Mechanisms of Ageing and Development 125 (2004) 315–324
Molecular imaging of the biological effects of quercetin
and quercetin-rich foods
Jan Øivind Moskaug, Harald Carlsen, Mari Myhrstad, Rune Blomhoff∗
Institute for Nutrition Research, Faculty of Medicine, University of Oslo, P.O. Box 1046, Blindern, 0316 Oslo, Norway
Abstract
The human diet contains several thousands of organic plant molecules (i.e. phytochemicals), many of which have significant bioactivities.
The specific physiological effects of these compounds are impossible to predict from in vitro studies using cell cultures and cell-free model
systems. Nutrigenomics, which may be defined as the application of genomic tools to study the integrated effects of nutrients on gene
regulation, however, holds great promise in increasing the understanding of how nutrients affect molecular events in an organism. Quercetin,
a phytochemical belonging to the flavonoids, has antioxidant activities, inhibit protein kinases, inhibit DNA topoisomerases and regulate gene
expression. The aim of the present review is to describe some of the many effects of quercetin, and how molecular imaging using transgenic
reporter mice may serve as a tool to study the integrated influence of quercetin and other dietary phytochemicals on gene expression in vivo.
We are using the bioluminescence emitted from firefly luciferase as the reporter since light originating from the inside of a cell or organism
can be detected externally in an intact living organism. Molecular imaging using reporter models is therefore a unique technology to study
the integrated effects of environmental insults and dietary substances on the influence of gene expression in disease development. We utilize
these in vivo models to elucidate the role of various flavonoids, such as quercetin, for modulating gene expression related to oxidative stress
and the antioxidant defence system.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Phytochemicals; Flavonoids; Nutrigenomics; NF-␬B; ␥-glutamylcysteine synthetase
1. Introduction
All living organisms have evolved in a hostile environment. Indeed, survival in challenging environments has
been the driving force in the continuous remodelling of all
multi-cellular organisms. Development of defence mechanisms and ability to withstand insults and subsequent
disease development is crucial in the process of evolution.
Studies of disease prevention is therefore closely linked to
effects of environmental factors such as nutrition, exposure
to radiation, ingestion of toxic substances and exposure to
the toxic gas oxygen. Common for the three latter factors
are production of substances capable of damaging cellular
lipid, protein, carbohydrates and DNA, whereas nutrition
may serve to improve cellular defense systems. Cellular
damage of radiation and exposure to many toxicants and
oxygen seems to be related to their ability to generate
reactive oxygen species (ROS), reactive nitrogen species
∗
Corresponding author. Tel.: +47-22-85-13-95; fax: +47-22-85-13-96.
E-mail address: rune.blomhoff@basalmed.uio.no (R. Blomhoff).
(RNS) and reactive iron species (RIS). Most of these ROS,
RNS and RIS are also formed during normal metabolism,
particularly as a consequence of oxygen consumption and
ATP production in the mitochondria. However, when these
reactive metabolites are formed in excess a condition called
oxidative stress will appear.
A general concept in development of defence mechanisms
seems to be adaptation through repeated exposure to the
harmful factor of limited duration and extent, thus preparing
the organism for more serious insults. An example of this is
the limited oxidative stress experienced during physical exercise that is currently used to explain the beneficial effect of
activity on various disease risks. Additionally, defence may
be developed as a consequence of exposure to substances
that are not toxic but are handled by the same detoxification systems as harmful substances. Nutrition may also play
an important role in disease prevention through ingestion of
substances that boost defence systems designed to inhibit
cellular damage by e.g. ROS. One of the most extensively
studied groups of such substances are the flavonoids which
are abundant in food items of plant origin. Of the flavonoids,
quercetin is among the substances that have been studied
0047-6374/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mad.2004.01.007
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in some detail. Most of these studies are built on cell cultures or cell-free model systems. It is of outmost importance,
however, to take into account that dietary factors affect a
living integrated organism in a physiological setting. Thus,
it is important also to employ model systems that respect
the dynamics and integrity of complex biological networks.
These aspects are central to the new emerging research field
of nutrigenomics (Muller and Kersten, 2003).
