NSAIDs are available. However the effect of
diet is less explored.
II/56 European Journal of Nutrition (2004) Vol. 43, Supplement 2
The possibility to use it as a basis for a claim may depend
on the availability of clinical data for the tissue
taken into consideration: COX-2 data are mainly arising
from colon (and prostate and breast) cancer,and it is not
sure if it is also valid for other types of cancers. Definition
of target groups may be particularly relevant for
this marker.
■ Potential targets
Several other potential targets (intercellular communication,
gene expression, signal transduction, etc), for
which numerous but incomplete data are available, are
listed in Table1. However at present, they are further
away from the true endpoint and as such can only be
used for mechanistic supporting evidence. This field
will particularly benefit from a development of research
and the use of high scale technologies for genomics and
proteomics.
Gut luminal markers
The gut microflora has been implicated in the aetiology
of colorectal cancer by a number of studies [reviewed by
Mallet and Rowland 71] and these observations form the
theoretical basis for use of gut flora biomarkers (faecal
biomarkers). They are composed of two main categories:
those examining the activity of bacterial enzymes
or bacterial metabolites and those based on
bioassays on faecal water. A number of these have been
used to investigate gut bacterial function [72, 73] and
some have potential mechanistic links to colorectal cancer
(CRC) aetiology.
■ Bacterial enzymes
A wide range of enzyme activities capable of generating
potentially carcinogenic metabolites in the colon is associated
with the gut microflora, including β-glucuronidase,
β-glucosidase, nitrate reductase and nitroreductase
(Table2). These are usually assayed in faecal
suspensions and appear to be present in many bacterial
types [74]. Of these enzymes, β-glucuronidase has been
the most extensively investigated as a biomarker of CRC
risk. It should be noted that these factors are associated
with the generation of carcinogens and promoters and
do not have a direct link with tumours.
β-Glucuronidase
■ Definition of target. Many carcinogenic compounds
are metabolised in the liver and then conjugated to glucuronic
acid before being excreted via the bile into the
small intestine. In the colon bacterial β-glucuronidase
can hydrolyse the conjugates, releasing the parent compound
or its activated, hepatic metabolite.
■ Relationship with cancer. The activity of β-glucuronidase
in the colon can alter the likelihood of tumour
induction in animal models of CRC. The use of a
β-glucuronidase inhibitor administered in conjunction
with the carcinogen azoxymethane (which undergoes
activation and conjugation in the liver) significantly reduces
the number of tumours formed in the rat colon,
indicating that microflora β-glucuronidase has a role in
tumour induction [75].Metabolic epidemiological studies
have shown that populations at high risk of CRC have
high levels of faecal β-glucuronidase activity. Furthermore,
faecal β-glucuronidase activity in colon cancer
patients is significantly higher than in healthy controls
[76].
■ Modulationbydiet. The activity of β-glucuronidase is
influenced by diet. High-risk diets for colorectal cancer
have consistently been shown to increase β-glucuronidase
activity relative to low risk diets [77]. Furthermore,
various types of fibre decrease the activity of
β-glucuronidase in rats [78–80].
■ Methodological considerations. β-Glucuronidase can
be assayed in fresh or frozen faecal samples by a simple
reproducible colorimetric procedure. It is usually expressed
as mmol p-nitrophenol released/min/g wet
weight faeces. There is considerable interindividual
variation in activity.
■ Conclusions. Although it represents a simple reproducible
marker, evidence for a role for β-glucuronidase
in human CRC is indirect and is remote from the final
endpoint (tumours).
■ Metabolites
A wide range of metabolites with potential genotoxic,
tumour promoting and anti-carcinogenic activities have
been identified in faeces. These are summarised in
Table3.
N-nitroso compounds
■ Definition of target. Nitrate, ingested via diet and
drinking water, is reduced by gut bacterial nitrate reductase
to its more reactive and toxic reduction product,
nitrite. Nitrite reacts with nitrogenous compounds in
the body to produce N-nitroso compounds (NOC). The
reaction can occur chemically in the acidic conditions
prevalent in the human stomach and can also be catalysed
at neutral pH by gut bacteria in the colon [81].
J. Rafter et al. II/57
PASSCLAIM – Diet-related cancer
Table1 Cellular effects: other potential targets
a. Miscellaneous
Target Definition Relationship with cancer Modulation by diet Methods References
• Gap junctions: channels • Dysruption of GJIC involved • Retinoids, carotenoids, and • Dye transfer after [240–244]
between adjacent cells in carcinogenesis. flavonoids (apigenin, microinjection in cell
permitting direct cell-cell • Reduced level of GJIC in tangeretin) enhance GJIC monolayers and tissues.
communication. most human and animal in vitro. • Expression of tissue-specific
• Important role of GJIC in the tumours. connexins by immunoblotting
control of tissue homeostasis • Transformed cells (in vitro or immunostaining.
and regulation of cell and in vivo) do not • Electrophysiology.
proliferation, differentiation communicate with • Metabolic cooperation.
and apoptosis. neighbouring normal cells. • Photobleaching.
• Modulation of GJIC at • Many tumour promotors • Scrape loading.
different levels of regulation: inhibit GJIC.
specific connexin gene • Transfection of connexin
expression, protein genes into communicationphosphorylation,
connexon deficient malignant cells
assembly, cell adhesion and reduces or eliminates the
channel gating. tumourigenicity of recipient
cells.
• Ornithine decarboxylase • ODC participates in • Retinoids, green-tea • ODC activity. [245–247]
(ODC) -catalysing formation carcinogenesis. polyphenols and flavonoids • Spermidine/spermine ratio.
of putrescine from ornithine: • Blocking ODC in vitro inhibits inhibit ODC activity in vitro.
a critical step in polyamine cell transformation.
biosynthesis. • ODC is now considered a
• Role of polyamines in cell putative proto-oncogene.
proliferation, differentiation
and malignant
transformation.
• Senescence of human cells • Human telomerase prevents • Dietary restriction inhibits • Enzyme activity. [186, 248–250]
(after 50–70 divisions) due telomere erosion and is able liver telomerase activity in • Fiber optical biosensor for
to telomere attrition. to synthetise new telomeric vivo. enzyme activity detection in
• Human telomeres consist of repeats. tumours.
repeated TTAGGG sequences • Telomerase is detected in
on the 3’end of single- more than 85 % of cancers,
strand-DNA. They cap and and is usually undetectable
protect the ends of in benign tumours and in
chromosomes against normal tissues surrounding
degradation, inappropriate tumours.
recombination and • Telomerase inhibition leads
interchromosomal fusions. to an immediate induction of
• During the successive rounds apoptosis.
of cellular division driven by
DNA polymerases,
incomplete replication of the
telomere results in a loss of
telomeric repeats.
• Lipoxygenases (5-LOX, • LOX products involved in • Inhibition of LOX by • Enzyme activity. [251–254]
8-LOX, 12-LOX, 15-LOX) development and curcumin, phenolic • LOH products.
catalyse the conversion of progression of human compounds of olive oil.
arachidonic acid to tumours.
hydroperoxyeicosatetraenoic • Abnormal expression and
acid (PHETE) and activities of LOX in pancreatic
leukotrienes. cancer.
• LOX inhibitors reduce
chemically-induced
carcinogenesis, and induce
apoptosis in human cancer
cells.
Gapjunctionalintercellularcommunication(GJIC)ODCTelomeraseLipoxygenases
II/58 European Journal of Nutrition (2004) Vol. 43, Supplement 2
■ Relationship to cancer. The term NOC covers a wide
range of compounds including N-nitrosamines, N-nitrosamides,
N-nitrosoguanidines and N-nitrosoureas,
the majority of which are highly carcinogenic, DNA
alkylating agents. However, the genotoxic or carcinogenic
activity of the NOC produced by the bacterial Nnitrosation
process in the large intestine has not yet
been established.
■ Modulation by diet. Faecal apparent total NOC
(ATNC) excretion is increased by red meat consumption
[82–84]. In conjunction with high meat intakes, wheat
Table1 Continued
b. Gene expression
Target Definition Relationship with cancer Modulation by diet Methods References
• In normal mammalian • Abnormal DNA methylation • Selenium, folate, vitamin • Comet assay. [255–258]
genome, DNA methylation in most cancers: global DNA B12, choline, methionine, • Microarrays.
only on cytosines 5’ to methylation accompanied retinoic acid and isoflavones • HPLC.
guanosines at CpG by gene-specific affect DNA methylation
dinucleotides. hypermethylation. status.
• Catalysed by • Hypermethylation of CpG
methyltransferases. islands associated with
• It regulates gene expression inactivation of DNA repair,
and chromosomal stability. cell cycle regulation,
inflammatory/stress
response and apoptosis.
• Nucleosomes (histone • Misregulation of chromatin • Sodium butyrate and diallyl • Immunoblotting of nuclear [259, 260]
octamere and 147 base pairs structure can cause incorrect disulfide are known to proteins using antibodies
of DNA) repress transcription gene activation or induce histone directed against total or
by blocking the binding of improper gene silencing. hyperacetylation via the specific acetylated lysines of
transcription factors and • Certain oncogenic inhibition of histone de- different histones.
basal transcription transcription factors acetylase. • Histone acetylase and
machinary to promoter promote oncogenesis by deacetylase assays.
regulatory sequences of misregulating chromatin
genes. structure. Conversely,
• Hypoacetylation of lysine tumour suppressors (Rb,
residues in the flexible P53 ...) utilise chromatin
N-terminal histone tails is remodeling as part of their
correlated with normal function.
transcriptional repression,
whereas hyperacetylation is
correlated with activation.
• The Rel/NF-κB family: a • Misregulation of the • Curcumin, green tea • Western blotting. [261, 262]
group of structurally-related Rel/NF-κB signal polyphenols, genistein,
and tightly-regulated transduction pathway in resveratrol and apigenin
transcription factors that human cancers. inhibit the induction of
control the expression of a • Inhibition of Rel/NF-κB NF-κB.
multitude of genes. activity reverses all or part
• They exist in cytoplasmic of the malignant state.
complexes with inhibitory • Tumour promotors induce
proteins of the IκB family, AP1 activity, in vitro.
and translocate to the
nucleus to act as
transcription factors when
activated.
• Activator protein-1 (AP-1) • Many of the genes of the fos • Resveratrol inhibits TPA and • DNA binding. [263, 264]
transcription factor, a and jun family are expressed UVC- induced AP-1 activity.
heterodimeric protein in transformed rapidly- • Human faecal water
complex composed of the growing cells. components can induce AP-1
products of members of the DNA-binding activity and
fos and jun gene family, augment AP-1-responsive
binds to DNA sequences gene expression in human
and induces the expression cultured colonocytes.
of various genes.
Chromatinremodeling
DNAmethylationHistoneacetylation
NF-κBAP-1
J. Rafter et al. II/59
PASSCLAIM – Diet-related cancer
bran,resistant starch and vegetable consumption had no
effect on faecalATNC excretion or concentration [82,84,
85].
■ Methodologicalconsiderations. N-nitroso compounds
in faeces can be monitored using a group-selective
method that determines ATNC in faeces and biological
fluids by a procedure employing a thermal energy
analyser [81, 86].
Secondary bile acids
■ Definition of target. The primary bile acids, chenodeoxycholic
acid and cholic acid, are subject to extensive
metabolism, predominantly 7-α-dehydroxylation,
by the intestinal microflora [87], which converts cholic
to deoxycholic acid (DCA) and chenodeoxycholic to
lithocholic acid (LCA). These are termed secondary bile
acids.
■ Relationship to cancer. The secondary bile acids exert
a range of biological and metabolic effects in vitro and
in animal models including cell necrosis, hyperplasia
Table1 Continued
c. Signal transduction
Target Definition Relationship with cancer Modulation by diet Methods References
• Prenylation: a type of lipid • Prenylation is catalysed by • Several monoterpenes, • Measure of the enzymatic [265, 266]
modification involving the farnesyl protein including limonene and activity.
covalent addition of either transferase (FPT): the first perillyl alcohol inhibit
farnesyl (15-carbon) or more step in the processing of farnesyl protein transferase.
commonly geranylgeranyl the oncogenic Ras proteins.
(20-carbon) isoprenoids via • Ras is often overexpressed
thioether linkages to in tumours.
cysteine residues at or near
the C terminus of
intracellular proteins.
• Many signal transduction • The substrates of protein • Various polyphenolic • Enzyme activities. [267]
events involve kinases include transcription compounds (EGCC, • Western blotting.
phosphorylation steps. regulatory factors, for flavonoids, resveratrol)
Activation of protein kinases example, the proteins inhibit MAPK pathways.
is a key mechanism in encoded by the jun, fos,
regulating signals for cell myb, rel and ets protoproliferation.
oncogenes.
• The mitogen-activated
protein kinases (MAPKs)
convert various exogenous
signals into intracellular
responses through serial
phosphorylation cascades.
Three distinct and parallel
MAP kinases cascades
(ERK, JNK and p38) have
been identified.
• Central role in proliferation, • In contrast to most tumour • Apigenin (and NSAIDs) • Western blotting. [268–270]
differentiation and cell suppressor genes (Rb, p53), upregulates p27 in vitro.
death. It inhibits cyclin/ which are recessive at the
cyclin-dependent kinase cellular level, p27 is
complexes, thereby blocking happloinsufficient for
cell cycle progression. tumour suppression.
• Regulated post- • Skp2, the specific recognition
transcriptionally, at the factor for p27 ubiquitination
level of both protein has oncogenic properties.
translation and stability,
through phosphorylations
and ubiquitination involving
the ubiquitin/proteasome
pathway.
FarnesylproteintransferaseProteinkinasesP27Tumoursuppressorfunction
II/60 European Journal of Nutrition (2004) Vol. 43, Supplement 2
Table2 Faecal bacterial enzymes
Target Definition of target Relationship with cancer Modulation by diet Methodology Reference
β-Glucuronidase Hydrolyzes biliary conjugates Populations at high risk of High risk diets for CRC Analysed by chromogenic [77, 271]
(GN)* in the colon releasing CRC have high GN activity. increase GN in human and substrate.
potential carcinogens. animal studies. Simple, rapid. Can be assayed
in frozen samples.
β-Glycosidase Hydrolyzes glycoside Some plant aglycones Poor relationship to high Analysed by chromogenic [77, 271]
conjugates of plant have mutagenic and risk diets. substrate.
compounds. carcinogenic activity. Simple, rapid. Can be assayed
Others act as anti-genotoxins. in frozen samples.
Nitrate reductase Reduces nitrate to more Nitrite is precursor of faecal No consistent response to Spectrophotometric analysis, [272]
reactive nitrite. NOC. dietary change. needs fresh faecal sample
and strict anaerobic
conditions.
Nitroreductase Reduces nitro compounds to Some amine products No consistent response to Spectrophotometric analysis, [272, 273]
amines. carcinogenic. dietary change. needs fresh faecal sample
and strict anaerobic
conditions.
IQ Converts IQ to 7-OHIQ. IQ is a known dietary Increased by high risk CRC Radiolabel chromatography [274]
oxidoreductase carcinogen requiring diets in rats. assay, anaerobic conditions
activation. The bacterial needed.
enzyme converts IQ to a
direct acting genotoxin.
* For further details see main text
Table3 Faecal metabolites
Target Definition of target Relationship with cancer Modulation by diet Methodology Reference
Ammonia Formed by bacterial Tumour promoter in rats; High-risk diets for CRC Simple chromogenic assay. [271, 275]
catabolism of protein and induces colon cell increase faecal ammonia in
urea in colon. proliferation. rats.
N-Nitroso Formed by bacterial action Many NOC are potent Increased by high red meat Difficult, expensive analysis [83, 86]
compounds in colon from nitrite and carcinogens (but activity of diets. (Thermal Energy Analyser).
(NOC)* amino compounds. faecal NOC not known).
Diacylglycerols Produced by gut bacteria Activate protein kinase C. Faecal DAG increased by Lipid extraction, then [88, 276, 277]
(DAG) from phospholipids and Humans with high fat high fat diets in rats and commercial kit.
triglycerides. content in faeces have high mice. DAG decreased by
DAG in gut and epithelial wheat bran in humans.
hyperproliferation.
Fecapentaenes Glycerol ethers containing a Direct acting mutagens in Faecal excretion of Difficult, hplc method [88, 278–280]
pentaene moiety. Produced Ames test. Associated with fecapentaenes is lower in
by gut bacteria in vitro. increased cell proliferation, vegetarians.
Detected in faeces in some tumour promotion in some
subjects. but not all studies.
Inconsistent associations
with CRC.
Secondary Formed by bacterial Tumour promoters, inducers High fat intake increases Analysis by GLC or HPLC, [88, 89, 96]
Bile acids* metabolism of cholic and of cell proliferation, faecal bile acid time consuming extraction
chenodeoxycholic acids. genotoxic. Concentration concentrations. required.
of faecal bile acids higher
in populations and individuals
at high CRC risk.
Short chain Major SCFA are acetic, Butyric acid has trophic SCFA increased (usually) in Analysis by GLC. Simple, [281]
fatty acids propionic and n-butyric effect on colonic epithelium. subjects consuming diets rapid.
(SCFA)* acids. Produced by In vitro butyric acid induces high in fibre, resistant starch
fermentation of apoptosis and differentiation or non-digestible
carbohydrates in colon. of colon cancer cell lines, but oligosaccharides.
increases cell proliferation
in normal colon in vivo.
* For further details see main text
J. Rafter et al. II/61
PASSCLAIM – Diet-related cancer
and tumour promoting activity in the colon, induction
of DNA damage, and apoptosis [reviewed by 88]. It has
also been suggested that secondary bile acids influence
colorectal cancer by selecting for apoptosis-resistant
cells or by interacting with various secondary messenger
signalling systems. Epidemiological studies indicate
that concentrations of secondary bile acids are higher in
populations at high risk of CRC and in case control studies
7-α-dehydroxylase activity is higher in cases than
controls. A number of human observational studies in
patients with adenomas or CRC have reported a correlation
between faecal bile acid (FBA) concentrations and
CRC risk [89–92]. Some studies [93, 94] have also suggested
that high DCA concentration and the DCA to
LCA ratio are associated with increased CRC risk. It
should be noted however that not all studies have confirmed
these relationships between bile acids and cancer
risk [95].
■ Modulation by diet. In human studies,high fat intake,
which correlates with CRC risk, increases FBA concentrations
[96], whereas increased consumption of wheat
bran (negatively correlated with CRC risk) reduces FBA
concentration.
■ Methodological considerations. Measurement of FBA
is usually performed by soxhlet extraction of faecal samples
followed by gas chromatographic analysis.
Short chain fatty acids
■ Definition of target. The short chain fatty acids
(SCFA) acetate, propionate and butyrate are the principal
end products of carbohydrate fermentation. These
are absorbed from the colonic lumen and metabolised
by various body tissues [97]. Butyrate is preferentially
metabolised by colonocytes [98].
■ Relationship to cancer. Butyrate is the most intensively
studied SFCA. Interest in butyrate’s role as a possible
antineoplastic agent has arisen from its potential to
inhibit proliferation of cells in vitro, including colon tumour
cell lines [99]. Moreover, at physiological concentrations,
sodium butyrate induces apoptosis in human
colon carcinoma cell lines [100].
In addition, reduced pH as a consequence of SCFA
production,has also been implicated as a protective factor
against colon cancer [101, 102]. This may be as a result
of altering the solubility of bile acids,or altering the
metabolic activity of colonic microflora.
■ Modulation by diet. There is evidence from in vitro
studies and animal models (where caecal SCFA concentrations
can be measured) that the type of carbohydrate
has an important influence on the amount and proportions
of SCFA produced,with starch and wheat bran being
particularly associated with elevated butyrate production
[103]. In human studies, inulin has been shown
to enhance excretion of total SCFA in human faeces,
whereas wheat bran increased absolute or relative proportions
of butyrate in faeces [104].Where the butyrate
is produced relative to proximal and distal regions of the
colon is important and should be a methodological con-
sideration.
