Polyphenols and glutathione synthesis regulation1– 4
Jan Ø Moskaug, Harald Carlsen, Mari CW Myhrstad, and Rune Blomhoff
ABSTRACT
Polyphenols in food plants are a versatile group of phytochemicals
with many potentially beneficial activities in terms of disease prevention. In vitro cell culture experiments have shown that polyphenols possess antioxidant properties, and it is thought that these activities account for disease-preventing effects of diets high in
polyphenols. However, polyphenols may be regarded as xenobiotics
by animal cells and are to some extent treated as such, ie, they interact
with phase I and phase II enzyme systems. We recently showed that
dietary plant polyphenols, namely, the flavonoids, modulate expression of an important enzyme in both cellular antioxidant defenses
and detoxification of xenobiotics, ie, ␥-glutamylcysteine synthetase.
This enzyme is rate limiting in the synthesis of the most important
endogenous antioxidant in cells, glutathione. We showed in vitro
that flavonoids increase expression of ␥-glutamylcysteine synthetase and, by using a unique transgenic reporter mouse strain, we
showed increased expression in vivo, with a concomitant increase in
the intracellular glutathione concentrations in muscles. Because glutathione is important in redox regulation of transcription factors and
enzymes for signal transduction, our results suggest that polyphenolmediated regulation of glutathione alters cellular processes. Evidently, glutathione is important in many diseases, and regulation of
intracellular glutathione concentrations may be one mechanism by
which diet influences disease development. The aim of this review is
to discuss some of the mechanisms involved in the glutathionemediated, endogenous, cellular antioxidant defense system, how its
possible modulation by dietary polyphenols such as flavonoids may
influence disease development, and how it can be studied with in
vivo imaging.
Am J Clin Nutr 2005;81(suppl):277S– 83S.
KEY WORDS Polyphenols, blueberries, glutathione, ␥-glutamylcysteine synthetase, gene regulation
have antioxidant properties (ie, reductants) and may react directly with reactive chemical species, forming products with
much lower reactivity. Alternatively, compounds in a plantbased diet may increase the capacity of endogenous antioxidant
defenses and modulate the cellular redox state. Changes in the
cellular redox state, conveying physiologic stimuli through regulation of signaling pathways, may have wide-ranging consequences for cellular growth and differentiation (3). In addition, it
has been well documented that polyphenols modulate protein
kinase activities (4), serve as ligands for transcription factors (5),
and modulate protease activities (6).
Many dietary polyphenols are antioxidants, and the possibility
exists that they protect against oxidative damage by directly
neutralizing reactive oxidants. We recently measured antioxidant capacity (ie, electron- or hydrogen– donating capacity) systematically in a variety of dietary plants, including various fruits,
berries, vegetables, cereals, nuts, spices, and pulses used
throughout the world. Our results (7) demonstrated a 쏜 1000fold difference in total antioxidant capacity among dietary
plants. Interestingly, berries (particularly blackberries, blueberries, and elderberries) high in antioxidant capacity are among the
plants with the highest concentrations of polyphenols. This is
intriguing, because many berries have been shown to protect
against oxidative stress-related pathologic conditions in vivo.
For example, Joseph et al (8, 9) demonstrated that long-term
feeding of blueberries to rats retarded and even reversed the onset
of age-related neurologic dysfunctions, such as a decline in neuronal signal transduction, and cognitive, behavioral, and motor deficits. Stoner and coworkers (10, 11) showed that supplementation
with black raspberries in the diet reduced the multiplicity and incidence of esophageal tumors in N-nitrosomethylbenzylaminetreated rats.
OXIDATIVE STRESS AND ANTIOXIDANTS
The hallmark of oxidative stress is increased production of
reactive oxygen species (ROS), in amounts that exceed cellular
antioxidant defenses. The consequence of oxidative stress may
be oxidative damage of lipids, proteins, and DNA, with subsequent disease development and aging (1). ROS production may
result from exogenous factors such as radiation and drug exposure or endogenous factors such as increased mitochondrial respiration and oxidative enzymes in infections and inflammation.