The aim of the present report is to highlight some aspects
related to the cellular effects quercetin and other flavonoids
in a system biological context. We would particularly like
to emphasise molecular imaging using transgenic reporter
models as a tool to study the dietary influence on gene expression in vivo. Bioluminescence emitted from firefly luciferase originating from the inside of a cell or organism
can be detected externally. Thus, such molecular imaging
permits the external visualization of cellular and subcellular processes in an intact living organism. Molecular imaging using reporter models is therefore a unique technology
to study the integrated effects of environmental insults and
dietary substances on the influence of gene expression involved in disease development.
2. Quercetin as free radical scavenger and inhibitor
of ROS production
The chemical structure of flavonoids, including quercetin,
makes them capable of stabilizing free electrons obtained
from free radicals such as ROS in in vitro systems (Hanasaki
et al., 1994; Pietta, 2000). Our understanding of the structure/activity relationship is however limited. Recently,
Heijnen et al. (2001) have shown that particular hydroxyl
groups seem to be positively related to abilities of flavonoids
to scavenge peroxynitrite. Flavonoids with particular structures may also inhibit ROS production by chelating metal
ions that would otherwise contribute to ROS production
through Fenton reactions (Pietta, 2000). Flavonoids including quercetin are also known to inhibit superoxide anion
production by xanthine oxidase (Hanasaki et al., 1994). It
is however currently not clear to what extent this property
of flavonoids contributes to the antioxidant defence of cells
in vivo.
3. Quercetin as protein kinase inhibitor
Cellular growth and differentiation are regulated by signal
transduction pathways involving a large number of both tyrosine and serine/threonine protein kinases and phosphatases.
Some of these kinases are targets for kinase modulators potentially useful in disease treatment (Cohen, 2001). Protein
kinase modulators are also omnipresent in food items of
plant origin. Among the most extensively studied dietary
protein kinase inhibitors are quercetin, curcumin and the soy
isoflavon genistein. The inhibitory activity of quercetin is
associated with its ability to compete with binding of ATP
to the nucleotide binding site on the kinases. Binding of
quercetin to phosphoinositide 3-kinase has been studied on
the atomic level through crystallization of the kinase in the
quercetin bound state. These studies reveal that quercetin
and related kinase inhibitors bind in the ATP-binding pocket
of the kinase (Walker et al., 2000).
The outcome of cellular exposure to flavonoids with kinase modulatory activity such as quercetin is hard to predict,
both beneficial and deleterious effects can be envisaged in
terms of disease prevention. Potentially deleterious effects
of quercetin is demonstrated in a recent study by Spencer
et al. where they show that quercetin at concentrations ranging from 10 to 30 ␮M reduce phosphorylation of the kinases
Akt and ERK with subsequent induction of caspase 3 activation and death in primary neuronal cells (Spencer et al.,
2003). These concentrations are however an order of magnitude above of what has been measured in plasma of humans after intake of quercetin-rich food items (Scalbert and
Williamson, 2000). This applies to almost all studies with
quercetin as protein kinase inhibitors. Davies and collaborators have determined the specificity of quercetin towards
a number of kinases and found that CK2, AMPK and P13K
were most sensitive to quercetin inhibition and was able to
reduce the kinase activity to approximately 20% of control
values (Davies et al., 2000).
4. Quercetin as topoisomerase inhibitor
Topoisomerases are DNA-associated enzymes that are
required during processes such as DNA replication and
transcription where they cleave and then religate either one
(type I) or two (type II) strands of DNA thereby allowing these strands to pass through one another. A number
of compounds interfere in these processes by inhibiting
the religation of the DNA double strands. Such inhibition
can lead to increased numbers of DNA breaks (Finkel and
Holbrook, 2000). A number of polyphenols including
tannins (Kashiwada et al., 1993) and flavonoids such a
quercetin (Constantinou et al., 1995) and isoflavones such
as genistein (Halliwell, 1995; Talalay et al., 1995; Zhang
et al., 1992) have been identified as topoisomerase type II
inhibitors. Myricetin, quercetin, fisetin, and morin inhibited
both types I and II enzymes, while kaempferol inhibited
topo II without inhibiting topo I (Constantinou et al., 1995).