■ Methodologicalconsiderations. Measurement of SCFA
in faeces is by a simple gas chromatographic method,
but the value obtained represents the balance of bacterial
production and the extensive colonic absorption, so
may not reflect accurately the concentrations of SCFA in
the colonic lumen [97].
■ Conclusions for sections on Bacterial enzymes and
Metabolites. Gut bacterial enzymes and faecal metabolites
are relatively simple to measure routinely and in
general may be of use in assessing effects of diet on
modulating exposure of the colon to potential carcinogens,
rather than reflecting cancer risk.
■ Faecal water activities
Faecal water cytotoxicity
■ Target. There is considerable evidence that colon tumours
are a result of gut luminal factors damaging the
mucosa. Furthermore, free reactive and soluble factors
are more likely to affect the epithelium than substances
bound to the insoluble matrix such as fibre. Therefore,
an alternative approach to assaying enzymes or metabolites
in faeces is to assess toxicological activity of fractions
using short-term tests for toxicity, genotoxicity
and mutagenicity. Usually the aqueous phase of the human
faeces (faecal water) is used [105, 106], since this
will contain most of the free reactive species.For assessment
of faecal water cytotoxicity,the effect on proliferation
of human colon carcinoma cells in culture is used.
■ Relationship to cancer. Proliferative zone expansion
in the colonic crypts and an increased rate of epithelial
proliferation are considered to be an early step in carcinogenesis.
Stimulation of proliferative activity in
colonic epithelium may in part be mediated via cytotoxic
mechanisms, resulting in increased cell loss at the
epithelial surface and a compensatory rise in mitotic activity
of the crypts.Such considerations led to the development
of assays to assess cytotoxic activity in faecal
water towards colon cells in vitro [105].It is thought that
bile acids, especially secondary bile acids, make a major
contribution to faecal water cytotoxicity [105].In a comparison
of faecal water cytotoxicity in patients at low (no
colon adenomas), medium (small colorectal adenomas)
II/62 European Journal of Nutrition (2004) Vol. 43, Supplement 2
and high (large tubular adenomas) risk of CRC, no significant
differences between the groups were observed
[95].
■ Modulation by diet. Interventions using dietary
regimes associated with increased or decreased CRC
risk have been shown to modulate appropriately faecal
water cytotoxicity.For example,dietary calcium has frequently
been shown to reduce the cytotoxicity of faecal
water presumably by precipitating soluble bile acids
[106, 107]. Faecal water cytotoxicity was higher in subjects
on a high fat, low calcium, low fibre diet compared
with those on a low fat, high calcium, high fibre regime
[105]. In rats, high red meat consumption increases the
cytotoxicity of faecal water.This effect was independent
of the fat and bile acid content of the faecal water and
may be related to dietary haeme [108, 109].
■ Methodological considerations. Human colon cells
(e.g Caco-2 or HT29) are exposed to faecal water fractions
(prepared by high speed centrifugation of fresh
faecal samples) and inhibition of cell proliferation is
measured usually using a dye such as MTT [110].
Faecal water genotoxicity
■ Definition of target. Venturi et al [111] demonstrated
the presence of DNA damaging activity towards human
cultured colon cells in samples of faecal water from
healthy human subjects. A wide variation was found
ranging from negligible to high activity.The presence of
genotoxic activity in faecal water can be considered to
reflect exposure of the colonic mucosa to carcinogens.
■ Relationship to cancer. There is now convincing evidence
that CRC is induced by a series of mutational
events in a number of critical genes [112]. Sporadic colorectal
tumours have been shown to contain mutations
and deletions in oncogenes and tumour suppressor
genes such asApc,K-ras and p53.DNA damage has been
detected in biopsies of colon tissue derived from laboratory
animals and human subjects. Thus, the presence in
the colonic lumen of DNA damaging agents could represent
an important risk factor for CRC.There are as yet
no reports of validation studies for the endpoint in patients
at different risk of colorectal cancer.
■ Modulation by diet. In healthy subjects, a diet high in
fat and meat, but low in dietary fibre (hence considered
to be of high CRC risk) was associated with a significantly
increased faecal water genotoxicity by comparison
to a diet low in fat and meat [113].
■ Methodological considerations. Human colon carcinoma
cell lines (e.g.Caco-2 and HT29) are exposed to
faecal water fractions and genotoxicity is usually measured
using the single cell gel electrophoresis (Comet)
assay, which provides a simple, rapid and sensitive
method for measuring single strand breaks in DNA.The
intra- and interindividual variation in faecal water
genotoxicity has been studied in healthy individuals
[114].
■ Conclusions. Cytotoxicity and particularly genotoxicity
of faecal water have a good mechanistic link with
colon carcinogenesis and hence provide potentially
valuable, non-invasive methods for assessing colorectal
cancer risk in human subjects. However, there is a need
for more extensive validation of these endpoints.
■ Diagnostic markers in faeces
There are several tumour markers that can be detected
in faeces. Haemoglobin is currently used for mass
screening for colon cancer (faecal occult blood test).
Haemoglobin is not continuously, but intermittently
leaking through the intestinal wall, thereby compromising
the sensitivity of the test. Other stool tumour markers,such
as abnormally glycosylated mucins,are continuously
secreted.However immuno-chemical methods to
detect these mucins in stool samples have failed to accurately
detect neoplasia,probably due to luminal instability
[115].Most promising are exfoliated markers,such as
whole colonocytes and mutant DNA. Tumours exfoliate
large amounts of epithelial cells that can be recovered
from stool. Using RT-PCR, genes that are overexpressed
in tumours can be demonstrated in these cells. Even
more promising are neoplasm-specific DNA alterations,
as DNA appears to be stable in the gut lumen.Several investigators
have recovered mutant DNA in stools from
colon cancer patients, mostly using K-ras mutations as
marker [reviewed by 116]. More recently, the specificity
of this test was improved by targeting multiple DNA mutations
(including K-ras, APC and p53), to minimalise
false-positives and false-negatives [117]. Long DNA
(>200 base pairs) appeared to be the most informative
stool marker in this study. Faecal DNA testing holds a
promise for the future as a non-invasive tumour marker,
as a single stool specimen will be sufficient to accurately
detect both precursor adenomas and carcinomas.
■ Conclusions. Of the considerable number of potential
prognostic or predictive indicators of colon cancer
based on faecal markers, only faecal occult blood has
achieved widespread clinical application. However, it
has limited usefulness as an indicator of cancer risk for
assessment of functional foods.Validation of the newer
techniques should be the goal for future research.
J. Rafter et al. II/63
PASSCLAIM – Diet-related cancer
Angiogenesis and metastasis
■ Inhibition of angiogenesis
■ Definition of target. Tumours require blood supply to
develop and grow. They take over existing blood vessels
and stimulate production of new vessels from these – a
process termed angiogenesis.
■ Relationship to cancer. Angiogenesis is a crucial early
event in tumour progression, beginning in premalignant
lesions [see review by Ruoslahti 118]. The key initiators
of endothelial cell growth are the vascular endothelial
growth factors (VEGFs), which act through a
family of tyrosine kinase receptors (VEGFRs) expressed
by angiogenic vessels. Angiopoietins and platelet-derived
growth factors are also involved and subsequently
the vessels acquire supporting cells such as pericytes,
smooth muscle cells and extracellular matrix.
Another group of receptors, integrins, expressed by
endothelial cells in angiogenic vessels, also play an important
role in the process of angiogenesis by mediating
cell adhesion and transmitting signals from the extracellular
matrix to cell types that regulate cell growth and
survival. It has been found in neuroblastoma that the
level of expression of the integrins reflects the grade of
malignancy.
Given that in the absence of a blood supply,a tumour
will become hypoxic and die, angiogenesis provides a
logical target for therapeutic and preventative agents.A
number of potential anti-angiogenic agents are currently
undergoing clinical trials. These substances include
drugs, peptides and antibodies that target vascular
growth factors and integrins and also compounds
such as endostatin, identified in activity based assays.
■ Modulation by diet. In vitro assays have been used to
demonstrate potential anti-angiogenic effects of
NSAIDs [119] and of various dietary components such
as genistein [120] and conjugated linoleic acids (CLA
[121]). In vivo, dietary CLA was shown to suppress angiogenesis
in a mouse matrigel model [121]. In the CLA
fed mice, the injected gel pellets contained fewer infiltrating
cells forming only limited branching networks.
Dietary CLA also decreased serum levels of VEGF. In a
study of a novel oestrogen metabolite, 2-methoxyoestradiol,
Fotsis etal. [122] demonstrated that, when
administered orally in mice, it strongly inhibited the
neovascularisation of solid tumours and suppressed
their growth.
■ Methodological considerations. A range of in vitro assays
for assessing angiogenesis inhibition have been
used including
ț Reduction in proliferation of endothelial cells stimulated
by bFGF or VEGF.
ț Inhibition of endothelial differentiation into capilliary
like structures on Matrigel.
ț Suppression of bFGF orVEGF-stimulated invasion of
collagen by endothelial cells.
There are a number of in vivo mouse models based on
modulation of angiogenesis after stimulation with various
growth factors such as fibroblast growth factor –2
[119] or sub-cutaneous injection with Matrigel followed
by measuring infiltration of the gel with blood vessels
[121].
Potential biomarkers of angiogenesis for human
studies: There is considerable interest in the use of
plasma VEGF and bFGF as indicators for prognosis in
cancer patients with a range of tumour types, including
colorectal, myeloma and lung. If substantiated, this
could provide a biomarker for intervention studies, although
severe ethical problems could arise. At present
the results are variable, with some studies indicating a
good correlation with other prognostic indicators and
others showing poor associations [123–125].
■ Conclusions. Inhibition of angiogenesis represents a
potentially useful target for dietary compounds and
there is a wide range of in vitro and in vivo assays that
can be used to screen potential candidates.However,the
efficacy of such substances in patients may be questioned
on the basis of current experience with drugs.
Furthermore, given the importance of angiogenesis for
normal tissues, there is likely to be little or no use for
functional foods with anti-angiogenic activity for the
general population. In other words, target groups must
be carefully specified in order to avoid anti-angiogenesis
diets being deleterious.
■ Inhibition of metastasis
■ Definitionoftarget. The metastatic escape and spread
of cells from a tumour to distant organs is a great barrier
to cancer cure. The process involves a complex series of
interactions between the cancer and its environment.
■ Relationship to cancer. Epithelial cells normally lie
between thin sheets of basement membranes and are
surrounded by an extracellular matrix, which they secrete
themselves. In order to become successfully invasive,
tumour cells must adhere to the basement membrane
(BM), produce and secrete proteolytic enzymes
that are capable of degrading the extracellular matrix
(ECM) and BM,adhere to and degrade the ECM components
of host cells and regulate genes encoding for proteolytic
enzymes for ECM degradation and adhesion.
Degradation is achieved by enzymes termed Matrix
Metalloproteinases (MMPs). MMP activity is inhibited
by natural tissue inhibitors of MMPs (TIMPS) and the
II/64 European Journal of Nutrition (2004) Vol. 43, Supplement 2
balance between MMP and TIMP activity is a strong indicator
of metastasis. MMP’s are also involved in some
of the later events in the metastatic process, namely intravasation
(in which the cancer cells enter the blood or
lymphatic vessels), extravasation (passage of the cell
from the vessels into tissues) and proliferation of the
cells at the secondary tissue [126].
■ Modulation by diet. N-3 fatty acids have been shown
to inhibit invasion of kidney tumour cells using the Matrigel
invasion assay [127]. Animal models have been
used to evaluate effects of food components on metastasis.
For example retinoids suppress the development
of bone metastasis of prostate cancer cells [128] and
modified pectin inhibited metastasis of human colorectal
and breast carcinoma cells [129]. Thus they provide
a method for assessing functional foods for potential
anti-metastatic activity, although the relevance to
the human situation remains to be established. There is
some evidence from less specialised animal models that
dietary change can modulate late events in cancer. For
example, probiotic treatment of rats given dimethylhydrazine
to induce colon tumours, resulted in a reduction
in tumour size and a lower proportion of malignant
tumours [130].
■ Methodological considerations. The Matrigel Invasion
assay (MIA) is an established in vitro assay to assess cell
invasion.Matrigel contains components of the basement
membrane and only cells with invasive properties will
pass through this layer. In this assay, cells from a
metastatic tumour line are seeded onto matrigel-coated
tissue culture inserts containing an 8-micron pore size
membrane.The matrigel occludes the pores of the membrane
blocking non-invasive cells from migrating
through the membrane. In contrast, invasive cells are
able to detach themselves from and migrate through the
matrigel matrix treated membrane in response to a
chemoattractant present in the underlying medium.
There are also a number of animal models (usually
based on athymic mice) that are used to study the whole
process of metastasis, in which the tumour cells are inoculated
subcutaneously or orthotopically and the number
of spontaneous metastases to secondary sites such
as lung liver and bone are assessed [131]. A wide range
of tumour cell lines have been used as the source of the
inoculum including colorectal tumour cells, melanoma,
prostate cell lines,T cell lymphomas and mammary adenocarcinomas
[126, 132–135].
In humans, detection of circulating tumour cells in
bone marrow and lymph nodes by immunocytochemistry
is a well-defined prognostic marker for colorectal
cancer progression, especially metastasis [136]. The
presence of micrometastases in bone marrow appeared
as an independent indicator of an aggressive tumour
with a poor outcome [137]. More recently, the detection
of tumour cells in the blood circulation is proving a
promising biomarker of tumour presence and progression
in humans. Longitudinal studies have shown that
the levels of circulating epithelial cells in peripheral
blood are directly linked to the tumour burden and response
to therapy in e.g. prostate cancer [138]. In animals,
clusters of tumour cells were found to be a more
relevant prognostic marker than single cells [139]. A
new technique,called immunomagnetic cell separation,
was recently used to show that cell clusters were also present
in peripheral blood samples of colon carcinoma patients
[140]. It is believed that the vast majority of these
cells originate from the primary tumour [141].
■ Conclusions. Inhibition of metastasis represents a potentially
useful target for dietary intervention and there
is a wide range of in vitro and in vivo assays that can be
used to screen potential candidates.
■ Tumour cells in circulation
As blood samples can be obtained easily and repeatedly,
this makes the detection of tumour cells in blood circulation
a very promising biomarker of tumour presence
and progression.
Carcinogen metabolising enzymes
Metabolism is often the major process responsible for
the elimination of a chemical, and hence is of key importance
as a target of functional effects.Metabolism includes
phases of biotransformation and of toxicokinetics.
Biotransformation is the process whereby a
substance is changed from one chemical to another
(transformed) by a chemical reaction within the body
[142].During this process a large number of phase I and
phase II enzymes metabolically convert lipophilic xenobiotics
to hydrophilic metabolites. This facilitates their
cellular elimination (phase III) and excretion,thus minimising
exposure to them.
■ Biotransformation of carcinogens
■ Definition of target. Phase I enzymes (e.g. cytochrome
P450, flavin-dependent monooxygenases, oxidoreductases)
convert hydrophobic compounds to reactive
electrophiles by oxidation, hydroxylation and
reduction reactions to prepare them for reaction with
water-soluble moieties. Phase II enzymes (e.g., glutathione
S-transferases [GST],UDP- glucuronosyltransferases
[UGT], sulphotransferases, aryl amine acetyltransferases)
primarily catalyse conjugation reactions.
J. Rafter et al. II/65
PASSCLAIM – Diet-related cancer
■ Relationshipwithcancer. Biotransformation enzymes
play a major role in regulating the toxic, mutagenic and
neoplastic effects of chemical carcinogens (Table4).
Phase I enzymes, particularly the cytochrome P450s,
have the propensity to result in bioactivation,compared
with the inactivation that often results from Phase II
reactions. This has led to the concept that selective
induction of phase II enzymes over phase I will contribute
to cancer chemoprevention. An example is the
agent [4-methyl-5-(2-pyrazinyl)–3H–1, 2-dithiole-3thione]
(oltipraz) which may protect against liver cancer
caused by high aflatoxin B1 exposure in Qidong,People’s
Republic of China [143, 144]. The studies have shown
that intermittent, high-dose oltipraz inhibited phase I
activation of aflatoxins, whereas sustained low-dose
oltipraz increased phase II conjugation of aflatoxin B1,
yielding higher urinary levels of aflatoxin-mercapturic
acid [145].There are numerous other reports indicating
that the balance of metabolising enzymes are directly
associated with the magnitude of toxic response for selected
chemicals [146].Genetic enzyme polymorphisms
for genes encoding phase I and phase II metabolism are
Table4 Carcinogen metabolism
Target Relationship with cancer Modulation by diet Methodology Reference
Phase I
Cytochrome P450 (CYP) Activate carcinogens Broccoli induces CYPs in rat tissues; CYP540 can activate and deactivate [147, 282–292]
CYP1A1 polycyclic dietary fibers differently modulate reactive xenobiotics, therefore
CYP1A2 aromatic hydrocarbons CYPs in rat tissues; oltipraz inhibits possible inhibition effects on their
CYP2A6 heterocyclic amines, CYPs that activate aflatoxin; own are not considered to be
CYP2B6 nitrosamines flavonoids modulate CYPs, Ffied beneficial. Several polymorphisms
CYP2D6 cyclophosphamide meat induces CYP1A2 are known
CYP2E2 tobacco specific nitroamine
CYP3A4 vinylchloride,
aflatoxin
Flavin-containing Oxidise xenobiotics with broad Gaps in knowledge, reviewed in [293] Bioactivation of substrates may not [293–296]
monooxygenases (FMO) substrate specificity. Metabolic be excluded. Polymorphism leading
EC 1.14.13.8 conversion may result in detoxication. to gene product with higher rates of
FMO3 Substrates are tertiary, secondary metabolic conversion has been
FMO2 and primary amines. reported.
FMO1
NAD(P)H Quinone Can either bioactivate or detoxify Numerous plant food extracts or Both bioactivation and detoxification [297–304]
oxidoreductases quinones; contributes to reducing ingredients induce QR in cell models of substrates is possible.
(NQO, QR) superoxide radical formation. Lower
EC 1.6.99.2. NQO1 activity may make individuals
vulnerable to leukemia secondary to
benzene exposure
Phase II
Glutathione Catalyse conjugation of reduced Induction of GST has been shown Species differences in gene [169, 178, 191,
S-transferases (GST) glutathione to activated substrates. for a high number of food regulation. Bioactivation of selected 305–308]
EC 2.5.1.18. Metabolic conversion usually results ingredients, metabolites in animal xenobiotics by GSTs is possible.
GSTA in detoxication experiments, cell cultures and in Genetic polymorphisms (deletions,
GSTM humans. sequence alterations) are common
GSTP
GSTT
UDP-Glucuronosyltrans Glucuronidation of endo-, xenobiotics Green tea induces UGTs and Glucuronide formation can also result [309–314]
ferases (UGTs) usually results in termination or enhances glucuronidation of in bioactivation. Allelic variants of
EC 2.4.1.17. reduction of biological activity. 2-amino-1-methyl-6-phenyl imidazo UGT1A1 may cause specific clinical
UGT1A Induction of UGTs is associated with [4.5b]pyridine (PhIP) . Feeding green conditions.
UGT2A decreased cancer related activities tea lowers incidence of preneoplastic Putative phytoprotectants (e. g.
UGT2B in some animal experiments lesions in animal experiments. isoflavones) are UGT substrates
Arylamine Catalyse three reactions involving the Gaps of knowledge: Ethanol has Depending on structure of the [181, 184, 290,
acetyltransferases transfer of an acetyl moiety, resulting been shown to increase acetylation substrate, the reactions can lead to 315, 316]
(NAT) EC 2.3.1.5. in N-acetylation (NAT), O-acetylation rate [Lester 1964, reviewed in [315]) detoxification or bioactivation, e. g.
NAT2 (OAT), N,O-transacetylation (AHAT). of heterocyclic amines.
