Article

pubs.acs.org/JAFC

Coffee Modulates Transcription Factor Nrf2 and Highly Increases the

Activity of Antioxidant Enzymes in Rats
Silvio J. V. Vicente,*,† Emília Y. Ishimoto,‡ and Elizabeth A. F. S. Torres‡
†
Department of Ecotoxicology, Santa Cecília University, Santos, SP, Brazil
‡
Department of Nutrition, School of Public Health, University of São Paulo, São Paulo, SP, Brazil

ABSTRACT: This study investigated the effect of a 28 day administration of coffee brew on the activity of antioxidant enzymes
in rats. After this period of 2.0 mL/day dosages of this beverage, the activities of hepatic superoxide dismutase, catalase, and
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glutathione peroxidase increased 74.8, 59.4, and 135.2%, respectively, whereas the cytosolic level of Nrf2 increased 131.3%. At the
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same time, the total antioxidant capacity of the hepatic tissue increased 25.1%, improving the defensive status against oxidative
stress. At the end of the experiment, the levels of biomarkers alanine transaminase and aspartate transaminase remained equal to
the control group, and no changes were observed in the hepatic histoarchiteture of the animals, suggesting that the liver tissue
was not impaired by the exposure to coffee. The changes in enzyme activities and antioxidant capacity were statistically significant
(p < 0.05), indicating that coffee could be considered an important alternative against oxidative stress and its correlated
degenerative diseases.
KEYWORDS: coffee, hepatic antioxidant enzymes, Nrf2, ORAC, rats

■ INTRODUCTION
Chemoprevention has been accepted as one of the most
successful strategy to fight cancers. In the past decades, studies
have been developed to investigate natural and synthesized
substances capable of restraining carcinogenic processes.1 Some
coffee bioactive substances such as phenolic compounds
(including chlorogenic acids and phenolic acids released by
the hydrolysis of chlorogenic acids), caffeine and other
xanthines, Maillard reaction products, and the diterpenes
cafestol and kahweol have been evaluated, and most results
have suggested that the ingestion of these substances is
inversely related to the incidence of liver and colon cancers.2−7
The degree of roasting also affects coffee antioxidant capacity as
it promotes the formation of Maillard reaction products and the
partial conversion of trigonelline to N-methylpyridinium, both
with defensive capacities.5,7
In general, a tumor is initiated by a permanent modification
of DNA by the action of electrophilic or oxidant species. Once
procarcinogens are introduced in organisms, they usually
require an enzymatic activation to be converted into highly
reactive intermediates that can attack important molecules such
as DNA, RNA, and proteins. These enzymatic processes are
frequently promoted by phase I enzymes. In opposition, phase
II enzymes have the ability to neutralize dangerous species,
generating less reactive and more soluble substances that can be
eliminated from the organisms.1,3,6,8,9 Antioxidant enzymes
such as superoxide dismutase (SOD), catalase (CAT), and
glutathione peroxidase (GPx) also play important defensive
roles, reducing the levels of dangerous species such as O2−
(superoxide anion radical), H2O2 (hydrogen peroxide), and
O22− (peroxide anion). This fact decreases the downstream
formation of other reactive oxygen species (ROS), neutralizing
or reducing their deleterious actions.2,10 Therefore, consid-
erable efforts have been made to identify substances that can
modulate the transcription and increase the activity of these
protective enzymes.
Different studies have produced strong evidence that the key
point for the transcription of phase II and antioxidant enzymes
is the protein nuclear factor-E2-related (Nrf2) factor. Under
normal (reducing) condition, Nrf2 remains anchored to its
inhibitor cytoplasmic complex Keap1−Nrf2 (Kelch-like ECH-
associated protein-1) for its polyubiquitination and subsequent
26S proteasome-mediated degradation. However, under
oxidative stress or exposure to inducers, Nrf2 is disrupted
from this complex, allowing its translocation to the nucleus of
the cells, where it binds to the antioxidant response element
(ARE) or electrophile response element (EpRE) present in the
promoter region of the mentioned enzymes, increasing their
transcription.3,6,8,9 Keap1 presents a dimeric structure formed
through its BTB/POZ (broad-complex, tramtrak, bric-à-brac/
pox virus-zinc finger) domains, sequestering one molecule of
Nrf2 when thiol groups of the cysteine residues C273 and C288
are reduced (−SH). Oxidative stress and inducers act on these
two cysteine residues to form intermolecular disulfides (−S−
S−) that cause a conformational change of Keap1, releasing
Nrf2 to migrate to the nucleus.5,9 A post-translational
phosphorylation also facilitates Nrf2 to dissociate from
Keap1-Nrf2, and this change would be promoted by kinases
such as protein kinase C (PKC), phosphatidyl inositol 3-kinase
(PI3K), and mitogen-activated protein kinase (MAPK).