Inhibition of topoisomerase II has been shown to increase
the frequency of chromosome translocations (Sperry et al.,
1989). Hence, it has been suggested that a high intake of
flavonoids by pregnant women may increase the risk of
giving birth to children with MLL (Ross, 2000), a form of
leukaemia characterized by translocations involving chromosome 11. Such genetic alterations are consistent with inhibition of DNA topoisomerases (Skibola and Smith, 2000;
Strick et al., 2000). Recently, Plaper and colleagues showed
that DNA gyrase, a prokaryote DNA topology modifying
J.Ø. Moskaug et al. / Mechanisms of Ageing and Development 125 (2004) 315–324
enzyme, is strongly inhibited by quercetin by competitive
binding to an ATP binding pocket (Plaper et al., 2003).
5. Quercetin as regulator of gene expression
What has been shown by our laboratory and others is that
flavonoids may contribute to the endogenous antioxidant defence of cells through gene regulation. Many genes involved
in detoxification and activation/inactivation of carcinogens
contain specific DNA sequences in their promoters that are
directly or indirectly regulated by flavonoids.
5.1. NF-κB transcription factors and AP-1
While quercetin and related plant nutrients often induce
expression of genes involved in detoxification (see below),
other transcription factors may be down-modulated by these
compounds. In this regard the family of NF-␬B transcription factors, and the transcription factor AP-1 have been
thoroughly studied particularly in vitro. NF-␬B, which was
discovered in 1986 (Sen and Baltimore, 1986) induces the
expression of many genes that play a critical role for both
the regulation of immune and inflammatory responses and
for the protection of cells from apoptosis. However, aberrant activation of NF-␬B, can be one of the primary causes
of a wide range of human diseases (Perkins, 2000). Several reports have indicated that NF-␬B is regulated by plant
derived substances such as quercetin and green tea extracts
(Muraoka et al., 2002), which may potentially ameliorate
disease states influenced by uncontrolled NF-␬B activation.
AP-1, which regulates the expression of genes associated
with cell growth and cellular stress has also been shown to be
regulated by phenolic nutrients such as quercetin, recently
demonstrated by Moon et al. (2003). Notably, the regulation
of NF-␬B and AP-1 by dietary nutrients is cell type specific
(Hofmann and Sonenshein, 2003; Rangan et al., 2002).
5.2. Xenobiotic responsive elements
Genes in the cytochrome P450 family are typically
genes with xenobiotic responsive elements (XRE) in their
promoters. XRE has been defined to be 5 -TNGCGTG-3
317
(Fujisawa-Sehara et al., 1987). Cytochrome P450 enzymes
containing XRE in their gene promoters may be regulated
in part by the aryl hydrocarbon receptor (AhR) (Mimura
and Fujii-Kuriyama, 2003). AhR mediate detoxification of
potent carcinogens such as PAHs exemplified by dioxin.
Flavonoids have a basic chemical structure with some common structures found in poly aromatic hydrocarbons (see
Fig. 1). It is therefore of no surprise that several studies have
found polyphenols to interact with cellular defence systems
such as phases I and II detoxification enzymes (Raucy and
Allen, 2001; Raucy, 2003). In particular quercetin has been
shown to bind directly to AhR as a natural ligand thereby
eliciting an induction of XRE-dependent gene transcription
(Ciolino et al., 1999). As AhR is important in transcriptional activation of cytochrome P450 enzymes it is possible
that cells exposed to quercetin obtained through the diet has
higher levels of phase I detoxification enzymes and thereby
are better prepared for subsequent toxic insults.
5.3. Antioxidant responsive elements/electrophile
responsive elements (ARE/EpRE)
Cellular defence can also be regulated by genes with
antioxidant responsive elements/electrophile responsive
elements (ARE/EpRE) in their promoters. ARE/EpREs
are specific nucleotide sequences found in many promoters of genes involved in cellular defence (see Table 1).
Comparison of ARE/EpRE motifs in several genes has allowed definition of a consensus sequence 5 -TA /C ANNA /G
TGAC /T NNNGCA /G -3 (Wasserman and Fahl, 1997).
Mulcahy and colleagues recently revealed a functional
variant of the motif in the promoter of the γGCSl gene.
The consensus ARE/EpRE may therefore be revised to
5 -RTKAYNNNGCR-3 (Erickson et al., 2002).
ARE/EpREs are bound by, among others, transcription
factors belonging to the cap’n’collar basic leucine zipper superfamily. p45 NF-E2 is the founding member of this family
and other members include NF-E2-related factors 1, 2 and
3 (Nrf1, Nrf2 and Nrf3) (Andrews et al., 1993; Kobayashi
et al., 1999; Luna et al., 1994; Nguyen and Pickett, 1992).