A possible mechanism for the protective effects of fruits and
vegetables with respect to disease is that bioactive compounds in
these food items reduce oxidative stress. Fruits and vegetables
contain several thousand structurally diverse phytochemicals, of
which a large fraction are polyphenols (2). Many polyphenols
POLYPHENOLS AND ␥GCSh GENE REGULATION
The most important endogenous antioxidant defense systems
are composed of the thiol-containing tripeptide glutathione and
1
From the Institute for Nutrition Research, University of Oslo.
Presented at the 1st International Conference on Polyphenols and Health,
held in Vichy, France, November 18 –21, 2004.
3
Supported by grants from the Norwegian Research Council, the Johan
Throne Holst Nutrition Research Foundation, and the Norwegian Cancer
Society.
4
Address reprint requests to R Blomhoff, Institute for Nutrition Research,
Faculty of Medicine, University of Oslo, PO Box 1046 Blindern, 0316 Oslo.
E-mail: rune.blomhoff@basalmed.uio.no.
2
Am J Clin Nutr 2005;81(suppl):277S– 83S. Printed in USA. © 2005 American Society for Clinical Nutrition
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small thiol-containing proteins such as thioredoxin, glutaredoxin, and peroxiredoxin. Of these, glutathione is found at millimolar concentrations in most cells and is the major contributor
to the redox state of the cell. Glutathione exists in cells in both a
reduced form (GSH) and an oxidized form (GSSG); it may also
be covalently bound to proteins through a process called glutathionylation (12, 13). The ratio of GSH to GSSG is determined by
the overall redox state of the cell. Glutathione is synthesized
enzymatically by ␥-glutamylcysteine synthetase (␥GCS) and
glutathione synthetase, with the former being the rate-limiting
enzyme (14).
One important task for cellular glutathione is to scavenge free
radicals and peroxides produced during normal cellular respiration, which would otherwise oxidize proteins, lipids, and nucleic
acids. One mechanism operating to counteract oxidative damage
involves transactivation of genes encoding enzymes that participate in glutathione metabolism and synthesis. Typically, these
enzymes belong to the phase I and II families of detoxification
genes. Analyses of promoter regions of these enzymes suggest
that several gene response elements may be involved in such
transcriptional regulation, including xenobiotic response elements and antioxidant/electrophil response elements (AREs/
EpREs) (15, 16). The latter is defined by a specific consensus
sequence of nucleotides (17) and responds to substances with
antioxidant properties (18).
Polyphenols are among the most abundant phytochemicals in
human food items and, of these, flavonoids are probably the most
well studied. The mechanism of action of flavonoids in cellular
processes is still not clearly understood. In a series of experiments, we showed that relatively low concentrations of flavonoids stimulated transcription of a critical gene for GSH synthesis in cells (19). Transcriptional control of the catalytic
subunit of ␥GCS, ␥GCSh, is mediated by a 5' flanking region
containing several response elements, including AP-1 sites, one
NF-␬B site, and several AREs/EpREs (20). Both onion extracts
and pure flavonoids transactivated ␥GCSh through antioxidant
response elements in the promoter in both COS-1 cells (19) and
HepG2 cells (Figure 1), with quercetin being the most potent
flavonoid. Structurally similar flavonoids were not as potent;
myricetin, with only one hydroxyl group more than quercetin,
was inactive, which emphasizes the apparent specificity of
␥GCSh induction.
Flavonoids in food items are conjugated to various sugar molecules, which likely influence their intestinal absorption, transport, and entry into cells. Conjugates of quercetin were found to
be totally inactive in tissue cell cultures (19), which emphasizes
the importance of in vivo experiments, in which the activity of
tested substances reflects bioavailability, metabolism, cellular
activity, and excretion of micronutrients. To this end, we developed a transgenic mouse strain with 앑3.8 kilobases of the ␥GCS
promoter upstream of luciferase incorporated into its genome.