Advances have accumulated in recent years, but some details
still need to be elucidated on the pathway that activates this
mechanism.8,9
Received: April 25, 2013
Revised: December 9, 2013
Accepted: December 13, 2013
Published: December 13, 2013

© 2013 American Chemical Society 116 dx.doi.org/10.1021/jf401777m | J. Agric. Food Chem. 2014, 62, 116−122
Journal of Agricultural and Food Chemistry
Two studies published experimental evidence that the
administration of phenolic acids (gentisic, gallic, ferulic, and
p-coumaric acids) to rats increased the hepatic and cardiac
activities of SOD, CAT, and GPx.11,12 Simultaneously, these
tissues showed higher levels of cytosolic Nrf2 and higher
expressions of the mRNA for these enzymes. In both cases, a
dose equal to 100 mg/kg of body weight (mg/kg BW) per day
of each phenolic acid dissolved in propylene glycol/saline was
administered to the animals over 14 days.
Aiming to evaluate the above-mentioned beneficial effect
using a natural beverage, we decided to use coffee brew as it
contains high levels of phenolic acids found as a family of
chlorogenic acids.13−15 Additionally, high levels of caffeine are
also found in coffee brew (from 0.9 to 1.3% w/w dry basis in
Coffea arabica; from 1.2 to 2.4% w/w dry basis in Coffea
robusta),16 and this xanthine has been reported as another
bioactive substance capable of modulating antioxidant
enzymes.2,3,6 Another important fact for choosing coffee is
that it is one of the most popular nonalcoholic beverages in the
world,2,5,6,17 and the eventual results of this study would have
wide application.
In a previous publication,18 we discussed the effect of a single
and small dose of coffee brew (from 0.5 to 2.0 mL) on the
activity of hepatic SOD, CAT, and GPx in rats, obtaining
increases of 19.1, 22.1, and 25.1%, respectively, in relation to
the control group. It was shown that doses containing from 2.0
to 8.0 mg/kg BW phenolic acids promoted measurable
beneficial effects. As cited in the present study, besides phenolic
acids, other bioactive compounds present in coffee certainly
collaborated to achieve this positive answer.2−6
Motivated by these encouraging results, we decided to
evaluate the effect of a repetitive administration of small doses
of coffee brew to rats, (1) measuring the effect of this exposure
in the activity of hepatic enzymes SOD, CAT, and GPx and in
the cytosolic concentration of Nrf2; (2) evaluating the changes
in the antioxidative status of the liver tissue; and (3) identifying
any damage to the liver through the quantification of the
hepatic biomarkers alanine transaminase (ALT) and aspartate
transaminase (AST) together with histological observations.
■
MATERIALS AND METHODS
Chemicals. Caffeic, ferulic, and p-coumaric acids, 5-caffeoylquinic
acid (5-CQA), 5-feruloylquinic acid (5-FQA), 2,2′-azobis(2-amidino-
propane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethyl-
chroman-2-carboxylic acid (Trolox), tris(hydroxymethyl)-
aminomethane hydrochloride (Tris-HCl) buffer, and fluorescein
were acquired from Sigma-Aldrich (USA). The commercial kits
Ransod and Ransel for SOD and GPx determinations were acquired
from Randox Laboratories (UK). Hydrogen peroxide (30%), formic
acid, and methanol were acquired from Merck KgaA (Germany). The
kits for the determination of AST and ALT were acquired from
LabTest Ltd.a. (Brazil). The Western blotting reagents were acquired
from Amersham Biosciences (USA).
