Nutrient-Gene Interactions—Research Communication
Berry Intake Increases the Activity
of the ␥-Glutamylcysteine
Synthetase Promoter in Transgenic
Reporter Mice1
types of antioxidants that may directly react with reactive
oxygen or nitrogen species forming products with much lower
reactivity. Alternatively, compounds in a plant-based diet may
increase the capacity of the endogenous antioxidant defenses.
Analyses of promoter regions have suggested that several response elements may be regulated by plant antioxidants. Dietary antioxidants may therefore protect against oxidative
damage both by directly neutralizing reactive oxidants and by
modulating genes related to oxidative stress (3).
Dietary antioxidants were recently assessed in a variety of
dietary plants including fruits, berries, vegetables, cereals, nuts
and pulses. Our results (4) demonstrated that there is more
than a thousand-fold difference between total antioxidants in
various dietary plants. Interestingly, berries, particularly blackberries, blueberries and elderberries, are among the plants with
the highest concentrations of antioxidants. These results are
intriguing because many berries have recently been shown to
protect against oxidative stress–related pathology in vivo. For
example, long-term feeding of strawberries and blueberries to
rats retards and even reverses the onset of age-related neurological dysfunctions such as decline in neuronal signal-transduction, and cognitive behavioral and motor deficits (5,6).
Furthermore, supplementation of strawberries or black raspberries reduced the multiplicity and incidence of esophageal
tumors in N-nitrosomethylbenzylamine-treated rats (7,8). The
chemopreventive effects of these berries may be explained by
their high content of ellagic acid (EA) because EA acid itself
has been shown to inhibit cancers in rodents (9 –14).
In the present report we tested the ability of antioxidantrich berries to modulate oxidative stress–related gene expression by using a novel transgenic luciferase reporter model. In
these studies we used the promoter for the gene encoding the
heavy subunit of the ␥-glutamylcysteine synthetase (GCSh),4
the rate-limiting enzyme in glutathione synthesis. The transcriptional control of this gene is mediated by several oxidative
stress related response elements including activator protein-1
sites, one nuclear factor-␬B site and several antioxidant response elements/electrophile response elements (15,16).
(Manuscript received 28 January 2003. Initial review completed 7
March 2003. Revision accepted 25 April 2003.)
Harald Carlsen,2 Mari C. W. Myhrstad,2 Magne Thoresen,*
Jan Øivind Moskaug and Rune Blomhoff3
Institute for Nutrition Research and *Department of Medical
Statistics, University of Oslo, Oslo, Norway
ABSTRACT A diet rich in fruit and vegetables is associated with decreased risk of disease. One possible mechanism for this is that dietary antioxidants positively regulate
protective genes. Toward our goal to identify bioactive
compounds with such functions in plants, we developed
transgenic mice that express luciferase controlled by the
␥-glutamylcysteine synthetase heavy subunit (GCSh) promoter. Mice that consumed a nonpurified diet ad libitum
were supplemented with juices or extracts of antioxidantrich berries for 42 h or 3– 4 wk. The treatments generally
increased luciferase activity in brain and skeletal muscle
and decreased it in liver compared with controls fed water.
The same overall pattern was also found in mice fed ellagic
acid (EA), a phenolic acid found in many berries. This
change in GCSh promoter activity after berry treatment
occurred in only ⬃50% of the mice, indicating that they
were either responders or nonresponders. Our results
demonstrate for the first time that berry extracts rich in
polyphenols and EA can induce GCSh in vivo. The induction
of protective enzymes may be important for the chemopreventive effects of fruits and vegetables.
J. Nutr. 133:
2137–2140, 2003.