NAT1 Substrates include carcinogenic Polymorphisms affect toxicological
aromatic amines and heterocyclic responses under specific exposure
amines (food contaminants) situations
II/66 European Journal of Nutrition (2004) Vol. 43, Supplement 2
well recognised susceptibility factors for sporadic tumours,
depending on the exposure situation and functional
consequences of the polymorphisms [147–150].
The induction of specific enzymes involved in the metabolism
of known risk factors can result in an enhancement
or reduction of exposure and accordingly
will modulate cancer related endpoints in model or animal
systems [151–154] and in humans [155],
■ Modulation of target by diet. A claim by which phytochemicals
from plant foods could be of benefit is that
they modulate biotransformation in such a manner that
carcinogens are less active and thus exert less harm in
the target cells of cancer, This is considered to be one of
the mechanisms by which fruits and vegetables act
chemoprotectively [156], One example is chemoprotection
conferred by cruciferous vegetables, due to their
high glucosinolate content and the capacity of glucosinolate
metabolites to modulate biotransformation enzyme
systems (e.g. cytochrome P450 and conjugating
enzymes) [157]. Glucosinolates (β-thioglycoside-N-hydroxysulfates)
are hydrolysed by the plant enzyme myrosinase
releasing the biologically active isothiocyanates
(ITC). ITCs induce expression of phase I and
phase II enzymes and also directly inhibit CYP450 [120].
Broccoli and Brussels sprouts increase metabolism of
cooked meat-derived heterocyclic aromatic amines,
which implies that they induce both CYP1A2 and phase
II enzymes involved in heterocyclic amine metabolism
[158]. The results of six cohort studies have shown inverse
associations between the consumption of Brassica
vegetables and risk of lung cancer, stomach cancer, and
all cancers taken together (reviewed in [159]). Two
thirds of 74 case-control studies have also shown an inverse
association for risks of several cancer sites including
the colon (reviewed in [159]).In a recent study [160]
dietary ITC intake from cruciferous vegetables was assessed
for 213 incident cases of colorectal cancer and
compared to 1194 controls. The additional analysis of
GSTM1, T1 or P1 genotypes revealed that there were no
overall associations between genotype and colorectal
cancer risk. However, among individuals with both
GSTM1 and T1 null genotypes, a 57% reduction in risk
among high versus low consumers of ITC was observed
for colon cancer.The results are compatible with the hypothesis
that ITCs from cruciferous vegetables modify
risk of colorectal cancer in individuals with low GST activity.
In the gut lumen the glucosinolate breakdown
product sulforaphane can inhibit DNA adduct formation
induced by a heterocyclic amine in a dose-dependent
manner, possibly acting through the induction of
phase II detoxification enzymes such as glutathione
transferases and UDP-glucuronosyltransferases [161].
■ Methodological considerations. Some plant food ingredients
(ITCs) induce phase I enzymes, others induce
only phase II enzymes,and some induce both [158–162].
The situation is uniquely complex because each plant
food contains a variety of glucosinolates and other phytochemicals
[163], which modulate biotransformation
systems in multiple ways. Therefore it is not easily predictable,
in which manner modulated biotransformation
results in risk reduction. More accurate knowledge
is needed on whether carcinogens cause specific types of
tumours and how these carcinogens are metabolically
transformed in the relevant human tissues. It is also necessary
to increase our understanding on how diet can
modulate the biotransformation in a favourable manner,
and how individual susceptibilities (genetic polymorphisms,
regulation of the target genes, baseline expression
levels) affect biotransformation and exposure.
The elucidation of these complex situations will require
experimental approaches of equal complexity, e.g. to
elucidate multiple genes and pathways of induction arising
from the impact of diet. Promising approaches to
achieve the analysis are the employment of new techniques
of genomics to reveal patterns of gene expression
(transcriptomics), revealing complete metabolic pathways
(metabonomics) and assessing the functional consequences
on cellular and organism levels. The criteria
for this approach,which are being developed in the field
of toxicogenomics [164] and are already applied in
pharmacogenetics [165] will also be an important scientific
basis for better understanding dietary chemopre-
vention.
■ Conclusion. It is very feasible,from a theoretical point
of view, to provide humans with foods, which enhance
the capacity of tissues to metabolise chemical carcinogens
in such a way that these are eliminated from the
body without being transformed to reactive intermediates.
The selective induction of Phase II over Phase I
could mediate such protection, provided that the resulting
conjugates are not more reactive than the substrates.
Taken together carcinogen metabolising enzymes could
be considered as potential“susceptibility markers”.
Glutathione S-transferases
The glutathione S-transferases (GSTs) are dealt with in
more detail below as they represent one of the more interesting
specific targets within the general target “biotransformation
of carcinogens”.
■ Definition of target. The glutathione S-transferases
(GSTs) are a multigene family of enzymes largely involved
in the detoxification of chemicals. They catalyse
the conjugation of the tripeptide glutathione via its sulphur
atom to many toxins containing an electrophilic
functional group, allowing these compounds to be excreted
from the body [166]. The best-characterised GST
isoenzymes in mammals have been grouped into four
J. Rafter et al. II/67
PASSCLAIM – Diet-related cancer
major cytosolic classes, termed Alpha (A), Pi (P), Mu
(M),Theta (T),and three membrane-bound GSTs [167].
■ Relationship with cancer. Oxidative molecular damage
in cells is linked to degenerative diseases including
cancer [168]. Glutathione S-transferases contribute to
resistance against oxidative stress [169] by inactivating
toxic and mutagenic alkene products of oxidative
processes and thus protect cells and tissues [170].An important
factor is that GSTs are inducible and that in rat
and mouse induction appears to be mediated through
the antioxidant response element (ARE),a transcription
enhancer [171, 172]. It contains sequences required for
basal expression of, for example rGSTA2, and its induction
is mediated by phenolic antioxidants, for example
green tea phenols [173]. These compounds also activate
genes via the AP-1 family of transcription factors,which
include Jun, fos, Maf, Nrl and Fra proteins [174]. AP-1binding
sites have been identified in the promotor regions
of the hGSTA1, hGSTA4 and hGSTP1 genes [175,
176]. Other transcription factor binding sites (NF-κB,
SP-1,AP-2, GRE) have been described for GSTP1 and/or
GSTA1.Whether or not an enhanced expression of GSTs
contributes to cancer prevention has not been demonstrated
in humans conclusively. But there are several
lines of evidence which support this assumption: 1) A
large number of animal experiments have shown that
cancer reducing activities of chemicals can be associated
with increases in phase II metabolism [177]; 2) In
human cells an enhanced GST-level is associated with
less damage by 4-hydroxynonenal [178] whereas lower
GST-levels are associated with more damage [179]; 3)
Individuals with null polymorphisms [180] for GSTM1,
GSTT1 or other types of genetic predispositions [181]
are at higher risks for developing tumours, especially in
the case of specific exposure situations [182–184].Alternatively,
sequence polymorphisms for GSTP1 protect
from benzo(a)pyrene mediated genotoxic damage
[185].
■ Modulationbydiet. Numerous diets,food ingredients
and related endogenous metabolites induce GSTs in animal
experiments [166, 186–190]. In humans, evidence
is also available on dietary or intervention mediated
regulation of GSTs [191–193]. Both inducing and suppressing
activities have been reported. One example is
the effects in human colon cells of butyrate, an abundant
fermentation product present in the human gut
[178, 194]. Also, genetic polymorphisms alter the preventive
effects of cruciferous vegetables, which are inducers
of GST [157].
■ Methodological considerations.
– Species differences in gene regulation: Different
mechanisms of transcriptional and posttranscriptional
events are important in controlling levels of expression
and these are to be expected in different
genes,cell types and tissues.For instance most of our
knowledge on the regulation of GSTs has been derived
from studies in rodents. However, the sequences
of the 5’-flanking regions of the human
genes are different from the rodents’, suggesting
species-differences in the regulation [195].
– Activation by phase II reactions: Not all phase II reactions
yield products that are less reactive than the
substrate prior to conjugation. For certain
haloalka(e)nes (ethylene dibromide,methylene chloride)
the conjugate is instable and rapidly decomposes
to reactive intermediates with mutagenic potential
[196].
– Shift in organotrophy: The modulation of biotransformation
does not necessarily provide the same protection
in all tissues.In some cases there may only be
a shift of metabolism,which can result in a shift of tumour
localisation. For example, in animal experiments
I-3-C decreased formation of aberrant crypt
foci in the colon,but enhanced the formation of GSTP
foci in the liver [197].
– Genetic polymorphisms of GSTs: Confounding situations
may be encountered on the basis of individual
susceptibilities. Null genotypes for GSTM1 and
GSTT1 occur in frequencies of approximately 50%
and 20% of the population and result in absence of
the respective enzymes. The primary hypothesis has
been that individuals with the GST-null genotypes
are at higher risk for cancer because of a reduced capacity
to eliminate activated carcinogens. Therefore
epidemiological studies have focused on interactions
between these polymorphisms and carcinogen exposure,
whereas only few human dietary interventions
have tested the effects of diet on biotransformation
enzymes in relation to the genetic
polymorphisms of the individuals. One was a controlled
feeding study to test if GSTM1genotype affects
response to a diet high in cruciferous vegetables
[163]. Serum GSTα concentration, an enzyme induced
by isothiocyanates, increased significantly in
response to cruciferous vegetable feeding,but only in
GSTM1-null individuals,indicating that the relationships
between cruciferous vegetable intake and cancer
risk are influenced by genetic polymorphisms of
GSTs [157].
■ Conclusion. It seems likely, that an induced expression
of the GSTs will result in the protection of cells from
genotoxic insult by specific chemicals, especially since
these particular enzymes are more involved in deactivation
rather than activation and they are inducible.Moreover,
many putative carcinogens are relevant substrates
for GSTs.However,the limitations in claiming such functions
from foods are based on the need for the experimental
verification of the hypothesis. To date there are
II/68 European Journal of Nutrition (2004) Vol. 43, Supplement 2
no data available showing that in humans the final consequence
of an induction of GST coupled to a favourable
overall modulation of biotransformation is the prevention
of tumours.
Genetic events
Genetic events, that is mainly damage to the genome of
a cell, constitute various types of lesions such as DNA
damage, DNA adducts, gene mutations, and cytogenetic
alterations.Associated with these is a deficiency of DNA
repair leading to genetic instability and to an even
higher incidence of genetic lesions (see Table5).
There is substantial evidence that genetic events are
involved in the initiation, promotion and progression
phases of carcinogenesis. The selected endpoints described
in the following sections are usually measured
in surrogate tissues (e.g. peripheral lymphocytes). The
endpoints largely serve as biomarkers of exposure, but
there are some probable lines of evidence showing that
causative associations are involved as well. Therefore,
several larger scale validation studies are presently underway
to assess causal relationships for individual endpoints
of genetic damage.
Guidelines considering several genetic endpoints are
available to provide concise guidance on the planning,
performing and interpretation of studies to monitor
groups or individuals exposed to genotoxic agents [198].
The guidelines contain important methodological considerations,
including ethical aspects, details on sampling
procedures, size and characteristics of studied
populations,how to assess individual exposures,and potential
confounders or effect modifiers, aspects of performing
the statistical analysis,as well as procedures for
publishing and archiving data, and ensuring sufficient
high quality of data. They provide a basis for studies to
monitor subjects of dietary intervention trials. Requirements
for reporting antioxidant, antimutagenic or anticarcinogenic
potential of test substances in in vitro experiments
and animal studies in vivo have been
identified as well [199].
Optimising vitamin and mineral intake by encouraging
dietary change, multivitamin and mineral supplements,
and fortifying foods might prevent genetic lesions
and therefore cancer and other chronic diseases
[200].
■ DNA strand breaks
■ Definition of target. Breaks of the DNA backbones
(single strand breaks). If two breaks occur on opposite
strands in close vicinity, these are called double strand
breaks.
■ Relationship of target with cancer. Many cancer-causing
compounds are genotoxic and some of these have
the potential to induce DNA strand breaks. However
strand breaks can also result from the enzymatic activity
of endonucleases in apoptosis or during DNA repair.
So, the significance of strand breaks is complex and reflects
DNA damage as well as DNA repair.
■ Modulationoftargetbydiet. The first studies showing
that dietary intervention in humans alters (lowers DNA
strand breaks) in lymphocytes were performed with
vegetable [201] and fruit juices [202].Meanwhile several
other studies are showing that other types of dietary intervention
have similar effects. Faecal water obtained
from humans consuming a diet high in fibre causes
fewer DNA strand breaks in cultured human colon cells
than a diet high in meat and animal fat, but low in dietary
fibre [113]. Moderate wine consumption protects
against hydrogen peroxide-induced DNA strand breaks
[203]. Instead of using only the surrogate lymphocytes,
studies using target tissues of cancer occurrence are
considered to be more relevant. To date, DNA strand
breaks in cells of potential target tissues have been
shown in animal studies, e.g. in the colon of rats after
oral ingestion of probiotics [204, 205], but methods are
now available to investigate effects of diet also in colon
cells of humans [206–209].
■ Methodological considerations. Guidelines considering
this endpoint are available to provide concise guidance
on the planning, performing and interpretation of
studies to monitor groups or individuals exposed to
genotoxic agents. They provide a basis for studies to
monitor subjects of dietary intervention trials [198].The
most frequently applied assay to detect strand breaks
and related events is the alkaline single cell gel electrophoresis
(Comet) assay [209] which has largely replaced
the technique of alkaline elution [210, 211]. Recent
recommendations for performing the in vivo
Comet assay are available [212].For a review of the clinical
applications of the Comet assay in different cell
types (lymphocytes, buccal and nasal cells, and sperm)
see also reference [213].
■ Conclusion. The determination of DNA damage, as is
now being performed with the comet assay, provides a
useful tool to investigate the effects of diet in a panel of
different human tissues, including tissues relevant for
diet-associated tumour occurrence. The modulation of
DNA damage reasonably well reflects also the modulated
exposure to genotoxic compounds that cause the
damage. Therefore the reduction of DNA damage will
indicate a reduced exposure, which in turn is related to
decrease of risk.
J. Rafter et al. II/69
PASSCLAIM – Diet-related cancer
■ DNA adducts
■ Definition of target. Modified DNA bases resulting
from the addition of DNA with an electrophile or free
radical are called DNA adducts.Adducts can also be generated
by the effect of ultraviolet radiation creating
thymine dimers. Depending on the type of chemical involved,
alkylated bases, oxidised bases, bulky adducts,
etheno- and propano-adducts can result as adducts.
Sometimes, the effect of an adduct can result in the
Table5 Genetic events
Target Relationship with cancer Modulation by diet Methodology Reference
DNA strand breaks Reduction of damage indicates a Vegetable and fruit juices decrease Can be measured in any tissue [201, 202,
(e. g. Comet assay) reduction of exposure to damage in peripheral lymphocytes and in exfoliated cells; body fluids 205, 317]
genotoxic compounds. Since a of humans. In animals DNA can be monitored for damaging
high proportion of cancers is most damage is decreased in colon cells capacity, reflecting exposure;
likely due to exogenous of rats consuming probiotics. multiple classes of damage can
compounds, reduced exposure is Faecal water genotoxicity can be be detected; analysis proceeds on
equivalent to reducing risks. modulated by diet. level of single cell
DNA adducts
Bulky DNA adducts from A recent meta-analysis has shown Specific inverse associations A number of different EU projects [155, 214,
polycyclic aromatic that current smokers with high between DNA adduct levels and are being performed to associate 318, 319]
hydrocarbons levels of bulky adducts have an antioxidant concentrations among DNA adducts, exposure and
increased risk of lung and bladder GSTM1 null subjects and smokers cancer in molecular
cancers, which also suggested have been reported. A six-month epidemiological studies.
that similar (aromatic) compounds vitamin intervention did not Methodological limitations are
may be involved in the aetiology reduce DNA damage (bulky that the level of measurement
of both types of cancer. adducts) in oral cells of heavy error for bulky adducts is high.
smokers. More standardised measurements
are needed in future investigations.
Oxidised DNA bases Oxidised DNA bases cause mutations Carotenoid-rich juices and Different methods are available to [318, 201,
(and oxidative stress) that are commonly observed in supplementation with antioxidants measure oxidised DNA bases, 202, 220,
mutated oncogenes and tumour have reduced oxidised bases namely oxidised pyrimidine and 221,
suppressor genes. Reduction measured with the comet assay. purine bases, using the comet 320–322]
indicates a reduction of exposure. Oxidative DNA-damage in humans assay with repair specific
An EU-funded Concerted Action is reduced by Brussels sprouts enzymes, 8-oxy-7- hydrodoxy(QLK1–1999–00568;
ESCODD) has consumption. In a recent guanosine, using HPLC with
been set up to establish a European epidemiological study, individual electrochemical detection
standards committee on oxidative dietary and lifestyle habits only (HPLC/ECD), GC/MS methods,
DNA damage. The project aims modestly affected lymphocyte DNA enzyme-linked immunosorbent
ultimately to establish the level of oxidation (measured in the assay (ELISA) techniques and
DNA damage in white blood cells modified comet assay) and 5-hydroxymethyl-29-deoxyuridine
from representative groups of men suggested that specific dietary (HMdU), a product of thymine
and women. One objective is to patterns, rich in fresh fruit and oxidation, using serum auto
resolve the problems in measuring vegetables, are not clearly related antibodies. Recent studies are
this marker and to reach a consensus to decreased oxidative damage in showing that the comet assay and
on the level of damage in normal peripheral lymphocytes in a HPLC are equally efficient for
cells. This is important from the Mediterranean population. detecting induced damage.
point of view of estimating cancer Standards are being evaluated in
risk and cancer-preventive effects the EU-funded Concerted Action
of dietary antioxidants. (QLK1–1999–00568; ESCODD).
Exocyclic DNA adducts Oxygen radicals attack lipids to High dietary omega-6 polyun- Presently an EU-funded project is [320,
(etheno- and propano-adducts) generate reactive intermediates saturated fatty acids drastically being performed (QLK4CT- 323–325]
that can couple to DNA resulting in increase the formation of etheno- 2000–00286) with the objective to
exocyclic bases. The promutagenic DNA base adducts in white blood analyse the repair functions, the
lesions can be related to increased cells. persistence and the mutational
probability of cancer risks if induced specificity of e-DNA adducts at
in oncogenes and tumour the gene level, so to better define
suppressor genes. For example the the mutagenic and carcinogenic
major lipid peroxidation product, potential of these miscoding
trans-4-hydroxy-2-nonenal, lesions in humans. An
preferentially forms DNA adducts immunohisto- chemical method
at codon 249 of human p53 gene, has been developed to detect
a unique mutational hotspot in malondialdehyde (MDA)-modified
hepatocellular carcinoma. DNA adducts in single human oral
mucosa cells.
II/70 European Journal of Nutrition (2004) Vol. 43, Supplement 2
breaking of a base into small residues or its removal
from DNA creating an abasic site.
■ Relationship of target with cancer. A recent metaanalysis
testing the hypothesis that the presence of a
Table5 Continued
Target Relationship with cancer Modulation by diet Methodology Reference
Mutations
Gene mutations The accumulation of multiple Using the HPRT (hypoxan-thine – The tissue-specific mutagenicity of [326, 327]
mutations, especially in guanine phosphoribosyl IQ was studied at the lacI locus in
protooncogens, tumour suppressor transferase) gene assay with the liver, colon and kidney of Big
genes and DNA repair genes is T-lymphocytes from 312 Blue(R) transgenic rats. This is
associated with cancer initiation, individuals, it was found that HPRT probably the most effeicient
promotion and progression. In fact mutant frequency (MF) was system to analyse mutations in vivo.
tumour progression selects for cells significantly decreased in relation However, the mutations identified
with specific mutations including to intake of vegetables, citrus fruits were different to the mutations
those which maintain genetic and berries, respectively, as well as identified in IQ-induced tumours,
stability. calculated vitamin C intake from which indicates the responses to be
diet. Only a borderline significant non-specific in relation to the
association was observed for effects in humans.
beta-carotene
Chromosomal aberrations Reduction in occurrence should Food mutagens can induce Putative anticarcinogens, namely [328–331]
indicate a reduction of exposure, cytogenetic lesions. Probiotics have the phytoestrogens coumoestrol
and possibly of cancer risk. A been shown to reduce CA in animal and genistein induce structural
present EU-funded concerted experiments, but otherwise there chromosomal aberrations in
action (BIOMED 2) of 13 European are gaps of knowledge on how cultured human peripheral blood
institutes, is characterising diet may reduce CA in humans lymphocytes
cytogenetic lesions, occupational
exposure and cancer incidence in
large cohorts, with the aim to
analyse the predictivity of
chromosome changes as indicators
of cancer risk
Micronuclei Reduction of occurrence indicates Different levels of micronuclei Micronuclei are easier to score [203, 234,
a reduction of exposure, and incidence were reported with age, (less time consuming) than 332–337]
possible of risk. “The International gender and vegetarian, versus chromosomal aberrations.