Venugopal and Jaiswal have demonstrated that overexpression of both Nrf1 and Nrf2 increased ARE/EpRE
driven gene expression. Furthermore, electrophoretic
Fig. 1. Basic chemical structure of flavonols and examples of substances found in the indicated food sources.
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Table 1
Enzymes containing ARE/EpRE enhancers in their gene promoters
Enzymes
References
NADPH-quinone oxidoreductase
(NQO1)
Glutathione S-transferase (GST)
UDP-glucuronosyl transferase
Thioredoxin
Heme oxygenase-1
Ferritin
␥-Glutamylcysteine synthetase
heavy subunit
␥-Glutamylcysteine synthetase
light subunit
Metallothionein 1
Inducible nitric oxide synthase
Cystine/glutamate exchange
transporter
Fra-1
Favreau and Pickett (1995)
Rushmore et al. (1990)
Prestera et al. (1995)
Kim et al. (2001)
Inamdar et al. (1996)
Tsuji et al. (2000)
Mulcahy and Gipp (1995)
Moinova and Mulcahy (1998)
Ren and Smith (1995)
Kuo et al. (2000)
Sasaki et al. (2002)
Yoshioka et al. (1995)
mobility super-shift assays (EMSA) established that both
Nrf1 and Nrf2 were capable of binding to the ARE/EpRE
found in the promoter of the NAD(P)H:quinone oxidoreductase1 (NQO1) gene (Venugopal and Jaiswal, 1996). More
direct evidence of these factors having a role in regulating
the expression of detoxification and antioxidant defence
genes come from the genetic manipulation of the nrf1 and
nrf2 genes in mice. The induction of phase II enzymes
was significantly affected in the nrf2-null mouse, and nrf1
mutants show reduced protection against the effects of oxidants and reduced levels of glutathione (Itoh et al., 1997;
Kwong et al., 1999).
Nrf1 and Nrf2 bind target DNA motifs as heterodimers
with the bZIP proteins MafF, MafG and MafK. In addition,
Nrf1 has been reported to dimerize with c-Jun, activating
transcription factors 2 and 4 (ATF2, ATF4) and Nrf2 can
form heterodimers with c-Jun, ATF4, polyamine-modulated
factor (PMF) and peroxisome proliferator-activated receptor ␥ (PPAR␥) (Hayes and McMahon, 2001). Both the
ARE/EpRE sequence context and the inducer compound
may influence which of the bZIP dimers that are recruited
to the promoters (Nguyen et al., 2000). It has also been
demonstrated that the different Nrf1- and Nrf2-containing
complexes differ in their specificity and magnitude of transactivation (Dhakshinamoorthy and Jaiswal, 2000).
Three major signal transduction pathways have been
implicated in the regulation of ARE/EpRE motifs; those
mediated by (1) mitogen-activated protein kinase (MAPK)
cascade, (2) phosphatidylinositol 3-kinase (PI3K) and (3)
protein kinease C (PKC):
(1) ARE/EpRE regulated reporter activity stimulated by
the inducers tert-butylhydroquinone (tBHQ) and sulforaphane was mediated by extracellular regulated
kinase (ERK) in hepatoma cell lines. Furthermore,
activation of p38 MAPK pathway by tBHQ and
␤-naphtoflavone was correlated with a down-regulation
of basal and inducible ARE/EpRE-mediated gene
expression in the same cell lines (Yu et al., 1999).
The p38 pathway may therefore exert a repressive effect upon the ARE/EpREs. The third MAPK pathway
c-Jun-N-terminal kinase (JNK), is activated in response
to sodium arsenite and mercury chloride (Yu et al.,
2000). At present it is unclear how the MAPKs influence ARE/EpRE activation, and none of the proteins
implicated in the ARE/EpRE response have shown to
be MAPK substrates. However, Nrf1 and Nrf2 contain
potential proline-directed serine/threonine residues and
are possible substrates.
(2) Other studies have implicated PI3K in transducing a response by phenolic antioxidants and oxidative stress to
the ARE/EpRE motifs. This kinase is an integral component of the insulin signalling pathway (Lee et al., 2001;
Li et al., 2002).
(3) Nrf2 was recently reported to be phosphorylated at a
serine residue by PKC in response to tBHQ treatment.