KEY WORDS: ● ␥-glutamylcysteine synthetase ● transgenic reporter mice ● luciferase ● antioxidants ● berries
MATERIALS AND METHODS
Materials. Luciferin (Biothema, Dalarö, Sweden), dithiothreitol
(Sigma, St. Louis, MO), Tricine (Sigma), ATP (Roche Diagnostics,
Ottweiler, Germany), CoA (Roche Diagnostics), EDTA (Merck,
Darmstadt, Germany) and Mg2SO4 (Merck) were used in the luciferase assay. Ellagic acid (LKT Laboratories, Minneapolis, MN),
blackberries (Rubus fruticosus) (Polarica Poland, Swidwin, Poland),
blueberries (Vaccinius myrtillus) (Polarica Poland) and berry juices
(blueberry: Vaccinius myrtillus, crowberry: Empetrum hermaphroditum,
elderberry: Sambucus nigra) (Helios, Slemmestad, Norway) were fed
to the mice. None of the juices contained added sugar and were low
in energy content (163 kJ/100 g).
A diet rich in fruits and vegetables reduces the incidence of
major diseases such as cancer, cardiovascular disease, diabetes,
cataract and inflammatory diseases (1,2). Because the active
compounds and the mechanisms involved in this protective
effect are poorly understood, the recommendations are that
eating a variety of fruits and vegetables will provide the best
protection (1,2).
Fruits and vegetables contain several hundred different
1
Supported by grants from the Norwegian Research Council, The Johan
Throne Holst Nutrition Research Foundation and The Norwegian Cancer Society.
2
These authors contributed equally to this paper.
3
To whom correspondence should be addressed.
E-mail: rune.blomhoff@basalmed.uio.no.
4
Abbreviations used: EA, ellagic acid; GCSh, ␥-glutamylcysteine synthetase
heavy subunit; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione.
0022-3166/03 $3.00 © 2003 American Society for Nutritional Sciences.
2137
2138
CARLSEN ET AL.
Generation of transgenic mice. A linearized reporter construct
(Hind III and Bam HI digestion) of a luciferase driven by 3.8 kb of the
GCSh promoter (GCSh-luc) (15) was injected into fertilized zygotes
(17). Founder M1 was selected for further work and crossed with wild
type F1 mice to yield heterozygous GCSh-luc mice in the (C57 BL/6J
⫻ CBA/J)F1 background. Wild-type C57 BL/6J and CBA/J mice were
obtained from Bomholtgård, Ry, Denmark.
Experiments with mice
Long-term feeding. Transgenic mice age 7– 8 mo were divided
into two groups (n ⫽ 10/group) and had unrestricted access to water
or berry juice as the sole drinking source for 3– 4 wk. They consumed
nonpurified diet (Standard diet no. 1, B&K Universal, Hull, UK)
throughout the study. Luciferase activity in homogenates from kidney, lung, spleen, abdominal skeletal muscle, brain (one hemisphere),
liver and small intestine was measured. The berry juice was a mixture
of blueberry, elderberry and crowberry (1:1:1). The same experiment
was conducted three times and the luciferase activities from each
experiment were normalized to the mean of the three control groups,
combined and analyzed statistically.
Short-term feeding. Frozen blueberries or blackberries were
blended in double distilled H2O (103 g berry/L H2O) to give a
homogenous solution. Mice (10/group) were given four doses of 250
␮L blueberry or blackberry solution by oral gavage over a period of
40 h. The control group consisted of 10 mice that were fed water. The
luciferase activities in abdominal muscle, tibialis anterior, gastrocnemius, brain and liver were measured and normalized to the mean
control activity. Luciferase activities in the three skeletal muscles
analyzed in the short-term experiment were correlated with one
another; we therefore presented the data in Figure 1 B as the means
of the three muscle types. The two berry groups were combined
(because they did not differ from one another) and compared with
the control group for statistical analyses.
Ellagic acid treatment. Mice were given a single dose of EA (6
mg/kg in 300 ␮L) or a similar amount of water (8 mice/group) by
gavage, and homogenates were assessed for luciferase activity after
18 h. In all experiments, mice were killed by cervical dislocation;
organs were removed, rinsed in PBS and frozen in liquid nitrogen. All
animal experiments were performed according to national guidelines
for animal welfare.