Coffee Description and Preparation. Medium roast (degree 3)
Brazilian coffee (C. arabica var. Bourbon blended with 15−30% of
Coffea canephora var. Robusta) produced in Minas Gerais state
(Brazil), packed under vacuum in 500 g aluminized bags with an
external cardboard box, was acquired from local stores. Packs were
kept under vacuum, at 4 °C (refrigerator) and in the dark to preserve
coffee characteristics during the tests. All tests were done using fresh
coffee brews prepared with 80 g of coffee powder per liter of mineral
water at 90 °C and filtered through paper filter as recommended by
the Brazilian Association of Coffee Industry (ABIC).17
Animal Characteristics and Treatment. Male rats (Ratus
novergicus var. Wistar) with 200 ± 10 g of BW were provided by
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the Animal Facility of the Medical School, University of São Paulo
(FM-USP). A total of 12 animals were housed in individual plastic
cages for 4 days (adaptation period), with free access to food (purified
diet AIN-76) and water. The temperature was kept at 22 ± 2 °C, and
the animals were exposed to 12 h light/dark cycles. On day 5, they
were randomly distributed in two groups (n = 6) and given 2.0 mL of
water (group 1) or 2.0 mL of coffee brew (group 2) per day by gavage.
After 28 days, the animals were deprived of food for 12 h, anesthetized,
and sacrificed by exsanguination (cardiac puncture). The liver tissue
was washed with sterile ice-cold 0.9% NaCl solution and immediately
frozen in liquid nitrogen. The procedures involving the animals were
conducted at the Institute of Tropical Medicine (IMT-USP) in
compliance with Brazilian laws and approved by the Committee of
Ethics in Research of the IMT-USP under number CEP-IMT-10/07.
Liver Homogenate Preparation. The liver homogenates were
prepared as described by Vicente et al.18 Basically, 1.0 g of washed liver
tissues was homogenized with 3 mL of phosphate buffer (0.1 M, pH
7.0) and centrifuged at 1000g (20 min at 4 °C); the supernatant was
collected and centrifuged at 11200g (20 min at 4 °C), and then the
supernatant was collected, diluted 1:10 (v/v) with phosphate buffer,
and centrifuged at 76900g (60 min at 4 °C). The final supernatant was
collected and stored at −80 °C in the dark until the end of the
analytical determinations (within 48 h of sacrifice). The preparation of
the homogenate was done within 2 h of sacrifice.
Instrumentation. The activities of antioxidant enzymes and the
biomarkers AST and ALT were determined using a spectrophotometer
model 1650 (Shimadzu, Japan). The antioxidant capacity was
measured by oxygen radical absorbance capacity (ORAC) using a
fluorimeter model FL-55 (Perkin-Elmer, UK). Both instruments were
equipped with a 10 mm temperature-controlled cell. Free phenolic
acids and caffeine were quantified by high-performance liquid
chromatography (HPLC) using an equipment supplied by TSP
(USA) and a C18 Microsorb MV column (Varian Inc., USA) with 250
× 4.6 mm (5 μm of particle size). The major chlorogenic acids were
quantified by HPLC-DAD-MS using a chromatograph model 1200 SL
(Agilent Technologies, USA) equipped with a diode array detector
(DAD), a mass spectrophotometer detector (MS), and a Zorbax SB-
C18 column (Agilent Technologies, USA) of 50 × 2.1 mm (1.8 μm
particle size). The electrophoresis for the Western blotting analysis
was developed using a Minive kit (Amersham Biosciences, USA), and
the chemiluminescence was measured using an ECL-Plus ImageQuant
CAS 4000 (GE Healthcare, USA).
Bioactive Compounds in Coffee Brew. Some of the bioactive
substances (caffeic, ferulic, and p-coumaric acids) capable of
modulating the transcription of antioxidant enzymes are present in
coffee as a family of esters called chlorogenic acids formed through
different combinations of these hydroxycinnamic acids with quinic
acid. Taking into account that both chlorogenic acids and free phenolic
acids have been reported as active substances, we decided to measure
these two forms. The major chlorogenic acids present in coffee brew
were quantified by HPLC-DAD-MS according to the method of
Corrêa.19 This analysis was done by injecting 20 μL of coffee brew,
starting with an isocratic elution of 85% of phase A (deionized water
with 0.1% of formic acid) and 15% of phase B (methanol) at 0.32 mL/
min until 2 min. Between 2 and 6 min, a gradient from 15 to 30% of
phase B was developed, ending with second gradient from 30 to 35%
of phase B between 6 and 12 min. Operation conditions for MS were
ionization voltage, 3000 V; N2 flow, 12.0 L/min at 30 psig; and
temperature, 350 °C. A previous standardization of this method was
done (recovery, limit of detection, and limit of quantification),19 the
quantification was performed at 324 nm using external standard curves
(five points, triplicate measurements), and the results were expressed
in micrograms per milliliter. Because coffee brew does not present free
phenolic acids, before the analysis of these substances a previous
hydrolysis was done according to the method of Nardini et al.,20 using
2 N NaOH containing 10 mM EDTA and 1% ascorbic acid to prevent
phenolic acid decomposition. Next, these substances were quantified
by HPLC as suggested by Kowalski and Wolski,21 injecting 20 μL of
hydrolyzed coffee brew. Water/methanol/acetic acid (75:24:1 v/v)
was used as mobile phase at 0.8 mL/min, and the determinations were
dx.doi.org/10.1021/jf401777m | J. Agric. Food Chem. 2014, 62, 116−122
Journal of Agricultural and Food Chemistry Article

done at 323 nm for caffeic and ferulic acids and at 309 nm for p- Windows software package, with a significance level of p < 0.05. All
coumaric acid. A previous standardization of this method was done tests were done in triplicate, and the results are presented as means ±
(recovery, limit of detection, and limit of quantification),15 the standard deviations.