We used this mouse strain to test the ability of polyphenol-rich
berries to modulate ␥GCSh gene expression, with a novel in vivo
whole-body or ex vivo whole-organ imaging technique (20) and
analysis of gene expression in tissue homogenates and sections
(21) (Figure 2). For monitoring of basal ␥GCSh promoter activity in the transgenic mice, D-luciferin was injected intravenously and the mice were placed on their dorsal sides in a lightsealed chamber and underwent imaging with an ultrasensitive
video camera. Strong luminescence was detected from the whole
FIGURE 1. ␥GCS promoter–mediated transactivation of luciferase in
HepG2 cells. HepG2 cells were plated 2 d before mRNA isolation, at a
density of 앑60% confluence. Cells were treated with quercetin dissolved in
dimethylsulfoxide (DMSO), or with dimethylsulfoxide as a control, for 17 h
before mRNA was isolated with oligo(dT)16 beads, according to the manufacturer’s protocol (Genovision, Oslo) (19). cDNA synthesis was performed
with an Omniscript kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. cDNA from each sample was analyzed with polymerase chain reaction amplification with specific primers for ␥GCSh and ␥GCS
and a Fast Start DNA Master Sybrgreen I kit (Roche Diagnostics, Ottweiler,
Germany) in a Light Cycler (Roche Diagnostics). After amplification, data
analyses were performed with the second-derivative maximum method. The
second-derivative maximum values were used to plot cycle number versus
log concentration of the standard plasmid. The actual numbers of ␥GCSh and
␥GCSl cDNA copies were related to that of ␤-actin in each sample. The
identity of the polymerase chain reaction products was confirmed with melting curve analyses and size determinations.
animal, reflecting ubiquitous expression of ␥GCSh and glutathione synthesis. Several organs were then excised and subjected to
ex vivo imaging (Figure 2). Interestingly, strong luminescence
was emitted from brain, muscle, and epididymis/testis, whereas
kidney and lung demonstrated medium luminescence and liver,
spleen, and heart showed much lower activity. When transgenic
animals were fed juice or homogenates made from antioxidantrich berries for 3– 4 wk, we observed that ␥GCSh promoter
activity was modulated in organs such as skeletal muscle, brain,
and liver (21). We also measured glutathione concentrations in
the organs in which modulation of the ␥GCSh promoter was
observed. The only organ in which we could detect statistically
significant increases in total glutathione concentrations was gastrocnemius muscle (Figure 3).
In vivo feeding experiments with polyphenol-rich diets revealed large differences in ␥GCSh promoter activity responses
among individual animals. Some animals responded and some
did not. One possible explanation for this phenomenon may be
related to differences in bacterial populations in the gut microbial
flora influencing the extent of enzymatic hydrolysis of polyphenol conjugates. Indeed, Aura et al (22) showed that conjugates of
flavonoids are hydrolyzed by the gut flora among humans, and
several studies demonstrated that sugar moieties influence enterocytic uptake and thus tissue distribution (reviewed in ref. 2).
MECHANISMS OF ACTION OF FLAVONOIDS
Gene regulation through binding of the transcription factor
Nrf2 to AREs/EpREs has been described in some detail (for
review, see ref 23). Although our studies indicate that the effects
POLYPHENOLS AND GLUTATHIONE
FIGURE 2. In vivo and ex vivo imaging of GCS promoter activity in
transgenic mice. Imaging of transgenic mice was performed with an ultrasensitive camera consisting of an image intensifier coupled to a chargecoupled device camera (C2400-47; Hamamatsu City, Japan) fitted with a
25-mm macro lens (Schneideroptics, Hauppauge, NY). Before imaging,
mice were anesthetized (Hypnorm/Dormicum; Roche, Basel, Switzerland)
and D-luciferin (120 mg/kg; Biothema, Dalaro, Sweden) dissolved in 200 ␮L
phosphate-buffered saline (pH 7.8) was injected intravenously in the tail
vein. Immediately afterward, the mice were placed in a light-sealed chamber
connected to the ultrasensitive camera. Luminescence emitted from each
mouse was integrated for 5 min, starting 2 min after the injection of luciferin
(20). Individual organs to be imaged were excised from the mice 3 min after
intravenous injection of D-luciferin (120 mg/kg). Organs were then placed in
a culture dish and immediately imaged as described. The pseudo-colored
images represent light intensity (white being strongest and blue weakest). All
images were processed with Image-Pro Plus 4.0 software (Media Cybernetics, Silver Spring, MD) integrated with the HPD-LIS module developed by
Hamamatsu.