Luciferase measurements. Luciferase activity in tissue homogenates was measured as described (18).
Quantitative mRNA analysis. mRNA was isolated from ⬃50
mg of tissue and cDNA was analyzed by PCR amplification as described (16). The PCR conditions were 4 mol/L MgCl2 for GCSh and
glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 10 pmol of
each PCR primer (Eurogentec, Seraing, Belgium) (GCSh primers:
5⬘-AGA AGG GGG AGA GGA CAA AC-3⬘ and 5⬘-AGT GAT
GGT GCA GAG AGC CT-3, G3PDH primers: 5⬘-TCA TCA ACG
GGA AGC CCA TCA CCA TCT TC-3⬘ and 5⬘-GTC TTC TGG
TTG GCA GTA ATG GCA TGG ACT-3⬘), 2 ␮L of LightCycler
DNA Master mix (Roche Diagnostics, Ottweiler, Germany), and
cDNA to a final volume of 20 ␮L. After 10 min preincubation at
95°C, 45 PCR cycles were performed with 15 s denaturation at 95°C,
5 s annealing at 55°C (GCSh) or 60°C (G3PDH), and 12 s extension
at 72°C. Pooled mRNA from abdominal muscle and gastrocnemius
from three responder mice was compared with corresponding mRNA
from three control mice and three nonresponder mice. Values were
normalized against the housekeeping gene G3PDH. The analysis was
performed twice.
Glutathione (GSH) assays. Tissues from mice were homogenized in homogenizing buffer and stored at ⫺80°C until analyses were
performed. For sample preparation, we used an HPLC-based kit
designed for homocysteine analyses (Bio-Rad Laboratories, Oslo,
Norway; #195– 4075). Chromatography was carried out using a
Hewlett Packard HPLC 1100 series system (Agilent, Oslo, Norway)
equipped with low pressure gradient equipment, pump, auto injector,
column oven and a fluorescence detector (excitation at 385 nm and
emission at 515 nm. Optimal separation was achieved using an
Analytical Cartridge (Hamilton, Bonaduz, Switzerland) and MicroGuard Cartridge (ChromTech Ltd, Cheshire, U.K.) in the HPLC
system with a mobile phase of 25% kit solution and 75% methanol.
Statistical analysis. The data are presented as means and quartiles. Nonparametric statistical methods (Mann-Whitney U tests)
were used initially for comparisons between the control and berrytreated groups because most of the variables were not normally
distributed and the number of observations limited. Spearman’s rank
correlation coefficients were calculated to evaluate relationships between different variables. Comparisons between responders, nonresponders and controls were by Students t-tests. Statistical analysis was
carried out using SPSS 11 (SPSS, Chicago, IL). Differences with
P-values ⬍ 0.05 were considered significant.
RESULTS
FIGURE 1 Effect of long- (panel A) and short-term (panel B) berry
feeding on ␥-glutamylcysteine synthetase heavy subunit (GCSh) promoter activity in transgenic reporter mice. Values are expressed as a
percentage of the mean of the water-fed controls. Box plots show
means, quartiles and extremes of 30 control and 30 berry fed mice
(panel A), and 10 controls and 20 berry fed mice (panel B). Asterisks
indicate a difference from the controls; **P ⬍ 0.01, *P ⬍ 0.05.
Berry juice induces GCS-promoter activity in skeletal
muscle and brain. Long-term feeding of berry juice to mice
increased luciferase activity in the abdominal skeletal muscle
compared with control mice (P ⫽ 0.009, Fig. 1A) and tended
to increase it in brain (P ⫽ 0.082). Luciferase activity tended
to be lower (15%; P ⫽ 0.192) in liver of treated mice (Fig.
1A). Minor differences between groups (⬍ 10%) were noted
in other organs (not shown).