quantification was performed using external standard curves (five
points, triplicate measurements), and the results were expressed in
micrograms per milliliter.
Caffeine is another substance capable of modulating the tran-
■ RESULTS AND DISCUSSION
Bioactive Compounds in Coffee Brew. As the main
scription of antioxidant enzymes, and it was quantified by HPLC as purpose of this study was to evaluate the effect of the
suggested by Vitorino et al.,22 with small adaptations. Briefly, coffee
brew was filtered (0.5 μm), and 20 μL was injected in the HPLC using
water/methanol/acetic acid (80:19:1 v/v) as mobile phase at 1.0 mL/
min. The wavelength 272 nm was selected for the detection of caffeine.
A previous standardization of this method was done, the quantification
was performed using external standard curves (five points, triplicate
measurements), and the results were expressed in micrograms per
milliliter.
Antioxidant Enzyme Activity and ORAC. The activity of SOD
was measured by visible spectroscopy (505 nm) according to the
method of Woolliams et al.,23 with small modifications for the use of
the kit Ransod.15 The liver homogenate was diluted 1:20 v/v with
phosphate buffer (75 mM, pH 7.4) for this assay. The activity of CAT
was determined by ultraviolet spectroscopy (230 nm) using the
method described by Aebi.24 The liver homogenate was diluted 1:5 v/
v with phosphate buffer to determine this enzyme. The activity of GPx
was also determined by ultraviolet spectroscopy (340 nm) according
to the method of Paglia and Valentine,25 with small modifications for
the use of the kit Ransel.15 The liver homogenate was not diluted for
this determination. These enzymatic activities were expressed in units
per milligram of protein, micromoles per minute per milligram of
protein, and units per milligram of protein, respectively. The
quantifications of the proteins for the calculations of the antioxidant
enzymes were done in accordance with Bradford26 using bovine serum
albumin (BSA) as standard.
The ORAC assay was developed by fluorescence spectroscopy
(excitation, 493 nm; emission, 515 nm) according to the methods of
Ou et al.27 and Prior et al.28 with small modifications.15 The liver
homogenate was diluted 1:40 v/v with phosphate buffer, and 300 μL
of this dilution was used in each test. The results were expressed as
micromoles of Trolox equivalent per liter (μmol TE/L).
Hepatic Biomarkers, Histopathological Analysis, and West-
ern Blotting of Nrf2. The hepatic biomarkers AST and ALT were
determined as described by Reitman and Frankel,29 with small
modifications for the use of the kits for these enzymes.15 Both
enzymes were determined by visible spectroscopy (505 nm) using a
calibration curve (five points, triplicate measurements, from 0 to 200 Figure 1. Typical chromatograms obtained during the quantification
IU/L). of free phenolic acids (A) and chlorogenic acids (B) in coffee brew.
For the histopathological analysis, a small section of the liver tissue
was collected after the sacrifice, washed with sterile ice-cold 0.9% NaCl
solution, fixed with 10% formaldehyde (48 h), embedded in liquid
paraffin, and sliced (5 μm thick), the paraffin was removed by heat,
and the tissue was stained with hematoxylin and eosin dyes (H&E) to
be observed under the microscope.30
The determination of Nrf2 was done as described by Yeh and Yen11
with small modifications, using the dye-binding assay proposed by
Bradford26 for the quantification of proteins (BSA as the standard).