of polyphenols are mediated through one or several Nrf2 binding
sites (ie, AREs/EpREs), several activities of flavonoids could be
responsible for the effect on the ␥GCSh promoter. Nrf2 is inactivated through cytosol sequestering through binding to the
cytoskeleton-associated protein Keap1. Binding of Nrf2 to
Keap1 is thought to be dependent on critical thiols in Keap1,
thiols that are possibly sensitive to the cellular redox state. Therefore, release and subsequent translocation of Nrf2 to the nucleus
are presumably sensitive to cellular oxidative stress, thiolreactive compounds, and antioxidants (24) (Figure 4). Because
ARE/EpRE-containing gene promoters are thought to be regulated by substances with antioxidant properties, a possible explanation for the different ␥GCSh promoter–inducing activities
could be that some flavonoids are better antioxidants than others.
However, when we measured the ability of flavonoids to reduce
Fe3ѿ in the ferric ion-reducing activity assay, which is commonly used to measure total antioxidant capacity in biologic
samples (25), we found that the antioxidant capacity did not
correlate with ␥GCSh-inducing activity in COS-1 cells. Myricetin had a higher antioxidant capacity than did kaempferol but it
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FIGURE 3. Measurement of GSH in mouse gastrocnemius. Mice were
gavage-fed several doses of a mixture of blueberries and blackberries in a
period of 40 h (21). Muscle tissue from the mice was homogenized, and total
glutathione concentrations were measured with an HPLC assay (catalog no.
195-4075; Bio-Rad Laboratories, Hercules, CA). Chromatography was performed with an analytic cartridge (Hamilton, Bonaduz, Switzerland) and a
MicroGuard cartridge (ChromTech, Cheshire, United Kingdom), with a mobile phase of 25% Bio-Rad solution/75% methanol and a fluorescence detector (excitation at 385 nm and emission at 515 nm). A nonparametric
Mann-Whitney U test was used for comparison of animals fed water or berry
juice for 3– 4 wk (**P 쏝 0.01).
did not transactivate the ␥GCSh promoter (19) (Figure 5), which
suggests that transcriptional ␥GCSh regulation depends on other
properties of the flavonoids.
Several activities of flavonoids should be considered in this
respect. First, flavonoid antioxidant scavenging of free radicals
often involves formation of a radical of the flavonoid itself.
Quercetin is oxidized to a quinone when serving as an antioxidant, and Boots et al (26) recently showed that such quinones
reacts with thiols. Therefore, it could be speculated that free
radical-oxidized quercetin reacts with thiols in Keap1, the key
regulatory protein in transcriptional regulation of antioxidantresponsive genes through Nrf2.
Second, the chemical properties of some antioxidants may
also give them prooxidant properties (27), and this should be
considered with respect to mechanisms for induction of cellular
antioxidant defenses. Flavonoids can auto-oxidize (27), and
products of auto-oxidation can possibly react with or otherwise
reduce cellular concentrations of glutathione. Quercetin and
myricetin are known to auto-oxidize at physiologic pH (28),
and subsequent reduction of glutathione concentrations can
possibly explain transcriptional up-regulation of both ␥GCS
subunits (29).
Third, it can be speculated that the effect is mediated by increased mitochondrial production of hydrogen peroxide and superoxide anion. Hodnick et al (30) showed that quercetin and
myricetin cause mitochondrial respiratory bursts. However,
myricetin was found to be the most efficient enzyme inhibitor
and producer of mitochondrial respiratory bursts, whereas kaempherol was the least efficient and quercetin was intermediate.
Therefore, there seems to be no correlation between the ability to
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FIGURE 4. Schematic illustration of ARE/EpRE-mediated regulation of ␥GCS. See text for details.
modulate mitochondrial respiration and the ␥GCSh promoter
induction observed in our studies (19).