Collaborative Project on non-vegetarian diet. Moderate Different types of staining methods
Micronucleus Frequency in Human wine consumption protects against can reveal the nature of origin of
populations (HUMN)” is collecting hydrogen peroxide-induced micronuclei. Determinations of
an international data base to micronuclei. Micronuclei are micronuclei in exfoliated cells of
determine associations with cancer modulated in lymphocytes and buccal and bladder mucosa are a
and ageing http://ehs.sph.berkeley. exfoliated buccal cells of promising alternative to
edu/holland/humn/ postmenopausal women with lymphocytes in human studies.
dietary changes in folate. Cigarette Presently larger scale international
smoking, intracellular vitamin validation and endpoint
deficiency, are associated with assessments are taking place.
micronuclei in epithelial cells of the
buccal mucosa
DNA Repair
BER Significantly increased risk for Significantly increased risk for Until recently, only two mammalian [338, 339]
orolaryngeal cancer was observed orolaryngeal cancer was observed DNA glycosylases specific for
for polymorph hOGG1 genotypes, for polymorph hOGG1 genotypes, oxidised bases had been
in smokers but not in non-smokers, in alcohol drinkers but not in never characterised, the E.coli
suggesting that hOGG1 may play drinkers smokers. endonuclease III (Nth) homolog
an important role in the repair of NTH1, and 8-oxoguanine-DNA
8-OH-dG adducts in the glycosylase (OGG1), pathoaerodigestive
tract and that the physiological effects. However,
hOGG1 (Ser326Cys) polymorphism mouse mutants lacking either
plays an important role in risk for NTH1 or OGG1 have no obvious
smoking- and alcohol-related phenotype, in spite of accumulation
orolaryangeal cancer of mutagenic and toxic base lesions
in their genomes. An explanation
is that repair is mediated by
additional DNA glycosylases.
J. Rafter et al. II/71
PASSCLAIM – Diet-related cancer
high level of bulky DNA adducts in tissues is associated
with an increased risk of cancer in humans reports that
current smokers with cancer have statistically significant
higher levels of adducts than controls, whereas results
were negative or contradictory in ex-smokers and
non-smokers. The authors conclude that current smokers
with high levels of adducts have an increased risk of
lung and bladder cancers [214].
■ Modulation of target by diet. DNA adducts of acetaldehyde
can be detected in peripheral white blood
cells of alcohol abusers [215]. Plasma levels of a combination
of antioxidant vitamins (vitamins C and E and βcarotene)
rose significantly during a randomised clinical
trial among heavy smokers, but the measurement of
DNA damage (immunological determination of polycyclic
aromatic hydrocarbon-DNA adducts) and oxidative
DNA damage (8-oxo- or hydroxydeoxyguanosine)
in mononuclear and oral cells was not statistically significantly
different in the group with antioxidant vitamin
intake than in the controls, thus providing no evidence
that vitamins prevent this type of DNA damage in
smokers [216]. The studies confirm earlier studies measuring
oxidative DNA damage in male smokers receiving
β-carotene supplementation [217].In another study,
antioxidant supplementation decreases oxidative DNA
damage in human lymphocytes [218]. Oxidised DNA
bases are reduced in lymphocyte-DNA of healthy, nonsmoking
males after consumption of vegetable juices
[201] and of a soy milk supplement [219]. However
when considering epidemiological studies instead of intervention
trials the associations are not as apparent. In
a recent epidemiological study [220],comparing dietary
and lifestyle determinants of oxidative DNA damage
(modified comet assay) basal levels of DNA oxidation
were positively association with coffee (P=0.01) and
tomato consumption (P=0.05). Instead, the consumption
of cruciferous vegetables tended to be negatively associated
with oxidative damage. A positive non-significant
association between the consumption of total vegetables
and fresh fruit and DNA damage was noted
(P=0.08 and P=0.10,respectively).In contrast,the estimated
intake of simple sugars was positively associated
with oxidative DNA damage, while vitamin E showed a
borderline positive association. The plasma levels of
several micronutrients did not appear to influence DNA
damage. Thus, the results indicated that individual dietary
and lifestyle habits only modestly affected the levels
of lymphocyte DNA oxidation and suggested that
specific dietary patterns, rich in fresh fruit and vegetables,
are not clearly related to decreased oxidative damage
in peripheral lymphocytes in a Mediterranean population
[221].
■ Methodological considerations. Guidelines considering
this endpoint are available to provide concise guidance
on the planning, performing and interpretation of
studies to monitor groups or individuals exposed to
genotoxic agents. They provide a basis for studies to
monitor subjects of dietary intervention trials [198].The
best established methods to detect DNA adducts are
sensitive chemical analytical detection methods,involving
32P post labelling of the DNA [222, 223]. Biological
determinations (e.g. using modifications of the comet
assay) are increasingly becoming available, which may
facilitate larger scale monitoring studies in the future
[220].
■ Conclusion. Adducts are specific indicators of DNA
damage caused by individual chemical groups (e.g.
“Bulky” DNA adducts by polycyclic aromatic hydrocarbons,
etheno-adducts by reactive aldehydes, oxidised
bases by reactive oxygen species). They therefore represent
an integrated marker of exposure to the specific
compounds,and of the ability of the individual to metabolically
activate carcinogens and to repair DNA damTable5
Continued
Target Relationship with cancer Modulation by diet Methodology Reference
NER Humans with a hereditary defect Gaps in knowledge DNA repair inhibitors hydroxyurea [216, 224,
in NER suffer from xeroderma and cytosine arabinoside enhance 340–342]
pigmentosum and are predisposed the sensitivity of the alkaline
to skin cancer caused by sunlight single-cell gel/electrophoresis
exposure. Cancer susceptibility is ‘Comet’ assay. The Comet assay is
associated with defective DNA more and more being used to
repair. Genetic instability is determine genetic instability.
associated with increased risk of
bladder cancer.
MMR Defects in MMR are associated in Gaps in knowledge MMR removes nucleotides [225, 343,
the hereditary nonpolyposis mispaired by DNA polymerases. 344]
colorectal cancer (HNPCC) and with Defects result in ‘microsatellite
a variety of other sporadic cancers. instability’, which are measured to
identify MMR deficiencies.
II/72 European Journal of Nutrition (2004) Vol. 43, Supplement 2
age. A reduction of adducts therefore reflects a lower
level of exposure and with that a lower risk.A number of
international studies are being performed to determine
in which manner specific types of DNA adducts are
causally related to cancer risks. The outcome of these
studies will be of high value for using the parameters as
a basis for possible claims.
■ DNA repair
■ Definitionoftarget. Base excision repair (BER) is primarily
responsible for repairing small base modifications
and abasic sites caused by endogenous damage
and environmental insult. Nucleotide excision repair
(NER) is the repair process which incorporates the excision
of damaged and altered bases (e.g. bulky adducts,
cyclobutane pyrimidine dimers) as part of an oligonucleotide
fragment. Mismatch repair (MMR) is the DNA
repair process by which mispaired bases are excised as
single nucleotides [224]. Interstrand cross-links and
DNA double strand breaks are repaired by homologous
recombination and/or end-joining repair. O6-methylguanine
methyltransferase, a single repair protein, reverts
damage by removing the non-native methyl group
from O6-methyl guanine [225].
■ Relationshipoftargetwithcancer. Inherited defects in
any of these pathways, in general, predisposes to malignancy
[225]. Examples are defective NER, which is the
cause for Xeroderma pigmentosum (XP), a syndrome
characterised by a severe predisposition to skin cancers
of various types (squamous cell carcinomas and basal
cell carcinomas).Defects in MMR in humans predispose
to cancer of the colon, but also to uterine, ovarian and
gastric cancer (reviewed in [224]). No human disorders
caused by inherited BER deficiencies have been identified,
but according to mouse models, the knockout of
BER enzymes (glycosylases) show only mild phenotypes,
although mutagenesis and cancer susceptibility
are probably increased [225].X-ray-sensitive persons afflicted
with ataxia telangiectasia have a defective ataxia
telangiectasia (ATM) protein kinase, which predisposes
to lymphoma [reviewed in 224]. Latent genetic instability
has been associated with an increased risk for several
cancers. Genetic instability, as measured by the comet
assay is associated with an increased estimated relative
risk of bladder cancer [216].
■ Modulationoftargetbydiet. Repair enzymes have not
been well characterised for regulation via transcriptional
activity.There are few data available related to exogenous
factors involved in regulation of these different
proteins, with perhaps the exception of alkyl methyltransferase
albeit the physiological consequences are
not well elucidated [226].There are large gaps in knowledge
pertaining to the potential regulation of repair enzymes
by diet, and hardly any information is available.
■ Methodologicalconsiderations. DNA repair deficiency
is related not only to cancer, but also probably to ageing
and exposure to oxidative stress. New knock-out mouse
models reflecting specific repair deficiencies are becoming
more and more available to study these associations.
There are hardly any studies on modulation of transcriptional
activity of DNA repair genes (e.g. potential
induction by dietary factors), although they would be
highly needed as a tool to study the possibility of repair
induction.
■ Conclusion. Large gaps in knowledge exist. Whether
or not it would be possible to make the potential claim
that DNA repair can be activated by enhanced transcription
of the respective genes has hardly been investigated
so far.
■ Cytogenetic effects
■ Definition of target. Chromosomal structural aberrations
are deletions, insertions, translocations, inversions,ring
structures and abnormalities of chromosome
number. Micronuclei originate from chromosome deletions
and chromosome loss. Nucleoplasmic bridges
originate from dicentric chromatids and chromosomes.
■ Relationship of target with cancer. A large number of
studies are available which demonstrate that deletions
in tumour suppressor genes and DNA repair genes
[227], resulting in the loss of a functional gene product,
are involved in the transformation of normal, dividing
cells into tumour cells. The alterations and the sequences
by which these alterations occur as well as the
subsequent functional responses have been reasonably
well investigated in, for example colon cancer [228].
Chromosomal aberrations and micronuclei in peripheral
lymphocytes are used as surrogate markers, although
the long-term effects of an increased frequency
of chromosomal aberrations in individuals are still uncertain.
In a recent study, new support for the associations
between cytogenetic damage and cancer was obtained
[229]. An increased risk of cancer in healthy
individuals with high levels of chromosomal aberrations
(CAs) in peripheral blood lymphocytes has been
described in recent epidemiological studies [230]. This
association did not appear to be modified by sex, age,
country, or time. The study evaluated whether CAs predicted
cancer because they were the result of past exposure
to carcinogens or because they were an intermediate
endpoint in the pathway leading to disease.A nested
case-control study was performed on 93 incident cancer
cases and 62 deceased cancer cases coming from two
J. Rafter et al. II/73
PASSCLAIM – Diet-related cancer
prospective cohort studies performed in Nordic countries
(Denmark,Finland,Norway,and Sweden) and Italy.
For each case, four controls matched by country, sex,
year of birth, and year of CA test were randomly selected.
Occupational exposure and smoking habit were
assessed by a collaborative group of occupational hygienists.
Logistic regression models indicated a statistically
significant increase in risk for subjects with a high
level of CAs compared to those with a low level in the
Nordic cohort (odds ratio, 2.35; 95% confidence interval,1.31–4.23)
and in the Italian cohort (odds ratio,2.66;
95% confidence interval, 1.26–5.62). These estimates
were not affected by the inclusion of occupational exposure
level and smoking habit in the regression model.
The risk for high versus low levels of CAs was similar in
subjects heavily exposed to carcinogens and in those
who had never, to their knowledge, been exposed to any
major carcinogenic agent during their lifetime,supporting
the idea that chromosome damage itself is involved
in the pathway to cancer. The results have important
ramifications for the understanding of the role played by
sporadic chromosome damage for the origin of neoplasia-associated
CAs.
■ Modulation of target by diet. One study has addressed
the effects of age and lifestyle factors on the accumulation
of cytogenetic damage as measured by chromosome
painting, and has found that stable chromosome
aberrations show a greater accumulation with age than
do unstable aberrations, suggesting that lifestyle factors
contribute to the accumulation of cytogenetic damage
[231]. Hardly any studies in humans exist showing that
diet may contribute to reducing the occurrence of chromosomal
aberrations. Micronuclei have been used in
this context more frequently, for example intervention
with vitamin E had no significant impact on spontaneous
genetic damage in human lymphocytes [232].
Moderate wine consumption protects against hydrogen
peroxide-induced DNA damage, measured as micronuclei
[203]. The micronuclei index is enhanced in older
men, deficient in homocysteine, folate and vitamin B12
[233]. A highly significant (p=0.001) positive association
between plasma levels of vitamin B12 and frequencies
of SCE was reported in smokers [234].After correction
for age, gender and GSTM1 genotype, a significant
association (p=0.026) between the MTRR66GG variant
genotype and higher micronucleus rates was observed
[234], whereas another study has shown that folate and
vitamin B12 supplementation reduces genome damage
in young adults [235]. In vitro studies have shown that
genomic instability in human cells is minimised when
folic acid concentration in culture medium is
>227nmol/l. Intervention studies in humans show
among other effects that micronucleus formation is
minimised when plasma concentration of vitamin B12 is
>300pmol/l and plasma homocysteine is <7.5micromol/l.These
concentrations are achievable at intake levels
in excess of current recommended dietary intakes
which may be particularly important in those with extreme
defects in the absorption and metabolism of these
vitamins,for which ageing is a contributing factor [236].
■ Methodological considerations. Cytogenetic lesions
are determined mainly in surrogate tissues such as peripheral
blood lymphocytes,and rarely in the target tissues
of dietary related cancers, mainly since it is technically
not easy to isolate dividing cells from the tissues
[237]. Alternative approaches include the determinations
of micronuclei in exfoliated epithelial cells of the
buccal cavity [238] or of the urinary bladder excreted in
the urine. Guidelines considering this end point are
available to provide concise guidance on the planning,
performing and interpretation of studies to monitor
groups or individuals exposed to genotoxic agents.They
provide a basis for studies to monitor subjects of dietary
intervention trials [198].
■ Conclusion. Cytogenetic lesions are indicators of
DNA damage caused by exposure.Also,they occur as the
consequence of genetic instability in cancer patients.
They therefore represent an integrated marker of exposure
and of disease. A reduction of cytogenetic lesions
reflects a lower level of exposure and with that a lower
risk, although systematic studies for these associations
are not available.A reduction of cytogenetic lesions as a
consequence of retardation of disease has also not been
reported. A number of international studies are being
performed to determine in which manner specific types
of cytogenetic lesions are causally related to cancer
risks. The outcome of these studies will be of high value
for using the parameters as a basis for possible claims.
Overall concluding remarks
This working group has identified 18 targets/markers in
the chain of events from initial exposure to overt malignant
tumour whose modification ‘have the potential’ to
be used for ‘anticancer’ claims for food components
(Table6). In this area, the one true marker is the malignant
human tumour, which for practical reasons is usually
not accessible to claims. In its absence, we must rely
on alternative markers (late and intermediate markers
of disease). At present, the strongest markers available
are precancerous lesions (e.g. polyps) in humans and
precancerous lesions and tumours in animal models.
The only marker that presently can be used for a type B
claim for food components is ‘polyp recurrence’. However,even
this marker has several limitations (e.g.not all
polyps progress to cancer) and the claim should specify
only ‘reduces risk of polyps’. Type B claims cannot be
made on the basis of results in animal models.
II/74 European Journal of Nutrition (2004) Vol. 43, Supplement 2
All of the other markers (intermediate markers)
presently lack validation against the ‘true endpoint’, the
tumour,and thus cannot be used for type B claims.However,
the majority of these intermediate markers, which
reflect events earlier in the chain of events leading to the
tumour and are mechanistically well linked to the disease
process, have varying degrees of support from in
vitro/animal studies. They may, thus, be used for type A
claims, such as ‘reduces carcinogen exposure’, ‘inactivates
carcinogens’, ‘reduces DNA damage’, ‘modulates
cellular events important for cancer prevention’, ‘inhibits
COX-2’.
‘Reduction of risk of disease’ claims in the area of
‘diet-related cancer’ should be based primarily on human
intervention studies using relevant/acceptable endpoints.
In the absence of the true endpoint, the tumour,
which is not always practical to use, precancerous lesions
(e.g. polyps, aberrant crypt foci) are to be preferred.
However, in this field, the availability of additional
intermediate markers is particularly important in
order to make dietary intervention studies more attractive/accessible
to those wishing to make claims. An important
area for future research will be the validation of
these surrogate markers [239]. It should also be mentioned
that in certain instances, claims might be made
based on results from large epidemiological studies.For
further information, the reader is referred to the “set of
interim criteria for the scientific substantiation of health
claims on foods and food components” [see 345].
The working group emphasises that in the area of
‘diet-related cancer’ when a claim is made, it should be
specified who may benefit from the effect, e.g. the
healthy population, high-risk persons or patients and
evidence should be presented from the target group.
Susceptibility to the claimed effect is of particular importance
in this area.We believe that a scientifically substantiated
mechanism is essential for any claim, especially
if evidence is presented using intermediate
markers as endpoints. We would also emphasise that in
human dietary intervention trials, an appropriate duration
and sample size to demonstrate the intended effect
be employed.
■ Acknowledgements The authors are grateful to the members of
the Biomarkers group of the French network on nutrition and cancer
research (NACRe network) for reading and commenting on this paper.
Prof.G. Speit, University Clinic Ulm, Germany and Prof.M.
Fenech, CSIRO Health Science and Nutrition, Adelaide, Australia are
gratefully acknowledged for valuable comments on the ‘Genetic
Events’ section of the review.
Table6 Concluding remarks
Possible claim Markers/targets Supportive data from Evidence from Target group Potential application
animal/in vitro studies human studies of marker for claims
Reduction in exposure to carcinogens Gut enzymes +++ ++ Healthy population A
Gut metabolites High risk persons
Faecal cytotoxicity
Faecal genotoxicity
DNA adducts
(DNA strand breaks)
(Cytogenetic effects)
Inactivation of carcinogens Biotransformation of ++ + Healthy population A
carcinogens (e.g. GSTs)
Reduction of DNA damage DNA strand breaks + + Healthy population A
DNA repair
Cytogenetic effects
(Apoptosis)
Modulation of cellular events Proliferation +++ ++ Healthy population A
Differentiation Patients
Apoptosis
COX-2
Prevention of precancerous lesions Aberrant crypt foci +++ ++ Healthy population B
Polyps High risk persons
Patients
Prevention of tumours Tumours in animals +++ – Healthy population B*
High risk persons
Inhibition of tumour progression Angiogenesis ++ + Patients A
Metastasis
* Animal basis for claim but must have additional data linking animal data to human
J. Rafter et al. II/75
PASSCLAIM – Diet-related cancer
1. Corpet DE, Tache S (2002) Most effective
colon cancer chemopreventive
agents in rats: a systematic review of
aberrant crypt foci and tumour data,
ranked by potency.Nutr Cancer 43:1–21
2. Steele VE, Moon RC, Lubet RA, Grubbs
CJ, Reddy BS, Wargovich M, McCormick
DL, Pereira MA, Crowell JA,
Bagheri D (1994) Preclinical efficacy
evaluation of potential chemopreventive
agents in animal carcinogenesis
models: methods and results from the
NCI Chemoprevention Drug Development
Program. J Cell Biochem (Suppl)
20:32–54
3. Corpet DE, Pierre D (2003) Point: From
animal models to prevention of colon
cancer.Systematic review of chemoprevention
in minmice and choice of the
model system. Cancer Epidemiol Biomarkers
Prev 12:391–400
4. Bird RP (1987) Observation and quantification
of aberrant crypts in the
murine colon treated with a colon carcinogen:
preliminary findings. Cancer
Lett 37:147–151
5. Roncucci L, Pedroni M, Vaccina F, Benatti
P, Marzona L, De Pol A (2000)
Aberrant crypt foci in colorectal carcinogenesis.