Phosphorylation of Nrf2 was associated with the appearance of the transcription factor in the nucleus, suggesting a role of PKC in the ARE/EpRE-mediated gene
expression (Huang et al., 2000).
Apart from the phosphorylation events elicited by kinases,
it has been suggested that sulfhydryl group chemistry may
be important in regulation of ARE/EpRE-mediated gene expression. The actin-binding protein Keap1 (Kelch-like ECH
associated protein 1) was cloned in 1999 and identified as a
docking site for Nrf2 (Itoh et al., 1999). These two proteins
associate through a double glycine-rich domain of Keap1
and a hydrophilic region in the Neh2 domain of Nrf2. Keap1
is responsible for sequestering Nrf2 in the cytoplasmic compartment of the cell through its association with actin filaments in the cytosol. Dinkova-Kostova and her colleagues
have identified several critical cysteine residues of Keap1
that are modified by electrophilic agents to induce the release of Nrf2 (Dinkova-Kostova et al., 2002). Under basal
conditions, Keap1 may also negatively regulate Nrf2 by
enhancing its rate of proteasomal degradation (McMahon
et al., 2003). Talalay et al. have recently suggested that a
large number of substances capable of inducing phase II
enzymes do so by reacting with thiol groups by alkylation, oxidation, reduction or thiol interchange. The activity of phase II inducers does not seem to depend on their
structure but may depend on their common chemical reactivity towards thiol groups (Talalay et al., 2003). The current model for regulation of phase II genes is illustrated in
Fig. 2.
An ARE/EpRE-like element has been identified in the
5 -flanking region of the nrf2 gene and over expression of
Nrf2 increased nrf2 promoter activity. Thus, Nrf2 appears
to auto regulate its own expression, leading to persistent nuclear accumulation of Nrf2 and prolonged induction of phase
II genes in response to chemo preventive agents (Kwak et al.,
2002). Any ARE/EpRE motif in the 5 -flanking sequence of
the nrf1 gene has to our knowledge not been identified.
J.Ø. Moskaug et al. / Mechanisms of Ageing and Development 125 (2004) 315–324
319
Fig. 2. Schematic representation of cellular effects of flavonoids. When flavonoids are taken up by cells they may or may not be metabolized depending
on their chemical structures. Flavonoids may then bind to the aryl hydrocarbon receptor and stimulate xenobiotic metabolism, modulate protein kinase
activities or interfere with cytosolic retention of Nrf2 and thereby influence transcription from ARE/EpRE containing gene promoters.
Nrf1/2 regulation of ARE/EpREs may be an important
pathway by which dietary components such as flavonoids
regulate cellular defence mechanisms. We have recently
shown that flavonoids such as quercetin increase expression
of the rate limiting enzyme in synthesis of the most important endogenous cellular antioxidant, glutathione (GSH)
(Myhrstad et al., 2002). Glutathione exists in thiol-reduced
and disulfide-oxidized (GSSG) forms. It acts as the main
cellular redox buffer and shift in the ratio of GSH:GSSG
may oxidize redox sensitive cysteines. Glutathione is required for numerous cellular functions and acts as storage
form of cysteine and provides reducing equivalents for many
cellular reactions. In addition, glutathione participates in
detoxification at several different levels, and may scavenge
free radicals, reduce peroxides, or be conjugated with electrophilic compounds (Anderson, 1998). Enzymes involved
in the metabolism of glutathione include glutathione peroxidase, glutathione reductase, GSTs, glutathione S-conjugate
efflux pumps, glutathione synthetase, ␥-glutamylcysteine
synthetase (GCS) and ␥-glutamyltranspeptidase. These enzymes function in an integrated fashion to allow cellular
adaptation to oxidative stress (Hayes and McLellan, 1999).
In addition to its antioxidant and free radical scavenging
properties GSH is crucial in conjugation and elimination
of potentially carcinogenic xenobiotics in phase II reactions. Increased intracellular levels of GSH may thus be
one way by which dietary compounds protect cells and
thereby whole organisms against deleterious factors in our
environment.