Briefly, 10 μL of the cytosolic fraction was separated by 10% SDS−
polyacrylamide gel electrophoresis (20 V, 300 mA). After the
electrophoresis, the proteins were electrotransferred from the gel to
a PVDF membrane (25 V, 400 mA) using 25 mM Tris-HCl buffer
with 20% of methanol; the membrane was washed with Tris-HCl
buffer containing 0.05% (v/v) Tween-20, and free binding sites were
blocked for 2 h with defatted dried milk (7% w/v). Next, the
membrane was incubated overnight at 4 °C with rabbit polyclonal Figure 2. SOD (U/mg of protein), CAT (μmol/min/mg of protein),
antibody against Nrf2, washed three times with Tris-HCl buffer, and GPx (U/mg of protein) average activities in the liver tissue of rats
incubated with IgG secondary antibody coupled with horseradish (n = 6) showing the differences between groups 1 (control) and 2
peroxidase for 1 h at room temperature, and washed with Tris-HCl (coffee). (∗ indicates statistically significant difference from the control
buffer; chemiluminescence was measured. The quantifications were group at p < 0.05.)
adjusted for the corresponding β-actin level (loading control protein).
Statistical Calculations. Statistical calculations (Kolmogorov− administration of coffee brew on the activity of antioxidant
Smirnov and Student’s t test) were done using the SPSS 16.0 for enzymes, it was important to quantify the levels of the bioactive
118 dx.doi.org/10.1021/jf401777m | J. Agric. Food Chem. 2014, 62, 116−122
Journal of Agricultural and Food Chemistry
Figure 3. Cytosolic levels of Nrf2 (relative concentrations) in the liver
tissue of groups 1 and 2 (n = 6): (top) original blotting; (bottom)
average results of the densitometric analysis. (∗ indicates statistically
significant difference from the control group at p < 0.05.)
Figure 4. Total antioxidant capacity measured by ORAC (μmol TE/
L) in the liver tissue of groups 1 and 2 (n = 6). (∗ indicates statistically
significant difference from the control group at p < 0.05.)
Figure 5. AST and ALT average levels in the liver tissue of groups 1
and 2 (n = 6). (No statistically differences from the control group were
observed at p < 0.05.)
substances in this beverage. After the hydrolysis of coffee brew
followed by chromatographic tests, a total of 793.3 μg of free
phenolic acids/mL of coffee was found (685.1 ± 30.8 μg/mL of
caffeic acid, 97.9 ± 8.0 μg/mL of ferulic acid, 10.3 ± 1.3 μg/mL
of p-coumaric acid), confirming that this beverage is a rich
source of these substances originally present as different
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chlorogenic acids (Figure 1A). The total concentration of
phenolic acids was shown to be 28.7−39.7% lower than the
results issued in other publications,13,14 and these differences
could be related to the use of automatic brewing machines in
the mentioned studies (longer contact between coffee powder
and water). Another possible reason could be the origin of
coffee samples. As shown by Farah and Donangelo,31 the
variety Bourbon widely cultivated in Brazil showed the second
lowest result for chlorogenic acids among 19 coffees from
different origins, probably due to the mild Brazilian climate
given that phenolic acids are secondary metabolites produced as
a defensive response against stressful environmental conditions.
There is a lot of controversial data regarding whether
chlorogenic acids or free phenolic acids are actually absorbed.
Some studies affirm that chlorogenic acids are absorbed in the
intestine being found intact in plasma and urine.32,33 However,
most publications mention an extensive hydrolysis of
chlorogenic acids promoted by esterases in the colon to yield
quinic acid and free hydroxycinnamic acids that are rapidly
transformed to different derivatives such as glucuronide and
sulfate conjugates.13,34−36 For this reason, the major chloro-
genic acids present in coffee brew were quantified (Figure 1B),
resulting in 1155.2 μg/mL (1002.3 ± 87.0 μg/mL of 3-, 4-, and
5-CQA, 152.9 ± 10.3 μg/mL of 5-feruloylquinic acid). The
results obtained in the present study were similar to the ones
issued by Boettler et al.5 (1040 μg/mL of 5-CQA) and
Corrêa19 (864.0 μg/mL of 3-, 4-, and 5-CQA isomers, 128.1
μg/mL of FQA isomers), all converted to 80 g/L of Brazilian
Arabica coffee powder. In view of these results, a single dose of
2 mL/day of coffee brew given to rats with 200 g of BW
represented approximately 8 mg of phenolic acids/kg of BW
per day (or approximately 12 mg of chlorogenic acids/kg of
BW per day), which fits the purpose of this study to administer
small doses of the active substances.
Caffeine is another bioactive compound present in coffee.