Cell and crypt dynamics.
Cell Prolif 33:1–18
6. Bird RP, McLellan EA, Bruce WR (1989)
Aberrant crypts, putative precancerous
lesions,in the study of the role of diet in
the aetiology of colon cancer. Cancer
Surv 8:189–200
7. Corpet DE, Tache S, Peiffer G (1997)
Colon tumour promotion: is it a selective
process? Effects of cholate, phytate,
and food restriction in rats on proliferation
and apoptosis in normal and
aberrant crypts. Cancer Lett 114:
135–138
8. Wargovich MJ, Jimenez A, McKee K,
Steele VE, Velasco M, Woods J, Price R,
Gray K, Kelloff GJ (2000) Efficacy of
potential chemopreventive agents on
rat colon aberrant crypt formation
and progression. Carcinogenesis 21:
1149–1155
9. Roncucci L,Stamp D,Medline A,Cullen
JB, Bruce WR (1991) Identification and
quantification of aberrant crypt foci
and microadenomas in the human
colon. Hum Pathol 22:287–294
10. Modica S, Roncucci L, Benatti P, Gafa L,
Tamassia MG, Dardanoni L, Ponz dL
(1995) Familial aggregation of tumours
and detection of hereditary non-polyposis
colorectal cancer in 3-year experience
of 2 population-based colorectalcancer
registries. Int J Cancer 62:
685–690
11. Narisawa T,FukauraY,Hasebe M,Ito M,
Aizawa R, Murakoshi M, Uemura S,
Khachik F, Nishino H (1996) Inhibitory
effects of natural carotenoids, alphacarotene,
beta-carotene, lycopene and
lutein, on colonic aberrant crypt foci
formation in rats. Cancer Lett 107:
137–142
12. Latham P, Lund EK, Johnson IT (1999)
Dietary n-3PUFA increases the apoptotic
response to 1.2- dimethylhydrazine,reduces
mitosis and suppresses
the induction of carcinogenesis in the
rat colon. Carcinogenesis 20:645–650
13. Rao CV, Hirose Y, Indranie C, Reddy BS
(2001) Modulation of experimental
colon tumorigenesis by types and
amounts of dietary fatty acids. Cancer
Res 61:1927–1933
14. Verghese M, Rao DR, Chawan CB
(2001) Dietary inulin suppresses
azoxymethane-induced preneoplastic
aberrant crypt foci and colon tumours
in Fisher 344 rats. J Nutr 132
(9):2809–2813
15. Dolara P, Caderni G, Lancioni L, GianniniA,AnastasiA,Fazi
M,Castiglione G
(1997) Aberrant crypt foci in human
colon carcinogenesis. Cancer Detect
Prev 21:141–147
16. Takayama T,Katsuki S,TakahashiY,Ohi
M, Nojiri S, Sakamaki S, Kato J, Kogawa
K, Miyake H, Niitsu Y (1998) Aberrant
crypt foci of the colon as precursors of
adenoma and cancer.N Engl J Med
339:1277–1284
17. Adler DG, Gostout CJ, Sorbi D, Burgart
LJ, Wang L, Harmsen WS (2002) Endoscopic
identification and quantification
of aberrant crypt foci in the human
colon. Gastrointest Endosc 56:657–662
18. Konstantakos AK, Siu IM, Pretlow TG,
Stellato TA, Pretlow TP (1996) Human
aberrant crypt foci with carcinoma in
situ from a patient with sporadic colon
cancer. Gastroenterology 111:772–777
19. Magnuson BA, Carr I, Bird RP (1993)
Ability of aberrant crypt foci characteristics
to predict colonic tumour incidence
in rats fed cholic acid.Cancer Res
53:4499–4504
20. YamadaY,Mori H (2003) Pre-cancerous
lesions for colon colorectal cancers in
rodents: a new concept. Carcinogenesis
24:1015–1019
21. Caderni G, Femia AP, Giannini A,
Favuzza A, Luceri C, Salvadori M,
Dolara P (2003) Identification of
mucin-depleted foci in the unsectioned
colon of azoxymethane-treated rats:
correlation with carcinogenesis.Cancer
Res 63:2388–2392
22. Moroson BC, Busey HJR, Day DW, Hill
MJ (1983)Adenomas of the large bowel.
Cancer Surv 2:451–477
23. Neugut AI, Jacobson JS, De VI (1993)
Epidemiology of colorectal adenomatous
polyps. Cancer Epidemiol Biomarkers
Prev 2:159–176
24. Gilbertsen VA, Nelms JM (1978) The
prevention of invasive cancer of the rectum.
Cancer 41:1137–1139
25. Stryker SJ, Wolff BG, Culp CE, Libbe
SD, Ilstrup DM, MacCarty RL (1987)
Natural history of untreated colonic
polyps. Gastroenterology 93:1009–1013
26. Radice P, Cama A, Mariani-Constantini
R (1996) Molecular genetics of polyposis
and herediatry colorectal cancer.Forum
Trends Exp Clin Med 6:275–291
27. Baron JA,Beach M,Mandel JS,van Stolk
RU, Haile RW, Sandler RS, Rothstein R,
Summers RW, Snover DC, Beck GJ,
Frankl H, Pearson L, Bond JH, Greenberg
ER (1999) Calcium supplements
and colorectal adenomas. Polyp Prevention
Study Group.Ann N Y Acad Sci
889:138–145
28. Bonithon-Kopp C,Kronborg O,Giacosa
A, Rath U, Faivre J (2000) Calcium and
fibre supplementation in prevention of
colorectal adenoma recurrence: a randomised
intervention trial. European
Cancer Prevention Organisation Study
Group. Lancet 356:1300–1306
29. Alberts DS, Martinez ME, Roe DJ,
Guillen-Rodriguez JM,Marshall JR,van
Leeuwen JB, Reid ME, Ritenbaugh C,
Vargas PA, Bhattacharyya AB, Earnest
DL, Sampliner RE (2000) Lack of effect
of a high-fiber cereal supplement on the
recurrence of colorectal adenomas.
Phoenix Colon Cancer Prevention
Physicians’ Network.N Engl J Med 342:
1156–1162
30. Schatzkin A, Lanza E, Corle D, Lance P,
Iber F,Caan B,Shike M,Weissfeld J,Burt
R, Cooper MR, Kikendall JW, Cahill J
(2000) Lack of effect of a low-fat, highfiber
diet on the recurrence of colorectal
adenomas. Polyp Prevention Trial
Study Group. N Engl J Med 342:
1149–1155
31. Arminskitc TC, McLean DW (1964) Incidence
and distribution of adenomatous
polyps of the colon and rectum
based on 1,000 autopsy examinations.
Dis Colon Rectum 7:249–261
32. Duffy MJ (2001) Clinical uses of tumour
markers: a critical review. Crit
Rev Clin Lab Sci 38:225–262
33. Konety BR,Getzenberg RH (2002)Vitamin
D and prostate cancer. Urol Clin
North Am 29:95–106, ix
34. Giovannucci E (2001) Insulin, insulinlike
growth factors and colon cancer: a
review of the evidence. J Nutr 131:
3109S–3120S
35. Berger DH (2002) Plasmin/plasminogen
system in colorectal cancer. World.
J Surg 26:767–771
References
II/76 European Journal of Nutrition (2004) Vol. 43, Supplement 2
36. Clarke LE, Leitzel K, Smith J, Ali SM,
Lipton A (2003) Epidermal growth factor
receptor mRNA in peripheral blood
of patients with pancreatic, lung, and
colon carcinomas detected by RT-PCR.
Int J Oncol 22:425–430
37. McLeod HL, Murray GI (1999) Tumour
markers of prognosis in colorectal cancer.
Br J Cancer 79:191–203
38. Hainaut P, Hollstein M (2000) p53 and
human cancer: the first ten thousand
mutations.Adv Cancer Res 77:81–137
39. Hartwell LH,Kastan MB (1994) Cell cycle
control and cancer. Science 266:
1821–1828
40. Bostick RM, Fosdick L, Grandits GA,
Lillemoe TJ, Wood JR, Grambsch P,
Louis TA, Potter JD (1997) Colorectal
epithelial cell proliferative kinetics and
risk factors for colon cancer in sporadic
adenoma patients. Cancer Epidemiol
Biomarkers Prev 6:1011–1019
41. Kelloff GJ, Boone CW, Steele VE, Crowell
JA,Lubet RA,Greenwald P,Hawk ET,
Fay JR, Sigman CC (1996) Mechanistic
considerations in the evaluation of
chemopreventive data. IARC Sci Publ
139:203–219
42. Milner JA, McDonald SS,Anderson DE,
Greenwald P (2001) Molecular targets
for nutrients involved with cancer prevention.
Nutr Cancer 41:1–16
43. Duchrow M,Gerdes J,Schluter C (1994)
The proliferation-associated Ki-67 protein:
definition in molecular terms.Cell
Prolif 27:235–242
44. Bravo R,Frank R,Blundell PA,Macdonald-Bravo
H (1987) Cyclin/PCNA is the
auxiliary protein of DNA polymerasedelta.
Nature 326:515–517
45. Liu SC, Klein-Szanto AJ (2000) Markers
of proliferation in normal and leukoplakic
oral epithelia. Oral Oncol 36:
145–151
46. Renehan AG, O’Dwyer ST, Haboubi NJ,
Potten CS (2002) Early cellular events in
colorectal carcinogenesis. Colorectal
disease 4:76–89
47. Kerr JF, Winterford CM, Harmon BV
(1994) Apoptosis. Its significance in
cancer and cancer therapy. Cancer 73:
2013–2026
48. Baker SJ, Reddy EP (1996) Transducers
of life and death: TNF receptor superfamily
and associated proteins. Oncogene
12:1–9
49. Kerr JF, Wyllie AH, Currie AR (1972)
Apoptosis: a basic biological phenomenon
with wide-ranging implications in
tissue kinetics. Br J Cancer 26:239–257
50. Johnson IT (2002) Anticarcinogenic effects
of diet-related apoptosis in the
colorectal mucosa. Food Chem Toxicol
40:1171–1178
51. Lowe SW, Lin AW (2000) Apoptosis in
cancer. Carcinogenesis 21:485–495
52. Butler LM, Hewett PJ, Fitridge RA,
Cowled PA (1999) Deregulation of
apoptosis in colorectal carcinoma: theoretical
and therapeutic implications.
Aust N Z J Surg 69:88–94
53. Gavrieli Y, Sherman Y, Ben-Sasson SA
(1992) Identification of programmed
cell death in situ via specific labeling of
nuclear DNA fragmentation. J Cell Biol
119:493–501
54. van Engeland M,Nieland LJ,Ramaekers
FC, Schutte B, Reutelingsperger CP
(1998) Annexin V-affinity assay: a review
on an apoptosis detection system
based on phosphatidylserine exposure.
Cytometry 31:1–9
55. Hofstra L, Liem IH, Dumont EA,
Boersma HH, van Heerde WL, Doevendans
PA, De Muinck E, Wellens HJ, Kemerink
GJ, Reutelingsperger CP, Heidendal
GA (2000) Visualisation of cell
death in vivo in patients with acute myocardial
infarction. Lancet 356:209–212
56. Riss TL (2001) Apoptosis as a biomarker
in chemoprevention trials.
Urology 57:141–142
57. Batth BK, Tripathi R, Srinivas UK
(2001) Curcumin-induced differentiation
of mouse embryonal carcinoma
PCC4 cells. Differentiation 68:133–140
58. Shamsuddin (2002) Anti-cancer function
of phytic acid. Int J Food Sci Technol
37:769–782
59. Sankaranarayanan R, Mathew B (1996)
Retinoids as cancer-preventive agents.
IARC Sci Publ 139:47–59
60. Osborne JE, Hutchinson PE (2002) Vitamin
D and systemic cancer: is this relevant
to malignant melanoma? Br J
Dermatol 147:197–213
61. Borghi R,Vene R, Arena G, Schubert D,
Albini A, Tosetti F (2003) Transient
modulation of cytoplasmic and nuclear
retinoid receptors expression in differentiating
human teratocarcinoma NT2
cells. J Neurochem 84:94–104
62. You H,YuW,Sanders BG,Kline K (2001)
RRR-alpha-tocopheryl succinate induces
MDA-MB-435 and MCF-7 human
breast cancer cells to undergo differentiation.
Cell Growth Differ 12:471–480
63. Smith WL, Dewitt DL (1996) Prostaglandin
endoperoxide H synthases-1
and –2.Adv Immunol 62:167–215
64. Dannenberg AJ, Altorki NK, Boyle JO,
Dang C,Howe LR,Weksler BB,Subbaramaiah
K (2001) Cyclo-oxygenase 2: a
pharmacological target for the prevention
of cancer. Lancet Oncol 2:544–551
65. Liu CH, Chang SH, Narko K, Trifan OC,
Wu MT, Smith E, Haudenschild C, Lane
TF, Hla T (2001) Overexpression of cyclooxygenase-2
is sufficient to induce
tumorigenesis in transgenic mice.J Biol
Chem 276:18563–18569
66. Oshima M, Dinchuk JE, Kargman SL,
Oshima H, Hancock B, Kwong E, Trzaskos
JM, Evans JF, Taketo MM (1996)
Suppression of intestinal polyposis in
Apc delta716 knockout mice by inhibition
of cyclooxygenase 2 (COX-2). Cell
87:803–809
67. Tiano HF, Loftin CD,Akunda J, Lee CA,
Spalding J, Sessoms A, Dunson DB, Rogan
EG, Morham SG, Smart RC, Langenbach
R (2002) Deficiency of either
cyclooxygenase (COX)-1 or COX-2 alters
epidermal differentiation and reduces
mouse skin tumorigenesis. Cancer
Res 62:3395–3401
68. Thun MJ (1996) NSAID use and decreased
risk of gastrointestinal cancers.
Gastroenterol Clin North Am 25:
333–348
69. Norrish AE, Jackson RT, McRae CU
(1998) Non-steroidal anti-inflammatory
drugs and prostate cancer progression.
Int J Cancer 77:511–515
70. Madaan S, Abel PD, Chaudhary KS,
Hewitt R, Stott MA, Stamp GW, Lalani
EN (2000) Cytoplasmic induction and
over-expression of cyclooxygenase-2 in
human prostate cancer: implications
for prevention and treatment. BJU Int
86:736–741
71. Mallett AK, Rowland IR (1990) Bacterial
enzymes: their role in the formation
of mutagens and carcinogens in the intestine.
Digestive Diseases 8:71–79
72. Goldin BR, Swenson L, Dwyer J, Sexton
M,Gorbach SL (1980) Effect of diet and
Lactobacillus acidophilus supplements
on human fecal bacterial enzymes.
Journal of the National Cancer Institute
64:255–261
73. Rowland IR, Rumney CJ, Coutts JT,
Lievense LC (1998) Effect of Bifidobacterium
longum and inulin on gut bacterial
metabolism and carcinogen-induced
aberrant crypt foci in rats.
Carcinogenesis 19:281–285
74. SaitoY,Takano T,Rowland IR (1992) Effects
of soybean oligosaccharides on
the human gut microflora in in vitro
culture. Microbial Ecology in Health
and Disease 5:105–110
75. Takada H, Hirooka T, Hiramatsu Y,
Yamamoto M (1982) Effect of betaglucuronidase
inhibitor on azoxymethane-induced
colonic carcinogenesis
in rats. Cancer Res 42:331–334
76. Kim DH,JinYH (2001) Intestinal bacterial
beta-glucuronidase activity of patients
with colon cancer. Arch Pharm
Res 24:564–567
77. Reddy BS, Mangat S, Weisburger JH,
Wynder EL (1977) Effect of high-risk
diets for colon carcinogenesis on intestinal
mucosal and bacterial beta-glucuronidase
activity in F344 rats.Cancer
Research 37:3533–3536
J. Rafter et al. II/77
PASSCLAIM – Diet-related cancer
78. Gestel G, Besancon P, Rouanet JM
(1994) Comparative evaluation of the
effects of two different forms of dietary
fibre (rice bran vs. wheat bran) on rat
colonic mucosa and faecal microflora.
Ann Nutr and Metabolism 38:249–256
79. Maziere S,Meflah K,Tavan E,Champ M,
Narbonne JF,Cassand P (1998) Effect of
resistant starch and/or fat-soluble vitamins
A and E on the initiation stage of
aberrant crypts in rat colon. Nutrition
and Cancer 31:168–177
80. Rao CV,Newmark HL,Reddy BS (1998)
Chemopreventive effect of squalene
on colon cancer. Carcinogenesis 19:
287–290
81. Massey RC, Key PE, Mallett AK, Rowland
IR (1988) An investigation of the
endogenous formation of apparent total
N-nitroso compounds in conventional
microflora and germ free rats.
Food Chemistry and Toxicology 26:
595–600
82. Hughes R (1999) The effects of diet on
colonic N-nitrosation and biomarkers
of DNA damage. University of Cam-
bridge
83. Hughes R, Cross AJ, Pollock JR, Bingham
S (2001) Dose-dependent effect of
dietary meat on endogenous colonic Nnitrosation.
Carcinogenesis 22:199–202
84. Silvester KR, Bingham SA, Pollock JRA,
Cummings JH, ONeill IK (1997) Effect
of meat and resistant starch on fecal extraction
of apparent N-nitroso compounds
and ammonia from the human
large bowel. Nutrition and Cancer 29:
13–23
85. Cummings JH,Beatty ER,Kingman SM,
Bingham SA, Englyst HN (1996) Digestion
and physiological properties of resistant
starch in the human large bowel.
Br J Nutr 75:733–747
86. Rowland IR, Granli T, Bockman OC,
Key PE, Massey RC (1991) Endogenous
N-nitrosation in man assessed by measurement
of apparent total N-nitroso
compounds in faeces. Carcinogenesis
12:1395–1401
87. MacDonald IA,BokkenheuserVD,Winter
J (1993) Degradation of steriods in
the human gut. J Lipid Res 24:675–700
88. Gill CIR, Rowland IR (2002) Diet and
cancer, assessing the risk. Br J Nutr
88:S73–S87
89. Imray CH, Radley S, Davis A, Barker G,
Hendrickse CW, Donovan IA, Lawson
AM, Baker PR, Neoptolemos JP (1992)
Faecal unconjugated bile acids in patients
with colorectal cancer or polyps.