6. Molecular imaging using reporter models: a unique
technology to study the integrated effects of environment
on gene expression in a physiological setting
Detailed knowledge about the cellular effects of dietary
compounds has mostly been obtained by studies on cell
tissue cultures in vitro. The integrated beneficial or deleterious effect of food components has at some point to be
established in complete organisms. As described above,
in vitro experiments demonstrate that quercetin and other
flavonoids have a plethora of effects due to their interference with major cellular pathways; nuclear receptor and
transcription factor interactions and protein kinase inhibition. To establish the importance of such interference in
complete organisms it is crucial to establish effects of the
substances on gene expression in vivo. This is particularly
important as dietary flavonoids are now available as supplements for self-administration. It should be of major concern
that manufacturers of dietary supplements advertise the
use of quercetin in capsules containing up to 500 mg and
suggest an intake of two capsules or more per day.
In an integrative approach to study effects of food components we have recently developed several transgenic
reporter animal models that express luciferase under the
control of various transcription factors, which potentially
are modulated by dietary components in plants.
A reporter gene is most frequently used to assess the
regulation of a specific DNA regulatory element which can
contain a complete promoter for a known gene, specific
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J.Ø. Moskaug et al. / Mechanisms of Ageing and Development 125 (2004) 315–324
parts of a promoter, or synthetic DNA binding regions
that are designed to monitor the activity of one particular
transcription factor. In transgenic mice this approach has
been successfully employed to investigate gene expression
patterns during early mouse development, and has been
employed in adult mice examining gene expression patterns
in various tissues. Such reporter mice are phenotypically
normal since the reporter gene ideally does not exert any
physiological effect on the animal. A reporter gene of interest in this respect is luciferase from the firefly. Luciferase
has a relatively short half-life making it suitable for assessment of dynamic changes in gene activity. Importantly,
animals express luciferase in sufficient amounts to detect
bioluminescence in vivo, first demonstrated by Contag
et al. (1998) using an ultra-sensitive imaging device, for
example the IVIS 100 system developed by Xenogen or
the C2400-47 system from Hamamatsu. This approach allows non-invasive imaging of gene regulation permitting a
temporal investigation of dietary intervention in mice.
Prior to the imaging, mice are anesthetized and the substrate for luciferase (D-luciferin) is injected into the tail
vein or intraperitoneally. Shortly afterwards the mice are
placed in a light-sealed chamber connected to the camera.
Gray scale images are obtained before luminescence imaging for reference. Luminescence emitted from the mouse is
integrated for typically 1–5 min starting 2–10 min after the
injection of luciferin depending on the mode of injection.
In the case of ex vivo imaging individual organs are excised from the mice 3 min following injection of luciferin.
Organs are then placed in a culture dish and immediately
imaged. Photon emission as a function of luciferase gene
expression can then be quantified. In this way animals can
be treated with pure compounds of dietary interest or complex diets, and the effect of gene expression be monitored.
Alternatively, transgenic mice can be exposed to oxidative
stress inducing agents and the effect of such stressors can be
monitored.
We have performed experiments with three different transgenic reporter mice; NF-␬B-luciferase mouse (Carlsen et al.,
2002, Fig. 3A), the AP-1 luciferase mouse obtained from
Flavell and Rincon (Rincon and Flavell, 1994, Fig. 3B) and
the GCSh-promoter luciferase mouse (Fig. 3C). The NF-␹Band GCSh-promoter luciferase models were developed in
our laboratory.
Fig. 3. In vivo imaging of transgenic mice. Three transgenic mice strains expressing luciferase regulated by various transcription factor binding sites
were imaged. The images taken with an ultra-sensitive video camera in complete darkness show basal activity from luciferase regulated by either NF-␬B
(A), AP-1 (B) or several binding sites in the ␥GCSh gene promoter (C) as illustrated by the constructs to the right.
J.Ø. Moskaug et al. / Mechanisms of Ageing and Development 125 (2004) 315–324
To characterize the NF-␬B luciferase mouse we treated
mice with classical NF-␬B inducers such as TNF-␣, IL-1␣,
or LPS and monitored the mice non-invasively, and found a
strong overall induction of NF-␬B-mediated luminescence
in a time dependent manner. Also, exposure of skin to a
low dose of UV radiation (360 J/m2 , which equals 1 human MED) increased luminescence in the exposed areas.
Furthermore, induction of chronic inflammation resembling
rheumatoid arthritis produced strong NF-␬B activity in the
affected joints, as revealed by in vivo imaging (Carlsen et al.,
2002). Thus, we have developed a versatile model for monitoring NF-␬B activation in vivo.