Short-term feeding of berries elevated GCSh promoter–
mediated luciferase activity 54% in brain (P ⫽ 0.046) compared with the control group (Fig. 1B). The increase in luciferase activity in the brain was more pronounced than that in
the muscle (35%; P ⫽ 0.397). Furthermore, activity tended to
be lower (21%; P ⫽ 0.338) in the liver of berry-treated mice.
Differences between the treated and control groups in kidney,
lung, spleen, and small intestine were all ⬍10% (data not
shown).
MODULATION OF OXIDATIVE STRESS–RELATED GENE EXPRESSION
Effect of a single dose of EA. Treatment with EA resulted
in similar differences in luciferase activity as in the mice fed
berries. When luciferase activities from gastrocnemius, tibialis
anterior and abdominal muscle were combined, that in treated
mice tended to be greater than in controls (35%; P ⫽ 0.152).
Brain activity also tended to be greater (64%; P ⫽ 0.200).
Liver luciferase did not differ between groups.
Identification of responder and nonresponder mice. In
berry-treated mice, we observed consistently positive and significant correlations in luciferase activity between abdominal
skeletal muscle and brain (long-term study, r ⫽ 0.500, P
⫽ 0.004; short-term study, r ⫽ 0.668, P ⫽ 0.002). Additionally, negative correlations were found between abdominal
skeletal muscle and liver (long-term study, r ⫽ ⫺0.795, P
⬍ 0.001; short-term study, r ⫽ ⫺0.769, P ⬍ 0.001), and brain
and liver (long-term study, r ⫽ ⫺0.370, P ⫽ 0.037; short-term
study, r ⫽ ⫺0.476, P ⫽ 0.046). Similar correlations were also
observed in the short-term study for tibialis anterior (liver, r
⫽ ⫺0.740, P ⫽ 0.001; brain, r ⫽ 0.745, P ⫽ 0.001) and
gastrocnemius muscles (liver, r ⫽ ⫺0.513, P ⫽ 0.030; brain, r
⫽ 0.752, P ⫽ 0.001). In control mice these correlations were
not evident, indicating that the berry treatment per se elicited
a coordinated response in muscle, brain and liver.
Based on the distribution of the data, transgenic mice were
apparently either responders or nonresponders to the berry
treatment. Those mice that had an activity in skeletal muscle
above the median were defined as responders (long-term treatment: 16 responders out of 32 mice; short-term treatment: 9
responders out of 18 mice). Interestingly, when data from the
above experiments were reanalyzed, GCSh promoter activity
in brain of responders was greater than in nonresponders in
both the long-term (84% increase, P ⫽ 0.001) and short-term
(97% increase, P ⫽ 0.04) feeding studies (Fig. 2). In liver,
2139
FIGURE 3 Comparison of ␥-glutamylcysteine synthetase heavy
subunit (GCSh) mRNA level and luciferase activity in skeletal muscle
from responder and nonresponder mice after short-term berry feeding.
Values are expressed as means ⫾ SD of mRNA levels [normalized to
glyceraldehyde-3-phosphate dehydrogenase (G3PDH)] and luciferase
activities (normalized to the mean of all controls) in controls (n ⫽ 3),
responders (n ⫽ 3) and nonresponders (n ⫽ 3).
luciferase activity was lower in the responder group than in the
nonresponder group (long term: P ⫽ 0.001, short term: P
⫽ 0.003). Luciferase activities in nonresponders did not differ
from the water-fed controls. Alternatively, when a similar
strategy was used to identify responders and nonresponders on
the basis of brain activities, luciferase activity was higher in
skeletal muscle in the responder group than in the nonresponder group (long term: P ⫽ 0.001, short term: P ⫽ 0.010).
Muscle glutathione. The short-term berry treatment increased GSH concentrations in gastrocnemius (87%; P
⫽ 0.001) and heart (38%; P ⫽ 0.012) but not in other muscle
types (tibialis anterior and abdominal). In the long-term study
in which we sampled only abdominal muscle, GSH did not
differ between groups.