Gut 33:1239–1245
90. Stadler J,Yeung KS,Furrer R,Marcon N,
Himal HS, Bruce WR (1988) Proliferative
activity of rectal mucosa and soluble
fecal bile acids in patients with
normal colons and in patients with
colonic polyps or cancer. Cancer Lett
38:315–320
91. Breuer NF, Dommes P, Jaekel S, Goebell
H (1985) Fecal bile acid excretion
pattern in colonic cancer patients.Dig
Dis Sci 30:852–859
92. Breuer NF, Jaekel S, Dommes P, Goebell
H (1986) Fecal bile acids in patients
with adenomatous polyps of the
colon. Case-control study. Digestion
34:87–92
93. Owen RW, Dodo M, Thompson MH,
Hill MJ (1987) Fecal steroids and colorectal
cancer. Nutr Canc 9:73–80
94. Kamano T,MikamiY,Kurasawa T,Tsurumaru
M, Matsumoto M, Kano M,
Motegi K (1999) Ratio of primary and
secondary bile acids in feces: possible
marker for colorectal cancer? Diseases
of the Colon and Rectum 42:668–672
95. de Kok T, Van Faassen A, Glinghammar
B, Pachen D, Rafter JJ, Baeten C,
Engels L, Kleinjans JCS (1999) Bile
acid concentrations, cytotoxicity, and
pH of fecal water from patients with
colorectal adenomas. Digestive Diseases
and Sciences 44:2218–2225
96. Thompson MH, Owen RW, Hill MJ,
Cummings JH (1985) Factors affecting
faecal bile acid concentrations: effect
of fat and fibre.Biochemical Soc Trans
13:392
97. Cummings JH, Pomare EW, Branch
WJ,Naylor CPE,Macfarlane GT (1987)
Short chain fatty acids in human large
intestine, portal, hepatic and venous
blood. Gut28:1221–1227
98. Roediger WEW (1982) Utilization of
nutrients by isolated epithelial cells of
the rat colon. Gastroenterology 83:
424–429
99. Salminen S, Bouley C, Boutron-Ruault
M-C, Cummings JH, Franck A, Gibson
GG, Isolauri E, Moreau MC, Roberfroid
MB, Rowland IR (1998) Functional
food science and gastrointestinal
physiology and function. Br J Nutr
80:147–171
100. Hague A, Paraskeva C (1995) The
short-chain fatty acid butyrate induces
apoptosis in colorectal tumour
cell lines. Eur J Cancer Prevention
4:359–364
101. Thornton JR (1981) High colonic pH
promotes colorectal cancer. Lancet
1:1081–1083
102. Van Dokkum W, De Boer BC, Van
Faassen A, Pikaar NA, Hermus RJ
(1983) Diet, faecal pH and colorectal
cancer. British Journal of Cancer 48:
109–110
103. Edwards CA, Rowland IR (1991) Bacterial
fermentation in the colon and its
measurement In: Dietary Fibre. A
component of Food,Nutritional Function
in Health and Disease. ILSI Eu-
rope
104. Kobayashi H, Fleming SE (2001) The
source of dietary fiber influences –
short chain fatty acid production and
concentrations in large bowel. Boca
Raton: CRC Press
105. Rafter JJ, Child P,Anderson AM,Alder
R, Eng V, Bruce WR (1987) Cellular
toxicity of fecal water depends on diet.
Am J Clin Nutr 45:559–563
106. Lapre JA, DeVries HT,Van der Meer R
(1993) Cytotoxicity of fecal water is
dependent on the type of dietary fat
and is reduced by supplemental calcium
phosphate in rats. J Nutr 123:
578–585
107. Van der Meer R, Termont DS, DeVries
HT (1991) Differential effects of calcium
ions and calcium phosphate on
cytotoxicity of bile acids.Am J Physiol
260:142–147
108. Sesink AL, Termont DS, Kleibeuker
JH, Van Der MR (2000) Red meat and
colon cancer: dietary haem, but not
fat, has cytotoxic and hyperproliferative
effects on rat colonic epithelium.
Carcinogenesis 21:1909–1915
109. Sesink AL, Termont DS, Kleibeuker
JH, Van Der MR (1999) Red meat and
colon cancer: the cytotoxic and hyperproliferative
effects of dietary heme.
Cancer Res 59:5704–5709
110. Glinghammer B, Venturi M, Rowland
IR, Rafter J (1997) Shift from a dairy
product-rich to a dairy product-free
diet: influence on cytotoxicity and
genotoxicity of fecal water – potential
risk factors for colon cancer.Am J Clin
Nutr 66:1277–1282
111. Venturi M, Hambly RJ, Glinghammar
B, Rafter JJ, Rowland IR (1997) Genotoxic
activity in human faecal water
and the role of bile acids: a study using
the alkaline comet assay. Carcinogenesis
18:2353–2359
112. Vogelstein B,Fearon ER,Hamilton SR,
Kern SE, Preisinger AC, Leppert M,
Nakamura Y, White R, Smits AM, Bos
JL (1988) Genetic alterations during
colorectal-tumour development. N
Engl J Med 319:525–532
113. Rieger MA,Parlesak A,Pool-Zobel BL,
Rechkemmer G, Bode C (1999) A diet
high in fat and meat but low in dietary
fibre increases the genotoxic potential
of ‘faecal water’. Carcinogenesis 20:
2311–2316
114. Osswald K, Becker TW, Grimm M,
Jahreis G, Pool Zobel BL (2000) Interand
intra-individual variation of faecal
water – genotoxicity in human
colon cells. Mutation Research 472:
59–70
115. Ahlquist DA, Gilbert J (1996) Stool
markers for colorectal cancer screening.Future
considerations.Dig Dis Sci
14:132–44
II/78 European Journal of Nutrition (2004) Vol. 43, Supplement 2
116. Ahlquist DA, Shuber AP (2002) Stool
screening for colorectal cancer: evolution
from occult blood to molecular
markers. Clin Chim Act 315:157–68
117. Ahlquist DA, Skoletsky JE, Boynton
KA, Harrington JJ, Mahoney DW,
Pierceall WE (2000) Colorectal cancer
screening by detection of altered human
DNA in stool: Feasibility of a
multitarget assay system. Gastroenterol
119:1219–1227
118. Ruoslahti E (2002) Specialisation of
tumour vasculature. Nat Rev Cancer
2:83–90
119. Dormond O, Foletti A, Paroz C, Ruegg
C (2001) NSAIDs inhibit alpha V beta
3 integrin-mediated and Cdc42/Racdependent
endothelial-cell spreading,
migration and angiogenesis. Nat Med
7:1041–1047
120. Adlercreutz H, Fotsis T, Lampe JW,
etal. (1993) Quantitative determination
of lignans and isoflavonoids in
plasma of omnivorous and vegetarian
women by isotope dilution gaschromatography
mass-spectrometry.
Scand J Clin Lab Invest 53:5–18
121. Masso-Welch PA, Zangani D, Ip C,
Vaughan MM, Shoemaker S, Ramirez
RA,Ip MM (2002) Inhibition of angiogenesis
by the cancer chemopreventive
agent conjugated linoleic acid.
Cancer Res 62:4383–4389
122. Fotsis T, Pepper M, Adlercreutz H,
Fleischmann G, Hase T, Montesano R,
Schweigerer L (1993) Genistein, a dietary-derived
inhibitor of in vitro angiogenesis.
Proc Natl Acad Sci USA
90:2690–2694
123. Minagawa N, Nakayama Y, Hirata K,
Onitsuka K, Inoue Y, Nagata N, Itoh H
(2002) Correlation of plasma level and
immunohistochemical expression of
vascular endothelial growth factor in
patients with advanced colorectal cancer.Anticancer
Res 22:2957–2963
124. Tamura M, Ohta Y, Nakamura H, Oda
M, Watanabe G (2002) Diagnostic
value of plasma vascular endothelial
growth factor as a tumour marker in
patients with non-small cell lung cancer.
Int J Biol Markers 17:275–279
125. Choi JH, Ahn MJ, Jang SJ, Park CK,
Park YW, Oh HS, Lee YY, Choi IY, Kim
IS (2002) Absence of clinical prognostic
value of vascular endothelial
growth factor and microvessel density
in multiple myeloma. Int J Hematol
76:460–464
126. Egeblad M, Werb Z (2002) New functions
for the matrix metalloproteinases
in cancer progression. Nat
Rev Cancer 2:161–174
127. McCabe A, Wallace J, Gilmore WS,
Strain JJ, McGlynn H (1999) The effects
of eicosopentanoic acid on matrix
metalloproteinase gene expression.
Lipids 34:217–218
128. Shaker MR, Yang G, Timme TL, Park
SH,Kadmon D,Ren CZ,Ji XR,Lee HM,
Sehgal I, Anzano M, Sporn MB,
Thompson TC (2001) Dietary 4-HPR
suppresses the development of bone
metastasis in vivo in a mouse model of
prostate cancer progression. Clin Exper
Metastasis 18:429–438
129. Nangia-Makker P, Hogan V, Honjo Y,
Baccarini S, Tait L, Bresalier R, Raz A
(2002) Inhibition of human cancer cell
growth and metastasis in nude mice
by oral intake of modified citrus
pectin. J Natl Cancer Inst 94:
1854–1862
130. McIntosh GH, Royle PJ, Playne MJ
(1999) A probiotic strain of L acidophilus
reduces DMH-induced large
intestinal tumours in male SpragueDawley
rats. Nutr Cancer 35:153–159
131. Tsunezuka Y, Kinoh H, Takino T,
Watanabe Y, Okada Y, Shinagawa A,
Sato H, Seiki M (1996) Expression of
membrane-type matrix metalloproteinase
1 (MT1-MMP) in tumour cells
enhances pulmonary metastasis in an
experimental metastasis assay. Cancer
Res 56:5678–5683
132. Hua J, Muschel RJ (1996) Inhibition of
matrix metalloproteinase 9 expression
by a ribozyme blocks metastasis in a
rat sarcoma model system.Cancer Res
56:5279–5284
133. Dyce M, Sharif SF, Whalen GF (1992)
Search for anti-metastatic therapy: effects
of phenytoin on B16 melanoma
metastasis. J Surg Oncol 49:107–112
134. Xing RH, Rabbani SA (1999) Regulation
of urokinase production by androgens
in human prostate cancer
cells: effect on tumour growth and
metastases in vivo. Endocrinology
140:4056–4064
135. Arlt M, Kopitz C, Pennington C, Watson
KL,Krell HW,BodeW,Gansbacher
B, Khokha R, Edwards DR, Kruger A
(2002) Increase in gelatinase-specificity
of matrix metalloproteinase inhibitors
correlates with antimetastatic
efficacy in a T-cell lymphoma model.
Cancer Res 62:5543–5550
136. Leather AJ, Gallegos NC, Kocjan G,
Savage F, Smales CS, Hu W, Boulos PB,
Northover JM, Phillips RK (1993) Detection
and enumeration of circulating
tumour cells in colorectal cancer.
Br J Surg 80:777–780
137. Lindemann F,Schlimok G,Dirschedl P,
Witte J,Riethmuller G (1992) Prognostic
significance of micrometastatic
tumour cells in bone marrow of colorectal
cancer patients. Lancet 340:
685–689
138. Moreno JG,O’Hara SM,Gross S,Doyle
G, Fritsche H, Gomella LG, Terstappen
LW (2001) Changes in circulating carcinoma
cells in patients with metastatic
prostate cancer correlate with disease
status. Urology 58:386–392
139. Mayhew E, Glaves D (1984) Quantitation
of tumorigenic disseminating
and arrested cancer cells. Br J Cancer
50:159–166
140. Molnar B,LadanyiA,Tanko L,Sreter L,
Tulassay Z (2001) Circulating tumour
cell clusters in the peripheral blood of
colorectal cancer patients.Clin Cancer
Res 7:4080–4085
141. Fehm T, Sagalowsky A, Clifford E,
Beitsch P,Saboorian H,Euhus D,Meng
S, Morrison L, Tucker T, Lane N,
Ghadimi BM, Heselmeyer-Haddad K,
Ried T, Rao C, Uhr J (2002) Cytogenetic
evidence that circulating epithelial
cells in patients with carcinoma
are malignant. Clin Cancer Res 8:
2073–2084
142. Wattenberg LW (1992) Inhibition of
carcinogenesis by minor dietary
constituents. Cancer Res 52 (Suppl):
2085s–2091s
143. Zhang BC, etal. (1997) Oltipraz
Chemoprevention Trial in Qidong,
Jiangsu Province, People’s Republic of
China.J Cellular Biochemistry Supplements
28/29:166–173
144. Kensler TW, Helzlsouer KJ (1995)
Oltipraz: Clinical opportunities for
cancer chemoprevention. J Cell
Biochem 22 (Suppl):101–107
145. Wang JS, Shen X, He X, Zhu YR,Zhang
BC,Wang JB,Qian GS,Kuang SY,Zarba
A, Egner PA, Jacobson LP, Munoz A,
Helzlsouer KJ, Groopman JD, Kensler
TW (1999) Protective alterations in
phase 1 and 2 metabolism of aflatoxin
B1 by oltipraz in residents of Qidong,
People’s Republic of China.J Natl Cancer
Inst 91:347–354
146. deBethizy JD, Hayes JR (1994) Metabolism,
a determinant of toxicity. In:
Hayes AW (ed) Principles and methods
of toxicology, 3 edn. Raven Press
Ltd., New York, pp 59–100
147. Sivaraman L, Leatham MP, Yee J,
Wilkens LR, Lau AF, Marchand LL
(1994) CYP1A1 genetic polymorphisms
and in situ colorectal cancer.
Cancer Res 54:5692–5695
148. Smith G, Stanley LA, Sim E, Strange
RC, Wolf CR (1995) Metabolic polymorphisms
and cancer susceptibility.
In: Ponder H, Bruce AJ (eds) Genetics
and cancer,a second look.Cold Spring
Harbor Laboratory Press, New York,
pp 27–65
149. Bartsch H, Hietanen E (1996) The role
of individual susceptibility in cancer
burden related to environmental exposure.
Environ Health Perspect 104:
569–577
J. Rafter et al. II/79
PASSCLAIM – Diet-related cancer
150. Sachse C,Smith G,Wilkie MJV,Barrett
JH, Waxman R, Sullivan F, Forman D,
Bishop DT, Wolfl CR, The Colon Cancer
Study Group (2003) A pharmacogenetic
study to investigate the role of
dietary carcinogens in the etiology of
colorectal cancer. Carcinogenesis 23:
1839–1849
151. Guengerich FP (2000) Metabolism of
chemical carcinogens. Carcinogenesis
21:345
152. Kaderlik KR, Minchin RF, Mulder GJ,
Ilett KF, Daugaard-Jenson M, Teitel
CH, Kadlubar FF (1994) Metabolic activation
pathway for the formation of
DNA adducts of the carcinogen 2amino-1-methyl-6-phenylimidazo
[4,
5-b]pyridine (PhIP) in rat extrahepatic
tissues. Carcinogenesis 15:
1703–1709
153. Murray BP,Edwards RJ,Murray S,Singleton
AM, Davies DS, Boobis AR
(1993) Human hepatic CYP1A1 and
CYP1A2 content, determined with
specific anti-peptide antibodies, correlates
with the mutagenic activation
of PhIP. Carcinogenesis 14:585–592
154. Turesky RJ (2002) Heterocyclic aromatic
amine metabolism,DNA adduct
formation, mutagenesis, and carcinogenesis.
Drug Metabolism Reviews
34:625–650
155. Palli D, Masala G, Vineis P, Garte S,
Saieva C,KroghV,Panico S,Tumino R,
Munnia A, Riboli E, Peluso M (2003)
Biomarkers of dietary intake of micronutrients
modulate DNA adduct
levels in healthy adults. Carcinogenesis
24:739
156. World Cancer Research Fund, American
Institute for Cancer Research
(1997) Food,nutrition and the prevention
of cancer: a Global perspective.
American Institute for Cancer Research,Washington
DC
157. Lampe JW, Peterson S (2002) Brassica,
biotransformation and cancer risk:
genetic polymorphisms alter the preventive
effects of cruciferous vegetables.
J Nutr 132:2991–2994
158. Murray S, Lake BG, Gray S, Edwards
AJ, Springall C, Bowey Ea, Williamson
G, Boobis AR, Gooderham NJ (2001)
Effect of cruciferous vegetable consumption
on heterocyclic aromatic
amine metabolism in man. Carcinogenesis
22:1413–1420
159. van Poppel G,Verhoeven DT,Verhagen
H, Goldbohm RA (1999) Brassica vegetables
and cancer prevention. Epidemiology
and mechanisms. Adv Exp
Med Biol 472:159–168
160. Seow A, Yuan JM, Sun CL, Van Den
Berg D, Lee HP, Yu MC (2002) Dietary
isothiocyanates, glutathione S-transferase
polymorphisms and colorectal
cancer risk in the Singapore Chinese
Health Study. Carcinogenesis 23:
2055–2061
161. Johnson IT (2002) Glucosinolates:
bioavailability and importance to
health. Int J Vitamins and Nutr Res
72:26–31
162. Slattery ML, Kampman E, Samowitz
W, Caan B, Potter JD (2000) Interplay
between dietary inducers of GST and
the GSTM-1 genotype in colon cancer.
Int J Cancer 87:728–733
163. Lampe JW,Chen C,Li S,Prunty J,Grate
MT, Meehan DE, Barale KV, Dightman
Da, Feng ZPJD (2000) Modulation of
human glutathione S-transferases by
botanically defined vegetable diets.
CancerEpidemiol.Biomarkers Prev 9:
787–793
164. Simmons PT, Portier CJ (2002) Toxicogenomics:
the new frontier in risk
analysis. Carcinogenesis 23:903
165. Roses AD (2000) Pharmacogenetics
and the practice of medicine. Nature
405:857–865
166. Hayes JD, Pulford DJ (1995) The glutathione
S-transferase supergene family.
regulation of GST* and the contribution
of the isoenzymes to cancer
chemoprotection and drug resistance.
Critical Reviews in Biochemistry and
Molecular Biology 30:445–460
167. Armstrong RN (1997) Structure, catalytic
mechanism, and evolution of
the glutathione transferases. Chem
Res Toxicol 10:2–18
168. Cerutti PA (1985) Prooxidant states
and tumour promotion. Science 227:
375–381
169. Hayes JD, Strange RC (1995) Potential
contribution of the glutathione Stransferase
supergene family to resistance
to oxidative stress. Free Rad Res
22:193–207
170. Berhane K,Widersten M, Engstrom A,
Kozarich JW, Mannervik B (1994)
Detoxication of base propenals and
other a,b-unsaturated aldehyde products
of radical reactions and lipid peroxidation
by humane glutathione
transferases. Proc Natl Acad Sci USA
91:1480–1484
171. Waleh NS, Calaoagan J, Murphy BJ,
Knapp AM,Sutherland RM,Laderoute
KR (1998) The redox-sensitive human
antioxidant responsive element induces
gene expresseion under low
oxygen conditions.Carcinogenesis 19:
1333–1337
172. Rushmore TH, Morton Mr, Pickett CB
(1991) The antioxidant responsive element.
J Biol Chem 18:11632–11639
173. Chen C, Yu R, Owuor ED, Kong AN
(2000) Activation of antioxidant-response
element (ARE), mitogen activated
protein kinases (MAPKs) and
caspases by major green tea polyphenol
components during cell survival
and death. Arch Pharm Res 23:
605–612
174. Pinkus R,Weinert LM,DanielV (1996)
Role of oxidants and antioxidants in
the induction of AP-1,NF-kB,and glutathione
S-transferase gene expression.
J Biol Chem 271:13422–13429
175. Desmots F, Rauch C, Henry C, Guillouzo
A, Morel F (1998) Genomic organization,
5’-flanking region and
chromosomal localization of the human
glutatione transferase A4 gene.
Biochem J 336:437–442
176. Whalen R,Boyer T (1998) Human glutathione
S-transferases. Seminars in
Liver Disease 18:345–358
177. Johnson IT, Williamson G, Musk SRR
(1994) Anticarcinogenic factors in
plant foods: A new class of nutrients?
Nutr Res Rev 7:175–204
178. Ebert MN, Beyer-Sehlmeyer G, Liegibel
UM, Kautenburger T, Becker TW,
Pool-Zobel BL (2001) Butyrate-induced
activation of glutathione Stransferases
protects human colon
cells from genetic damage by 4-Hydroxynonedienal.
Nutr Cancer 41:
156–164
179. Knoll N (2002) Modulation der GST
Aktivität in HT29Zellen und Konsequenzen
für die Genotoxizität von 4Hydroxynonena.