Exposure to UV radiation is believed to contribute significantly to ageing in the skin, possibly through increased
production of ROS, in a process termed photoageing (for a
recent review see Rittie and Fisher, 2002). UVB-mediated
AP-1 and NF-␬B activation has been shown to increase activities of metalloproteinases (Berneburg et al., 2000), enzymes that contribute to degradation of matrix proteins in
the skin. Indeed, skin may serve as a useful model to study
tissue alterations mediated by oxidative stress. The AP-1 luciferase mouse has been used in several studies of dietary
antioxidants (Barthelman et al., 1998) and other compounds
(Huang et al., 1997) topically applied to the skin. In these
studies it has been shown, by taking punch biopsies from the
321
Fig. 4. In vivo image of AP-1 luciferase mouse exposed to low levels
of UVB. Fur from the abdomen of AP-1 luciferase mouse was removed
and two regions of the abdominal skin was exposed to 360 J/m2 of UVB
before injection of luciferin and in vivo imaging. A regular bright field
image is superimposed on a dark field image.
Fig. 5. Ex vivo imaging. The images show basal luciferase activity from various complete organs excised from ␥GCSh-luciferase mice after anaesthesia
and injection of luciferin into the living animal. Hippocampus slices from ␥GCSh-luciferase mouse were prepared and cultured for 10 days before imaged
in the presence of luciferin in the medium.
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skin and bioluminescence measurements that UVB induce
AP-1 activity in skin tissue homogenates. Topical treatment
of mouse skin with epi-gallocatechin gallate reduced AP-1
driven luciferase activity (Barthelman et al., 1998). In vivo
imaging of these mice shows that AP-1 activity can be induced by very low doses of UVB (360 J/m2 ) (Fig. 4). Dietary
interventions with single or mixed antioxidants can then be
undertaken and effect on AP-1 activity can be measured in
vivo. It should be noted that basal AP-1 activity is not evenly
distributed in the skin of these mice. Close examination of
the mouse skin after removal of the fur shows that areas with
increased luciferase activity overlap exactly with pigmented
areas. Care should therefore be taken in selecting skin areas
when comparing the effect of various treatments.
We have recently shown that the ␥GCS-promoterluciferase mouse reports differences in promoter activity
dependent on dietary interventions. We gave ␥GCS mice
blueberry juice as the only liquid source and observed an
increase in promoter activity in brain and muscle tissue
homogenates and a decrease in activity in liver (Carlsen
et al., 2003). However, in these mice the basal activity of
the promoter assessed by in vivo imaging is too high to
observe differences using this method.
Imaging of luciferase activity can be used to discriminate
differences in activity of gene promoters in non-homogenous
tissues. ␥GCS-promoter activity in the transgenic mice is
particularly high in brain and testes and in these organs
different parts of the organ exhibit differential activity. Thus,
we can visualize the luciferase activity in the CA1, CA3 and
the dentate gyrus in hippocampal slices (Fig. 5).
Also, testes and epididymis displayed high activity probably reflecting the increased need for glutathione (Fig. 5). Evidence exists that differentiation of spermatozoa in the caput
of testis requires high production of super oxide anions and
hydrogen peroxide. It is plausible that such production also
requires an increased demand for antioxidants such as glutathione and hence high expression of ␥GCS. Several studies
have demonstrated that in fact the epididymis contains high
amounts of antioxidant enzymes (Jervis and Robaire, 2001;
Tramer et al., 1998). The ␥GCS promoter-luciferase mouse
may thus be useful for studies of antioxidant activity during
spermatogenesis.
7. Conclusion
Human diet, particularly that of plant origin, contains
several thousands of organic molecules with potentially
beneficial and disease preventive, or deleterious and disease
promoting effects. As combination of these compounds may
have a plethora of cellular effects, and even several effects
from a single compound, it is very hard to predict the net
outcome of taking these substances on for example on gene
expression. Flavonoids, as exemplified by quercetin, have
been shown to have many effects on cells in vitro. Nutrigenomics, which may be defined as the application of genomic
tools to study the integrated effects of nutrients on gene regulation, holds great promise in increasing the understanding
of how nutrients affect the whole organism in health and
disease (Muller and Kersten, 2003). Our approach to use
in vivo imaging of gene expression by bioluminescence is
thus one of many important complementary tools that will
contribute significantly to studies of the integrated effects
of bioactive dietary compounds.
References
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