GCSh mRNA in skeletal muscle. The GCSh mRNA level
in the responders tended to be greater than in the controls
(238%; P ⫽ 0.076) and the nonresponders (257%; P ⫽ 0.075).
The level in the nonresponder groups did not differ from the
control group. These results were consistent with the luciferase
activities in tissues of the same mice (Fig. 3).
DISCUSSION
FIGURE 2 Identification of responder and nonresponder mice
after long- (A) and short-term (B) berry feeding. Values are expressed as
means ⫾ SD of responders and nonresponders. Asterisks indicate a
difference from the controls; **P ⬍ 0.01, *P ⬍ 0.05.
The transcriptional regulation of the GCS genes by chemoprotective agents has been studied extensively in vitro,
showing that a number of phenolic substances can increase
GSH levels by altering GCS status. Recently, we demonstrated that a plant extract rich in flavonoids, in particular
quercetin, induced the GCSh promoter in COS-1 and HepG2
cells leading to a concurrent increase in GSH (16). Few
reports exist, however, of the regulation of GCSh in vivo using
naturally occurring compounds. Therefore, we used a transgenic reporter mouse model containing the complete GCSh
promoter coupled to luciferase to test the hypothesis that a
diet rich in polyphenols can transcriptionally regulate GCSh.
In the present study, we demonstrated that the GCSh
promoter activity in transgenic reporter mice generally is increased in brain and muscle, and decreased in liver in shortand long-term feeding experiments with berries. Interestingly,
we observed a substantial variation in the response to berry
treatment. Only about half of the mice responded. Thus, mice
were either responders or nonresponders with respect to treatment with berries. This phenomenon may be related to the
observation that some polyphenols require metabolism by
CARLSEN ET AL.
2140
phase I enzymes to create metabolites that modulate transcription (19). Mice are polymorphic with respect to phase I
cytochrome P450 enzymes, and various mouse strains, among
them CBA and C57 BL/6J, exhibit substantial differences in
their ability to metabolize xenobiotics (20). The mice used in
the present study were a cross between C57BL/6J and CBA.
Our data indicated that the transcriptional regulation of
GCSh is tissue specific and may therefore be dependent on the
transcriptional machinery present in various cell types. In
brain, berry feeding upregulated GCS. Increased antioxidant
defense in brain may slow down the aging process and improve
brain functions. Joseph and co-workers (5,6) demonstrated
that long-term feeding of strawberries and blueberries to rats
can retard, and even reverse the onset of age-related neurological problems such as declines in neuronal signal-transduction, and cognitive behavioral and motor deficits.
We observed that berry feeding induced GCSh activity in
skeletal muscle, in agreement with Galli and co-workers (21)
who found that a blueberry diet decreased markers of inflammation compared with control rats. A protective effect of
berries in skeletal muscles may be relevant for physical exercise
because several studies have shown increased markers of oxidative stress and decreased glutathione levels in skeletal muscle after exhaustive exercise (22,23).
In parallel with the induction of GCSh promoter activity in
muscles by berries, total glutathione was also increased in some
but not all types of muscles. In a previous study by Shepherd
and co-workers (24), induction in GCS mRNA by phytochemicals was often not reflected in elevation of GSH. Thus,
although GCS normally catalyzes the rate-limiting step in
GSH production, levels are also regulated by other mechanisms.
Plant phenols from berries are absorbed in the intact form
as demonstrated in recent studies in rats (25,26). The total
intake of plant phenols by humans is assumed to be ⬃1 g/d,
making these compounds an important part of the diet (27).
Thus, the induction of expression of protective enzymes by
plant phenols may be an important aspect of the chemopreventive effects of fruits and vegetables.
ACKNOWLEDGMENTS
We thank R. Timothy Mulcahy for the kind gift of the GCSh
promoter construct. We also thank Kari Holte for excellent help with
the animal work. Berry juices were generously provided by Helios
(Helios, Slemmestad, Norway).
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