The aforementioned explanations depend on potentially deleterious effects of flavonoids. How deleterious effects may be
translated into disease-preventing effects may not be obvious,
but the principle of hormesis has been suggested to explain how
repeated challenges, such as repeated low oxidative stress, may
adapt cells and organs and in the long term make them more
resistant to severe oxidative stress (31). The hormetic principle
has been proposed to explain why physical activity and exercise,
which increase oxidative stress in various tissues, can significantly decrease the risk of disease development (32). It is possible that repeated mild cellular oxidative stress induced by flavonoids through the diet boosts cellular antioxidant defense
systems and in the long term shifts these defense systems to a
FIGURE 5. Total antioxidant activity of pure flavonoids, as measured
with ferric ion-reducing activity assays. Pure flavonoids were dissolved in
ethanol and reducing activity values were determined as described by
Benzie et al (25), with automated measurements of absorbance changes at
600 nm (7).
higher steady state, which prevents disease development or reduces the impact of oxidative stress when disease occurs. Nrf2mediated transactivation is also influenced by phosphorylation
(33), making it possible that the protein kinase inhibitory activities of flavonoids may be involved.
CELLULAR EFFECTS OF ALTERED GLUTATHIONE
METABOLISM
Detoxification of xenobiotics requires sufficient glutathione
synthesis for conjugation and excretion of GSH-conjugated metabolites. Therefore, polyphenol-stimulated glutathione synthesis could well be beneficial in cellular handling of carcinogenic
substances, for example. The role of glutathione in detoxification
was recently reviewed (34).
The intracellular glutathione concentration and the redox status of the cell are likely to influence regulation of protein function
through glutathionylation, ie, covalent binding of glutathione to
the protein through disulfide bonds. Until recently, glutathionylation of proteins had been described for only a few cellular
proteins. A proteomic approach taken by Fratelli et al (35) identified 38 proteins that are glutathionylated in oxidatively stressed
T lymphocytes.
Two mammalian transcription factors known to be glutathionylated are AP-1 and NF-␬B (36), but the role of such covalent
modification in the cell nucleus is still under debate. Determination of the roles of transcription factors in the regulation of many
disease-associated genes in cancer and chronic inflammatory
diseases requires detailed studies to establish the influence of
cellular glutathione on their activity. Many transcription factors
contain thiol groups that have been shown to influence DNA
binding. Redox switching and regulation of transcription factors
were recently described in great detail, particularly for single-cell
organisms (37). Because glutathione is the fundamental redox
regulator in eukaryotic cells, it is conceivable that principles of
GSH-mediated redox switching of transcription factors can be
extrapolated from single cells to multicellular organisms.
POLYPHENOLS AND GLUTATHIONE
Neurodegenerative diseases such as Parkinson’s disease, involving dopaminergic neurons, are associated with neuronal oxidative stress. It was recently shown that the dopaminesynthesizing enzyme tyrosine hydroxylase is inhibited by
reversible glutathionylation during oxidative stress (38).
Cellular turnover of proteins is mediated to a large extent by
proteasomes. Recent evidence from Demasi and Davis (39)
showed that protein turnover attributable to proteasomes is regulated by intracellular glutathione. Those authors found that glutathionylation of 20 S proteasomes and hydrogen peroxideinduced oxidative stress inhibited the proteolytic activity of
proteasomes.
Enzymes such as protein kinases and phosphatases have been
shown to be modulated by critical thiol groups (for a recent
review, see ref 40), and protein kinase A can be regulated directly
through glutathionylation (41). Intracellular concentrations of
glutathione clearly play a role in the regulation of protein tyrosine
phosphatase SHP-1, which is oxidized and inactivated by hydrogen peroxide and can be re-reduced and activated by thiols such
as glutathione (42).
Intracellular glutathione also influences immune responses.
Production of major immune regulatory cytokines such as
interleukin-12 has been shown to be regulated by intracellular
glutathione (43). Even cell cycle transitions involving cyclin D1
appear to be redox regulated, because thiol-containing antioxidants delay the transition from G0 to G1 and S phases in embryonic mouse cells (44).
GLUTATHIONE AND DISEASE
Oxidative stress is a general term intimately linked to production of reactive oxygen, nitrogen, or iron species and the overall
redox state of the cell. Oxidative stress can be assessed by measuring GSH and GSSG, and it is frequently expressed as the ratio
between the two. Indeed, the ratio has been suggested as a clinical
marker in diseases in which oxidative stress is particularly important. Interestingly, GSH is oxidized to GSSG in an agedependent manner, possibly reflecting accumulating oxidative
stress (45). A decreased ratio between GSH and GSSG is also
associated with progression of tumors (46), and decreased total
glutathione concentrations were found among patients with
chronic diseases, including genitourinary, gastrointestinal, cardiovascular, and musculoskeletal diseases (47). Oxidative stress
in general and GSH concentrations in particular are also associated with neurodegenerative disorders and HIV. In Parkinson’s
disease, ROS concentrations are increased in parts of the substantia nigra and glutathione concentrations are decreased (48).