2002. FriedrichSchiller
University Jena
180. Katoh T, Nagata N, Kuroda Y, Itoh H,
Kawahara A, Kuroki N, Ookuma R,
Bell DA (1996) Glutathione S-transferase
M1 (GSTM1) and T1 (GSTT1)
genetic polymorphism and susceptibility
to gastric and colorectal
adenocarcinoma. Carcinogenesis 17:
1855–1859
181. de Jong MM,Nolte IM,te Meerman GJ,
van der Graaf WTA, de Vries EGE, Sijmons
RH, Hofstra RMW, Kleibeuker
JH (2002) Low-penetrance genes and
their involvement in colorectal cancer
susceptibility. Cancer Epidemiol Biomarkers
Prev 11:1332–1352
182. Brockton NT (2002) UGT1A1 polymorphisms
and colorectal cancer susceptibility.
Gut50:749
183. Ichiba M,Wang Y,Oishi H,Iyadomi M,
Shono N, Tomokuni K (1996) Smoking-related
DNA adducts and genetic
polymorphism for metabolic enzymes
in human lymphocytes. Biomarkers
1:211–214
184. Marchand LL,Hankin JH,Wilkens LR,
Pierce LM, Franke A, Kolonel LN,
Seifried A, Custer LJ, Chang W, LumJones
A, Donlon T (2002) Combined
effects of well-done red meat, smoking,
and rapid N-acetyltransferase 2
and CYP1A2 phenotypes in increasing
colorectal cancer risk.Canc Epid Biom
Prev 10:1259–1266
II/80 European Journal of Nutrition (2004) Vol. 43, Supplement 2
185. Hu X, Herzog C, Zimniak P, Singh
SV (1999) Differential protection
against benzo [a]pyrene-7.8-dihydrodiol-9.10-epoxide-induced
DNA damage
in HepG2 Cells stably transfected
with allelic variants of p class human
glutathone S-transferases. Cancer Res
59:2358–2362
186. Chou MW, Mikhailova MV, Nichols J,
Poirier LA, Warbritton A, Beland FA
(2000) Interactive effects of methyldeficiency
and dietary restriction on
liver cell proliferation and telomerase
activity in Fischer 344 rats pretreated
with aflatoxin B(1). Cancer Lett 152:
53–61
187. Primiano T, Sutter TR, Kensler TW
(1997) Antioxidant-inducible genes.
Adv Pharmacol 38:293–328
188. Stein J, Schröder O, Bonk M, Oremek
G, Lorenz M, Caspary WF (1996) Induction
of glutathione-S-transferasepi
by short-chain fatty acids in the intestinal
cell line Caco-2. Europ J Clin
Invest 26:84–87
189. Treptow-van Lishaut S, Rechkemmer
G, Rowland IR, Dolara P, Pool-Zobel
BL (1999) The carbohydrate crystalean
and colonic microflora modulate
expression of glutathione S-transferase
subunits in colon of rats. Eur J
Nutr 38:76–83
190. van Lieshout EMM, Bedaf MMG,
Pieter M, Ekkel C, Nijhoff WA, Peters
WHM (1998) Effects of dietary anticarcinogens
on rat gastrointestinal
glutathione S-transferase theta 1–1
levels. Carcinogenesis 19:2055–2057
191. Bogaards JJP,Verhagen H,Willems MI,
van Poppel G, van Bladeren PJ (1994)
Consumption of Brussels sprouts results
in elevated a class glutathione Stransferase
levels in human blood
plasma. Carcinogenesis 15:1073–1075
192. Kensler TW,Groopman JD,Wogan GN
(1996) Use of carcinogen-DNA and
carcinogen-protein adduct biomarkers
for cohort selection and as modifiable
end points in chemoprevention
trials. In: Steward BW, McGregor DB,
Kleihues P (eds) Principles of Chemoprevention.
International Agency for
Research on Cancer, Lyon, pp 237–248
193. Szarka CE,Pfeiffer GR,Hum ST,Everly
LC, Balshem AM, Moore DF, Litwin S,
Goosenberg EB, Frucht H, Engstrom
PF, Clapper ML (1995) Glutathione Stransferase
activity and glutathione Stransferase
m expression in subjects
with risk for colorectal cancer. Cancer
Res 55:2789–2793
194. Ebert MN, Klinder A, Schäferhenrich
A, Peters WHM, Sendt W, Scheele J,
Pool-Zobel BL (2003) Expression of
glutathione S-transferase (GST) in human
colon cells and inducibility of
GSTM2 by butyrate. Carcinogenesis
24:1637–1644
195. Morel F,SchulzWA,Sies H (1994) Gene
structure and regulation of expression
of human glutathione S-transferases
alpha. Biol Chem 375:641–649
196. Thier R,Taylor JB,Pemble EE,Griffeth
HW, Persmark M, Ketterer B, Guengerich
FP (1993) Expression of mammalian
glutathione S-transferase 5–5
in Salmonella typhimurium TA1535
leads to base-pair mutations upon exposure
to dihalomethanes. Proc Natl
Acad Sci USA90:8576–8580
197. Kim DJ, Han BS, Ahn B, Hasegawa R,
Shirai T, Ito N, Tsuda H (1997) Enhancement
by indole-3-carbinol of
liver and thyroid gland neoplastic development
in a rat medium-term multiorgan
carcinogenesis model. Carcinogenesis
18:377–381
198. Albertini RJ,Anderson D,Douglas GR,
Hagmar L, Hemminki K, Merlo F,
Natarajan AT, Norppa H, Shuker DEG,
Tice RR, Waters MD, Aitio A (2000)
IPCS guidelines for the monitoring of
genotoxic effects of carcinogens in humans.Mutation
Research 463:111–172
199. Verhagen H, Aruoma OI, van Delft
JHM, Dragsted LO, Ferguson LR,
Knasmüller S, Pool-Zobel BL, Poulsen
HE,Williamson G,Yannai S(2003) The
10 basic requirements for a scientific
paper reporting antioxidant, antimutagenic
or anticarcinogenic potential
of test substances in in vitro experiments
and animal studies in vivo.
Food Chem Toxicol 41:603–610
200. Ames BN,Wakimoto P (2002) Are vitamin
and mineral deficiencies a major
cancer risk? Nature Reviews Cancer
9:694–704
201. Pool-Zobel BL, Bub A, Müller H, Wollowski
I, Rechkemmer G (1997) Consumption
of vegetables reduces genetic
damage in humans: first results
of an intervention trial with carotenoid-rich
foods. Carcinogenesis 18:
1847–1850
202. Bub A, Watzl B, Blockhaus M, Briviba
K, Liegibel UM, Müller H, Pool-Zobel
BL, Rechkemmer G (2003) Fruit juice
consumption modulates antioxidative
status, immune status and DNA damage.
J Nutr Biochem 14:98
203. Fenech M, Stockley C,Aitken C (1997)
Moderate wine consumption protects
against hydrogen peroxide-induced
DNA damage. Mutagenesis 12:
289–296
204. Pool-Zobel BL, Münzner R, Holzapfel
WH (1993) Antigenotoxic properties
of lactic acid bacteria in the Salmonella
typhimurium mutagenicity assay.
Nutr Canc 20:261–270
205. Pool-Zobel BL, Neudecker C, Domizlaff
I, Ji S, Schillinger U, Rumney CJ,
Moretti M, Villarini M, ScassellatiSforzolini
G,Rowland IR (1996) Lactobacillus-
and Bifidobacterium-mediated
antigenotoxicity in colon cells of
rats: Prevention of carcinogen-induced
damage in vivo and elucidation
of involved mechanisms. Nutr Canc
26:365–380
206. Pool-Zobel BL, Lotzmann N, Knoll M,
Kuchenmeister F, Lambertz R, Leucht
U, Schröder HG, Schmezer P (1994)
Detection of DNA damage in gastric,
colonic and nasal mucosa cells derived
from the rat and from human biopsy
specimens. Environ Mol Mutagen 24:
23–45
207. Pool-Zobel BL,Abrahamse SL, Collins
AR, Kark W, Gugler R, Oberreuther D,
Siegel EG, Treptow-van Lishaut S,
Rechkemmer G (1999) Analysis of
DNA strand breaks, oxidized bases
and glutathione S-transferase P1 in
human colon cells. Canc Epid Biom
Prev 8:609–614
208. Schäferhenrich A, Sendt W, Scheele J,
Kuechler A, Liehr T, Claussen U, Rapp
A, Greulich KO, Pool-Zobel BL (2003)
Endogenously formed cancer risk factors
induce damage-of p53 in human
colon cells obtained from surgical
samples. Fd Chem Toxicol 41:655–664
209. Singh NP, Tice RR, Stephens RE,
Schneider EL (1991) A microgel electrophoresis
technique for the direct
quantitation of DNA damage and repair
in individual fibroblasts cultured
on microscope slides. Mutation Res
252:289–296
210. Sina JF,Bean CL,Dysart GR,TaylorVI,
Bradley MO (1983) Evaluation of the
alkaline elution/rat hepatocyte assay
as a predictor of carcinogenic/mutagenic
potential. Mutation Research
113:357–391
211. Pool BL, Brendler SY, Liegibel UM,
Tompa A, Schmezer P (1990) Employment
of adult mammalian primary
cells in toxicology: In vivo and in vitro
genotoxic effects of environmentally
significant N-nitrosodialkylamines in
cells of the liver, lung and kidney. Environm
Mol Mutagen 15:24–35
212. Hartmann A, Agurell E, Beevers C,
Brendler-Schwaab S,Burlinson B,Clay
P,Collins A,Smith A,Speit G,Thybaud
V, Tice RR (2003) Recommendations
for conducting the in vivo alkaline
Comet assay. Mutagenesis 18:45–51
213. Kassie F, Parzefall W, Knasmuller S
(2000) Single cell gel electrophoresis
assay: a new technique for human biomonitoring
studies. Mutat Res 463:
13–31
J. Rafter et al. II/81
PASSCLAIM – Diet-related cancer
214. Veglia F, Matullo G, Vineis P (2003)
Bulky DNA adducts and risk of cancer:
a meta-analysis. Cancer Epidemiol
Biomarkers Prev 12:157–160
215. Fang JL, Vaca CE (1997) Detection of
DNA adducts of acetaldehyde in peripheral
white blood cells of alcohol
abusers. Carcinogenesis 18:632
216. Schabath MB, Spitz MR, Grossman
HB,Zhang K,Dinney CP,Zheng PJ,Wu
X (2003) Genetic instability in bladder
cancer assessed by the Comet assay.
JNCI Cancer Spectrum 95:540
217. van Poppel G, Poulson HE, Loft S,Verhagen
H (1995) No influence of beta
carotene on oxidative DNA damage in
male smokers. J Natl Cancer Inst
87:310–311
218. Duthie SJ, Ma A, Ross MA, Collins AR
(1996) Antioxidant supplementation
decreases oxidative DNA damage in
human lymphocytes. Cancer Res 56:
1291–1295
219. Mitchell JH, Collins AR (1999) Effects
of a soy milk supplement on plasma
cholesterol levels and oxidative DNA
damage in men – a pilot study. Eur J
Nutr 38:143–148
220. Gedik CM, Boyle SP, Wood SG,
Vaughan NJ, Collins AR (2002) Oxidative
stress in humans: validation of
biomarkers of DNA damage. Carcinogenesis
23:1441–1446
221. Giovannelli L, Saieva C, Masala G,
Testa G, Salvini S, Pitozzi V, Riboli E,
Dolara P, Palli D (2002) Nutritional
and lifestyle determinants of DNA oxidative
damage: a study in a Mediterranean
population.Carcinogenesis 23:
1483–1489
222. Vulimiri SV, Smith CV, Randerath E,
Randerath K (1994) 32P-Postlabeling
of bile components: bulky adduct-like
behavior in polyethyleneimine-cellulose
thin layer chromatography. Carcinogenesis
15:2061–2064
223. Randerath K, Zhou GD, Monk SA,
Randerath E (1997) Enhanced levels in
neonatal rat liver of 7.8-dihydro-8oxo-2’deoxyguanosine
(8-hydroxydeoxyguanosine),
a major mutagenic
oxidative DNA lesion. Carcinogenesis
18:1419–1421
224. Friedberg EC (2001) How nucleotide
excision repair protects against cancer.
Nature Reviews Cancer 1:22–33
225. Hoeijmakers JHJ (2001) Genome
maintenance mechanisms for preventing
cancer. Nature 411:366–374
226. Margison GP, Povey AC, Kaina B, Santibanez
Koref MF (2003) Variability
and regulation of O6-alkylguanineDNA
alkyltransferase. Carcinogenesis
24:625
227. Hesketh R (1997) The Oncogene and
Tumour Suppressor Gene Facts Book,
2 edn
228. Fodde R, Smits R, Clevers H (2001)
APC signal transduction and genetic
instability in colorectal cancer. Nature
Reviews Cancer 1:55–67
229. Smerhovsky Z,Landa K,Rössner P,Juzova
D, Brabec M, Zudova Z, Hola N,
Zarska H, Nevsimalova E (2002) Increased
risk of cancer in radon-exposed
miners with elevated frequency
of chromosomal aberrations. Mutation
Res 514:176
230. Bonassi S, Hagmar L, Stromberg U,
Montagud AH,Tinnerberg H,Forni A,
Heikkila P, Wanders S, Wilhardt P,
Hansteen IL, Knudsen LE, Norppa H
(2000) Chromosomal aberrations in
lymphocytes predict human cancer
independently of exposure to carcinogens.
European Study Group on Cytogenetic
Biomarkers and Health. Cancer
Res 60:619–625
231. Ramsey MJ, Moore DH, Briner JF, Lee
DA,Olsen L,Senft JR,Tucker JD (1995)
The effects of age and lifestyle factors
on the accumulation of cytogenetic
damage as measured by chromosome
painting. Mutation Res 338:95–106
232. Fenech MF, Dreosti I, Aitken C (1997)
Vitamin-E supplements and their effect
on vitamin-E status in blood and
genetic damage rate in peripheral
blood lymphocytes. Carcinogenesis
18:359–364
233. Fenech MF,Dreosti I,Rinaldi JR (1997)
Folate,vitamin B12,homocysteine status
and chromosome damage rate in
lymphocytes of older men. Carcinogenesis
18:1329–1336
234. Zijno A, Andreoli C, Leopardi P, Marcon
F, Rossi S, Caiola S, Verdina A,
Galati R, Cafolla A, Crebelli R (2003)
Folate status,metabolic genotype,and
biomarkers of genotoxicity in healthy
subjects. Carcinogenesis
24:1097–1103
235. Fenech M, Aitken C, Rinaldi J (1998)
Folate, vitamin B12, homocysteine
status and DNA damage in young
Australian adults. Carcinogenesis 19:
1163–1171
236. Fenech M (2001) The role of folic acid
and Vitamin B12 in genomic stability
of human cells. Mutation Res 475:
57–67
237. Levine AJ, Salvan A, Talaska G,
Boeniger MF, Suruda A, Schulte PA
(1997) The utility of epithelial-cell
micronuclei in the assessment of intermittent
exposures. Biomarkers 2:
135–138
238. Desai SS, Ghaisas SD, Jakhi SD, Bhide
SV (1996) Cytogenetic damage in exfoliated
oral mucosal and circulating
lymphocytes of patients suffering
from precancerous oral lesions. Cancer
Lett 109:9–14
239. Schatzkin A, Gail M (2002) The
promise and peril of surrogate endpoints
in cancer research. Nature Reviews
Cancer 2:1–9
240. Yamasaki H, Omori Y, Zaidan-Dagli
ML, Mironov N, Mesnil M, Krutovskikh
V (1999) Genetic and epigenetic
changes of intercellular communication
genes during multistage
carcinogenesis.Cancer Detect Prev 23:
273–279
241. Chaumontet C, Bex V, GaillardSanchez
I, Seillan-Heberden C,
Suschetet M,Martel P (1994) Apigenin
and tangeretin enhance gap junctional
intercellular communication in
rat liver epithelial cells. Carcinogenesis
15:2325–2330
242. Bex V, Mercier T, Chaumontet C, Gaillard-Sanchez
I, Flechon B, Mazet F,
Traub O,Martel P (1995) Retinoic acid
enhances connexin43 expression at
the post- transcriptional level in rat
liver epithelial cells. Cell Biochem
Funct 13:69–77
243. Sies H, Stahl W (1997) Carotenoids
and intercellular communication via
gap junctions. Int J Vitam Nutr Res
67:364–367
244. Trosko JE, Chang CC (2001) Mechanism
of up-regulated gap junctional
intercellular communication during
chemoprevention and chemotherapy
of cancer. Mutat Res 480–481:219–229
245. Auvinen M, Paasinen A, Andersson
LC, Holtta E (1992) Ornithine decarboxylase
activity is critical for cell
transformation. Nature 360:355–358
246. Thomas T, Thomas TJ (2001) Polyamines
in cell growth and cell death:
molecular mechanisms and therapeutic
applications. Cell Mol Life Sci 58:
244–58
247. Tabib A, Bachrach U (1999) Role of
polyamines in mediating malignant
transformation and oncogene expression.
Int J Biochem Cell Biol 31:
1289–1295
248. Lavelle F, Riou JF, Laoui A, Mailliet P
(2000) Telomerase: a therapeutic target
for the third millennium? Crit Rev
Oncol Hematol 34:111–126
249. Ouellette MM, Lee K (2001) Telomerase:
diagnostics,cancer therapeutics
and tissue engineering. Drug Discov
Today 6:1231–1237
250. Schmidt PM, Lehmann C, Matthes E,
Bier FF (2002) Detection of activity of
telomerase in tumour cells using fiber
optical biosensors. Biosens Bioelectron
17:1081–1087
251. SteeleVE,Hawk ET,Viner JL,Lubet RA
(2003) Mechanisms and applications
of non-steroidal anti-inflammatory
drugs in the chemoprevention of cancer.
Mutat Res 523–524:137–44
II/82 European Journal of Nutrition (2004) Vol. 43, Supplement 2
252. Ding XZ, Hennig R, Adrian TE (2003)
Lipoxygenase and cyclooxygenase
metabolism: new insights in treatment
and chemoprevention of pancreatic
cancer. Mol Cancer 2:10
253. Li JK, Lin-Shia SY (2001) Mechanisms
of cancer chemoprevention by curcumin.
Proc Natl Sci Counc Repub
China B 25:59–66
254. de la Puerta R, Ruiz Gutierrez V, Hoult
JR (1999) Inhibition of leukocyte 5lipoxygenase
by phenolics from virgin
olive oil. Biochem Pharmacol 57:
445–449
255. Issa JP (1999)Aging,DNA methylation
and cancer. Crit Rev Oncol Hematol
32:31–43
256. Verma M, Srivastava S (2002) Epigenetics
in cancer: implications for early
detection and prevention. Lancet Oncol
3:755–763
257. KimYI,Baik HW,Fawaz K,Knox T,Lee
YM, Norton R, Libby E, Mason JB
(2001) Effects of folate supplementation
on two provisional molecular
markers of colon cancer: a prospective,randomized
trial.Am J Gastroenterol
96:184–195
258. Stover PJ GC (2002) Molecular and genetic
considerations for long-term nutrition
interventions. Asia Pacific J
Clin Nutr 11:S129–S136
259. Cairns BR (2001) Emerging roles for
chromatin remodeling in cancer biology.
Trends Cell Biol 11:S15–S21
260. Hinnebusch BF,Meng S,Wu JT,Archer
SY, Hodin RA (2002) The effects of
short-chain fatty acids on human
colon cancer cell phenotype are associated
with histone hyperacetylation.J
Nutr 132:1012–1017
261. Joyce D, Albanese C, Steer J, Fu M,
Bouzahzah B, Pestell RG (2001) NFkappaB
and cell-cycle regulation: the
cyclin connection. Cytokine Growth
Factor Rev 12:73–90
262. Gilmore T,Gapuzan ME,Kalaitzidis D,
Starczynowski D (2002) Rel/NF-kappa
B/I kappa B signal transduction in the
generation and treatment of human
cancer. Cancer Lett 181:1–9
263. Yu R,HebbarV,Kim DW,Mandlekar S,
Pezzuto JM, Kong AN (2001) Resveratrol
inhibits phorbol ester and UV-induced
activator protein 1 activation by
interfering with mitogen-activated
protein kinase pathways.Mol Pharmacol
60:217–224
264. DiSepio D, Sutter M, Johnson AT,
Chandraratna RA, Nagpal S (1999)
Identification of the AP1-antagonism
domain of retinoic acid receptors.Mol
Cell Biol Res Commun 1:7–13
265. Hill BT, Perrin D, Kruczynski A (2000)
Inhibition of RAS-targeted prenylation:
protein farnesyl transferase inhibitors
revisited. Crit Rev Oncol
Hematol 33:7–23
266. Haluska P, Dy GK, Adjei AA (2002)
Farnesyl transferase inhibitors as
anticancer agents. Eur J Cancer 38:
1685–1700
267. Kong AN, Yu R, Hebbar V, Chen C,
Owuor E, Hu R, Ee R, Mandlekar S
(2001) Signal transduction events
elicited by cancer prevention compounds.
Mutat Res 480–481:231–241
268. Philipp-Staheli J, Payne SR, Kemp CJ
(2001) p27(Kip1): regulation and
function of a haploinsufficient tumour
suppressor and its misregulation
in cancer. Exp Cell Res 264:
148–168
269. Gupta S,Afaq F, Mukhtar H (2002) Involvement
of nuclear factor-kappa B,
Bax and Bcl-2 in induction of cell
cycle arrest and apoptosis by apigenin
in human prostate carcinoma cells.
Oncogene 21:3727–3738
270. Bloom J,Pagano M (2003) Deregulated
degradation of the cdk inhibitor p27
and malignant transformation. Semin
Cancer Biol 13:41–47
271. Hambly RJ,Rumney CJ,Cunninghame
M, Fletcher JME, Rijken PJ, Rowland
IR (1997) Influence of diets containing
high and low risk factors for colon
cancer on early stages of carcinogenesis
in human flora-associated (HFA)
rats. Carcinogenesis 18:1535–1539
272. Goldin BR (1986) In situ bacterial metabolism
and colon mutagens. Ann
Rev Microbiol 40:367–393
273. MallettAK,Rowland IR (1988) Factors
affecting the gut microflora.In: Role of
the Gut Flora in Toxicity and Cancer.
Academic Press
274. Carman RJ, Van Tassel RL, Kingston
DGI (1988) Conversion of IQ,a dietary
pyrolysis carcinogen to a direct mutagen
by intestine bacteria of humans.
Mutation Res 206:335–342
275. Clinton SK, Bostwick DG, Olson LM,
Mangian HJ,VisekWJ (1988) Effects of
ammonium acetate and sodium
cholate on N-methyl-N’-nitro-Nnitrosoguanidine-induced
colon carcinogenesis
of rats. Cancer Res 48:
3035–3039
276. Pickering JS, Lupton JR, Chapkin RS
(1995) Dietary fat, fiber and carcinogen
alter fecal diacylglycerol composition
and mass. Cancer Res 55:
2293–2298
277. Reddy BS, Simi B, Engle A (1994)
Biochemical epidemiology of colon
cancer: effect of various types of
dietary fiber on colonic diaclglycerols
in women. Gastroenterology 106:
883–889
278. Hinzman MJ, Novotny C, Ullah A,
Shamsuddin AM (1987) Fecal mutagen
fecapentaene-12 damages mammalian
colon epithelial DNA.Carcinogenesis
8:1475–1479
279. Shamsuddin AM, Ullah A, Baten A,
Hale E (1991) Stability of fecapentaene-12
ans its carcinogenicity in F-
344 rats. Carcinogenesis 12:601–607
280. Schiffman MH,Van Tassell RL, Robinson
A, Smith L, Daniel J, Hoover RN,
Weil R, Rosenthal J, Nair PP, Schwartz
S (1989) Case-control study of colorectal
cancer and fecapentaene excretion.
Cancer Res 49:1322–1326
281. Macfarlane GT, Cummings JH (1991)
The colonic flora, fermentation and
the large bowel digestive function. In:
Phillips SF, Pemberton JH, Shorter RG
(eds) The Large Intestine: Physiology,
Pathophysiology and Disease. Raven
Press, New York, pp 51–91
282. Crofts F, Taioli E, Trachman J, Cosma
GN, Currie D, Tonniolo P, Garte SJ
(1994) Functional significance of different
human CYP1A1 genotypes.
Carcinogenesis 15:2961–2963
283. Dai R, Jacobson KA, Robinson RC,
Friedman FK (1997) Differential effects
of flavonoids on testosterone metabolizing
cytochrome P450s. Life Sciences
6:75–80
284. Kawata S, Tamura S, Matsuda Y, Ito N,
Matsuzawa Y (1992) Effect of dietary
fiber on cytochrome P450IAI induction
in rat colonic mucosa. Carcinogenesis
13:2121–2125
285. Langouet S, Coles B, Morel F, Becquemont
L, Beaune P, Guengerich FP, Ketterer
B, Guillouzo A (1995) Inhibition
of CYP1A2 and CYP3A4 by oltipraz
results in reduction of aflatoxin B1
metabolism in human hepatocytes
in primary culture. Cancer Res 55:
5574–5579
286. LeCluyse E, Madan A, Hamilton G,
Carroll K,DeHaan RPA (2000) Expression
and regulation of cytochrome
P450 enzymes in primary cultures of
human hepatocytes. J Biochem Mol
Toxicol 14:177–188
287. Roland N, Migpm-Baudon L, Flinois
JP, Beaune PH (1994) Hepatic and Intestinal
cytochrome P450, Glutathone
s-transferase and UDP Glucuronosyl
transferase are affected by six types of
dietary fiber in rats inoculated with
human whole fecal flora. J Nutr 124:
1581–1587
288. Rosenberg DW (1991) Tissue specific
induction of the carcinogen inducible
cytochrome P450 isoform, P450IA1 in
colonic epithelium. Arch Biochemistry
Biophysics 284:223–226
289. Sinha R, Rothman N, Brown ED, Mark
SD, Hoover RN, Caporaso NE, Levander
OA, Knize MG, Lang NP, Kadlubar
FF (1994) Pan-fried meat containing
high levels of heterocyclic aromatic
amines but low levels of polycyclic
aromatic hydrocarbons induces cytochrome
P4501A2 activity in humans.
Cancer Res 54:6154–6159
J. Rafter et al. II/83
PASSCLAIM – Diet-related cancer
290. Stillwell WG, Kidd LCR, Wishnok JS,
Tannenbaum SR, Sinha R (1997) Urinary
excretion of unmetabolized and
phase II conjugates of 2-amino-1methyl-6-phenylimidazo
[4, 5-b]pyridine
and 2-amino-3.8-dimethylimidazo
[4, 5-f]quinoxaline in humans:
relationship to cytochrome p4501A2
and N-acetyltransferase activity. Cancer
Res 57:3457–3464
291. Vang O,Jensen H,Autrup H (1991) Induction
of cytochrome p-450IAq,IA2,
IIB1, IIB2 and IIE1 by broccoli in rat
liver and colon. Chem-Biol Interac 78:
85–96
292. Wolf CR, Mahmood A, Henderson CJ,
McLeod R, Manson MM, Neal gE,
Hayes JD (1996) Modulation of the cytochrome
p450 system as a mechanism
of chemoprotection. In: Steward
BW, McGregor DB, Kleihues P (eds)
Principles of Chemoprevention.International
Agency for Research on Cancer,
Lyon, pp 165–174
293. Cashman JR (2001) Flavin monooxygenases.In:
Ioannides C (ed) John Wiley
and sons, Chicester, pp 67–93
294. Cashman JR (2002) Human and plant
flavin-containing monooxygenase Noxygenation
of amines: detoxication
vs. bioactivation. Drug Metabolism
Rev 34:513–521
295. Krueger SK, Williams DE, Yueh M-F,
Martin SR, Hines RN, Raucy JL, Dolphin
CT, Shepard EA, Phillips IR
(2002) Genetic polymorphisms of
flavin-containing monooxygenase
(FMO). Drug Metabolism Reviews
34:532
296. Ziegler DM (2002) An overview of the
mechanism, substrate specificities
and the structure of FMOs. Drug Metabolism
Reviews 34:503–511
297. Benson AM, Hunkeler MJ, Talalay P
(1980) Increase of NAD(P)H:quinone
reductase by dietary antioxidants:
possible role in protection against carcinogenesis
and toxicity. Proc Natl
Acad Sci USA77:5216–5220
298. Montano MM, Katzenellenbogen BS
(1997) A quinone reductase gene: a
unique estrogen receptor-regulated
gene that is activated by antiestrogens.
Proc Natl Acad Sci USA94:2581–2586
299. Smith MT (1999) Benzene,NQO1,and
genetic susceptibility to cancer. Proceedings
of the National Academy of
Sciences USA96:7624–7626
300. Tawfig N, Wanigatunga S, Heaney RK,
Musk SRR,Williamson G,Fenwick GR
(1994) Induction of the anti-carcinogenic
enzyme quinone reductase by
food extracts using murine hepatoma
cells. Eur J Cancer Prevention 3:
285–292
301. Tawfig N, Heaney RK, Plumb JA, Fenwick
GR, Musk SRR, Williamson G
(1995) Dietary glucosinolates as
blocking agents against carcinogenesis:
Glucosinolate breakdown products
assessed by induction of quinone
reductase activity in murine
hepa1c1c7 cells. Carcinogenesis 16:
1191–1194
302. UdaY,Price KR,Williamson G,Rhodes
MJC (1997) Induction of the anticarcinogenic
marker enzyme, quinone
reductase,in murine hepatoma cells in
vitro by flavonoids. Cancer Lett 120:
213–216
303. Wang W, Liu LQ, Higuchi CM, Chen H
(1998) Induction of NADPH: Quinone
reductase by dietary phytoestrogens
in colonic Colo205 cells. Biochem
Pharmacol 56:189–195
304. Yannai S, Day AJ, Williamson G,
Rhodes MJC (1998) Characterization
of flavonoids as monofunctional or bifunctional
inducers of quinone reductase
in murine hepatoma cell lines. Fd
Chem Toxicol 36:623–630
305. Clapper ML, Szarka CE (1998) Glutathione
S-transferases – biomarkers
of cancer risk and chemopreventive
response. ChemBiol Interac 111–112:
377–388
306. Hayes JD, McLeod R, Ellis EM, Ulford
DJ, Ireland LS, McLellan LI, Udah DJ,
Anson MM, Eal GE (1996) Regulation
of glutathione S-transferases and
aldehyde reductase by chemoprotectors:
studies of mechanisms responsible
for inducible resistance to aflatoxin
B1. In: Steward BW, McGregor
DB, Kleihues P (eds) Principles of
Chemoprevention. International
Agency for Research on Cancer, Lyon,
pp 175–188
307. Kirlin WG,Cai J,DeLong MJ,Patten EJ,
Jones DP (1999) Dietary compounds
that induce cancer preventive phase 2
enzymes activate apoptosis at comparable
doses in HT29 colon carcinoma
cells. J Nutr 129:1827–1835
308. Nijhoff WA, Mulder TPJ, Verhagen H,
van Poppel G, Peters WHM (1995)
Effects of consumption of Brussels
sprouts on plasma and urinary glutathione
S-transferase class-a and
-p in humans. Carcinogenesis 16:
955–957
309. Embola CW, Weisburger JH, Weisburger
MC (2001) Urinary excretion
of N-OH-2-amino-3-methylimidazo
[4, 5-f]quinoline-N-glucuronide in
F344 rats is enhanced by green tea.
Carcinogenesis 22:1095–1098
310. Embola CW, Sohn OS, Fiala ES, Weisburger
JH (2002) Induction of UDPglucuronosyltransferase
1 (UDP-GT1)
gene complex by green tea in male
F344 rats.Fd Chem Toxicol 40:841–844
311. Fisher MB, Paine MF, Strelevitz TJ,
Wrighton SA (2001) The role of hepatic
and extrahepatic UDP-glucuronosyltransferases
in human drug
metabolism. Drug Metabolism Reviews
33:273–297
312. Malfatti MA, Felton JS (2001) N-glucuronidation
of 2-amino-1-methyl-6phenylimidazo
[4, 5-b]pyridine
(PhIP) and N-hydroxy-PhIP by specific
human UDP -glucuronosyltransferases.
Carcinogenesis 22:1087–1093
313. Yang CS, Chen L, Lee MJ, Landau JM
(1996) Effects of tea on carcinogenesis
in animal models and humans. In:
American Institute for Cancer Research
(ed) Dietary Phytochemicals in
Cancer Prevention and Treatment,401
edn.Plenum Press,NewYork and London,
pp 51–62
314. Zhang Y, Hendrich S, Murphy PA
(2003) Glucuronides are the main
isoflavone metabolites in women. J
Nutr 133:399–404
315. Levy GN, Weber WW (2001) Arylamine
acetyltransferases. In: Ioannides
C (ed) Enzyme systems that
metabolise drugs and other xenobiotics.
John Wiley and Sons, Chicester,
pp 441–457
316. Grant DM (1993) Molecular genetics
of the N-acetyltransferases. Phramacogenetics
3:45–50
317. Martin FL, Venitt S, Carmichael PL,
Crofton-Sleigh C, Stone EM, Cole KJ,
Gusterson BA, Grover PL, Phillips DH
(1997) DNA damage in breast epithelial
cells: detection by the single-cell
gel (comet) assay and induction by human
mammary lipid extracts. Carcinogenesis
18:2299–2305
318. Jacobson JS,Begg MD,Wang LW,Wang
Q, Agarwal M, Norkus E, Singh VN,
Young TL,Yang D, Santella RM (2000)
Effects of a 6-month vitamin intervention
on DNA damage in heavy smokers.
Cancer Epidemiol Biomarkers
Prev 9:1303–1311
319. Perera FP, Mooney LA, Stampfer M,
Phillips DH, Bell DA, Rundle A, Cho S,
Tsai WY, Ma J, Blackwood A, Tang D
(2002) Associations between carcinogen-DNA
damage, glutathione Stransferase
genotypes,and risk of lung
cancer in the prospective Physicians’
Health Cohort Study. Carcinogenesis
23:1641–1646
320. Marnett LJ (2000) Oxyradicals and
DNA damage. Carcinogenesis 21:361
321. Mayne ST (2003) Antioxidant nutrients
and chronic disease: use of biomarkers
of exposure and oxidative
stress status in epidemiologic research.
J Nutr 133:933S–9940
II/84 European Journal of Nutrition (2004) Vol. 43, Supplement 2
322. Verhagen H, Poulson HE, Loft S, van
Poppel G,Willems MI,van Bladeren PJ
(1995) Reduction of oxidative DNAdamage
in humans by Brussels
sprouts. Carcinogenesis 16:969–970
323. Hu WW,Feng Z,Eveleigh J,Iyer G,Pan
J,Amn S,Chung F,Tang MS (2002) The
major lipid peroxidation product,
trans-4-hydroxy-2-nonenal, preferentially
forms DNA adducts at codon 249
of human p53 gene, a unique mutational
hotspot in hepatocellular carcinoma.
Carcinogenesis 23:1781–1789
324. Nair J, Vaca CE, Velic I, Mutanen M,
Valsta LM, Bartsch H (1997) High dietary
w-6 polyunsaturated fatty acids
drastically increase the formation of
etheno-DNA base adducts in white
blood cells of female subjects. Canc
Epid Biom Prev 6:597–601
325. Zhang Y, Chen SY, Hsu T, Santella
RM (2002) Immunohistochemical
detection of malondialdehyde-DNA
adducts in human oral mucosa cells.
Carcinogenesis 23:207–211
326. Loeb KR, Loeb LA (2000) Significance
of multiple mutations in cancer. Carcinogenesis
21:379–385
327. Bol SAM, Horlbeck J, Markovic J, de
Boer JG, Turesky RJ, Constable A
(2000) Mutational analysis of the liver,
colon and kidney of Big Blue(R) rats
treated with 2-amino-3-methylimidazo
[4,5-f]quinoline.Carcinogenesis
21:1
328. Kulling SE, Rosenberg B, Jacobs E,
Metzler M (1999) The phytoestrogens
coumoestrol and genistein induce
structural chromosomal aberrations
in cultured human peripheral blood
lymphocytes.Arch Toxicol 73:50–54
329. Renner HW, Münzner R (1991) The
possible role of probiotics as dietary
antimutagens. Mutation Res 262:
239–245
330. Goldman R, Shields PG (2003) Food
Mutagens. J Nutr 133:965S–9973
331. Tucker JD, Carrano AV, Allen NA,
Christensen ML,Knize MG,Strout CL,
Felton JS (1989) In vivo cytogenetic effects
of cooked food mutagens. Mutation
Research 224:105–113
332. Fenech M, Rinaldi J (1995) A comparison
of lymphocyte micronuclei and
plasma micronutrients in vegetarians
and non-vegetarians. Carcinogenesis
16:223–230
333. Fenech MF, etal. (2003) Intra- and inter-laboratory
variation in the scoring
of micronuclei and nucleoplasmic
bridges in binucleated human lymphocytes.
Results of an international
slide-scoring exercise by the HUMN
project. Mutation Res 534:45–64
334. Moore LE, Smith AH, HopenhaynRich
C, Biggs ML, Kalman DA, Smith
MT (1997) Micronuclei in exfoliated
bladder cells among individuals
chronically exposed to arsenic in
drinking water. Canc Epid Biom Prev
6:31–36
335. Pastor S,Gutierrez S,Creus A,Xamena
N, Piperakis S, Marcos R (2001) Cytogenetic
analysis of Greek farmers using
the micronucleus assay in peripheral
lymphocytes and buccal cells.
Mutagenesis 16:539–545
336. Piyathilake CJ, Macaluso M, Hine RJ,
Vinter DW, Richards EW, Krumdieck
CL (1995) Cigarette smoking,intracellular
vitamin deficiency, and occurrence
of micronuclei in epithelial cells
of the buccal mucosa. Cancer Epidemiol
Biomarkers Prev 4:751–758
337. Titenko-Holland N, Jacob RA, Shang
N, Balaraman A, Smith MT (1998) Micronuclei
in lymphocytes and exfoliated
buccal cells of postmenopausal
women with dietary changes in folate.
Mutation Res 417:101–114
338. Elahi A,Zheng Z,Park J,Eyring K,McCaffrey
T, Lazarus P (2002) The human
OGG1DNA repair enzyme and
its association with orolaryngeal cancer
risk. Carcinogenesis 23:1229–1234
339. Hazra TK, Izumi T, Kow YW, Mitra S
(2003) The discovery of a new family
of mammalian enzymes for repair of
oxidatively damaged DNA, and its
physiological implications. Carcinogenesis
24:155–157
340. Martin FL, Cole KJ, Orme MH, Grover
PL, Phillips DH, Venitt S (1999) The
DNA repair inhibitors hydroxyurea
and cytosine arabinoside enhance the
sensitivity of the alkaline single-cell
gel/electrophoresis ‘comet’ assay in
metabolically-competent MCL-5 cells.
Mutation Research 445:21–43
341. de Boer J,Hoeijmakers JHJ (2000) Nucleotide
excision repair and human
syndromes. Carcinogenesis 21:453
342. Berwick M,Vineis P (2000) Markers of
DNA repair and susceptibility to cancer
in humans: an epidemiologic review.
JNCI Cancer Spectrum 92:874
343. Aaltonen LA, Peltomäki P, Lee FS, Sistonen
P,Pylkkänen L,Meo JP,Järvinen
H, Powell SM, Stanley JJ, Petersen GM,
Kinzler W, Vogel B, de la Chapelle A
(1993) Clues to the pathogenesis of familial
colorectal cancer. Science 260:
812–815
344. Thibodeau SN, Bren G, Schaid D
(1993) Microsatellite instability in
cancer of the proximal colon. Science
260:816–819
345. Howlett J, Shortt C (2004) PASSCLAIM-Report
of the Second Plenary
Meeting: review of a wider set of interim
criteria for the scientific substantiation
of health claims.Eur J Nutr
43(Suppl 2):174–183