Some improvement among patients with early Parkinson’s disease has been observed with administration of glutathione (49).
Decreased concentrations of glutathione among HIV-infected
individuals may have dual effects. First, decreased concentrations are known to promote apoptosis in the CD4ѿ subtype of T
cells (50). Second, it has been shown that intracellular thiols
regulate HIV transcription (51) and that GSH increases transcriptional activation of HIV through NF-␬B binding sites in the HIV
long terminal repeat (52). These combined effects suggest that
decreased concentrations contribute significantly to HIV/AIDS
development and that supplementation with glutathione precursors (such as N-acetylcysteine) may be of some benefit for HIV/
AIDS patients. Corroborating this, Herzenberg et al (53) found
that GSH concentrations were associated with survival rates in
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HIV. Other viruses also seem to be dependent on the intracellular
redox state for replication. Expression of late influenza viral
proteins was found to be increased when cells were treated to
reduce intracellular glutathione concentrations (54).
Alteration of glutathione concentrations may also be associated with tumor development through dependence on glutathione S-transferases and glutathione peroxidases. These enzymes
are essential in detoxification of carcinogens and scavenging of
ROS. Polymorphisms in various classes of glutathione
S-transferases have been associated with increased development
of tumors of the lung, bladder, and colon (55) and head and neck
(56). Decreased activity of key enzymes involved in glutathione
synthesis, accompanied by decreased availability of cyst(e)ine
synthesis, contributes to mucosal glutathione deficiency in inflammatory bowl disease (57). Glutathione is also critical in the
lung, and altered concentrations have been associated with pulmonary diseases such as acute respiratory distress syndrome (58)
and chronic obstructive pulmonary disease (59).
CONCLUDING REMARKS
Dietary antioxidants have long been suspected to scavenge
reactive ROS and thereby avert deleterious effects on proteins,
lipids, and nucleic acids in cells. This has been put forward as one
of the major mechanisms for the disease-preventing effects of
fruits and vegetables. Our results (19, 21) add modulation of
intracellular GSH concentrations to the list of possible diseasepreventing effects of polyphenols, with the implication that they
modulate GSH-dependent cellular processes, such as detoxification of xenobiotics, glutathionylation of proteins, and regulation of redox switching of protein functions in major cellular
processes.
The potential beneficial effects of flavonoids have prompted
commercial interest in manufacturing supplements containing
high concentrations of flavonoids. However, their concentrationdependent cellular effects, some of which may be harmful,
should be of concern with respect to high-dose supplementation.
Flavonoids at high concentrations produce reactive radicals
through auto-oxidation and increasing mitochondrial respiratory
bursts (27, 30). Although the redox potentials of most flavonoid
radicals are lower than those of the superoxide and peroxyl radicals (60), the effectiveness of the radicals in generating lipid
peroxidation, DNA adducts, and mutations may still be significant in disease development (61). Also of concern is the observation that some flavonoids inhibit enzymes (such as topoisomerases) involved in DNA structure and replication (62), and it has
been suggested that high intake of flavonoids predisposes subjects to the development of certain childhood leukemias (63, 64).
Flavonoid supplementation as a general recommendation to increase cellular glutathione concentrations may also be troublesome, because glutathione has a major role in overall redox
regulation of cell functions and is not suitable as a therapeutic
target for substances that alter cellular concentrations by orders
of magnitudes. Therefore, the current recommendation is to increase fruit and vegetable consumption. Because the active compounds and the mechanisms involved in disease-preventing effects are still poorly understood, the recommendation is that
eating a variety of fruits and vegetables provides the best protection. It remains to be determined whether dietary polyphenols
modulate cellular glutathione concentrations among humans and
whether they contribute to regulation of major cellular signaling
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pathways, which would explain the indisputable fact that fruits
and vegetables protect against disease.
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