The level of this substance was also determined in coffee brew,
and an average concentration equal to 728.1 ± 20.5 μg/mL was
found. This value was 39.7% lower than the result issued by
Natella et al.,37 and the possible reasons were already discussed
(the use of automatic brewing machine and the nature of
coffee). This result indicated that 2 mL/day of coffee brew to
rats represented approximately 7 mg of caffeine/kg of BW per
day, which fits the intention to administer small doses of the
bioactive substances.
Antioxidant Enzymes, Nrf2, and Antioxidant Ca-
pacity. The activities of antioxidant enzymes in group 1
(control) showed average results equal to 16.3 ± 1.9 U/mg of
protein for SOD, 10.1 ± 1.0 μmol/min/mg of protein for CAT,
and 16.2 ± 2.4 U/mg of protein for GPx, well-suited to the
figures obtained in other studies with similar animal
models.1,11,12,38,39 At the same time, group 2 (coffee) average
results were 28.5 U/mg of protein for SOD, 16.1 μmol/min/
mg of protein for CAT, and 38.1 U/mg of protein for GPx,
showing increases of 74.8, 59.4, and 135.2%, respectively, in
relation to the control group (Figure 2). The results of groups 1
and 2 were compared (independent samples), and the
differences were statistically significant according to the Student
t test (p < 0.001 for all enzymes). This finding showed that
even a small quantity of a natural beverage containing phenolic
acids (released by the hydrolysis of chlorogenic acids in the
organism), caffeine, and other unquantified bioactive substances
was capable of producing notable changes in the activity of
hepatic SOD, CAT, and GPx. As filtered coffee was used, the
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Journal of Agricultural and Food Chemistry
Figure 6. Liver tissues (group 1, left; group 2, right) showing normal architectures and no apparent damage.
contribution of the diterpenes cafestol and kahweol was
negligible as they were absorbed by the paper filter.3
In a preceding publication, we showed that a single dose of
2.0 mL of coffee brew increased the activities of SOD, CAT,
and GPx, but this effect was transitory and the activities started
to return to basal levels a couple of hours after the
administration.18 In the present study, even after 12 h of
fasting, the enzymatic activities remained much higher than the
levels obtained after a single dose. This indicated that a daily
ingestion of a small dose of coffee brew produced an
accumulative effect that increased and maintained the activities
of antioxidant enzymes far above the basal levels.
Another remarkable issue was the concomitant increase in
the activities of SOD, CAT, and GPx, also observed in the
previous publication using a single dose of coffee.18 As these
enzymes work in a sequential process10,15 (SOD converts O2−
to O22− or H2O2 that is converted to H2O by CAT or GPx), the
simultaneous increases indicate an interesting solution found by
evolution to avoid excessive concentration of any dangerous
intermediate during hepatic detoxification. Increased levels of
SOD could efficiently reduce O2− naturally produced in the
mitochondrial domain to form O 2 2− (or H 2 O 2 ) and
subsequently water by the action of CAT and GPx, protecting
the cells against the attack of these and other downstream ROS.
Because Nrf2 acts in DNA promoter regions as a direct factor
for the transcription of the antioxidant enzymes as well as
itself,3,7−9 it was important to verify if coffee could also affect
the concentration and the expression of this protein. The
cytosolic level of Nrf2 was measured in groups 1 and 2,
showing an increase equal to 131.3% (relative concentrations)
in group 2 in relation to group 1 (Figure 3). Increased levels of
cytosolic Nrf2 certainly promote the translocation of this
protein to the nucleus, activating the transcription of ARE-
responsive genes.3,7−9 These results were compared and
indicated a statistically significant difference according to the
Student t test (p < 0.001). As Nrf2 plays an important role in
the transcription of antioxidant enzymes, its increased level
certainly collaborated to raise the expression of SOD, CAT, and
GPx, resulting in higher activities for these enzymes.
Using the same samples, the total antioxidant capacity of the
liver tissue was measured by ORAC. The control group showed
an average result equal to 689 ± 34 μmol TE/L, which rose to
862 ± 44 μmol TE/L in group 2 (Figure 4), resulting in a
statistically significant difference according to the Student t test
(p < 0.001). As the ORAC assay measures the total antioxidant
capacity of the samples, part of the observed effect must be
assigned to the increased activities of the antioxidant enzymes
and part to the phenolic compounds present in coffee as they
are potent antioxidant molecules through the formation of
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resonance-stabilized phenoxy radicals.39 Once more, the
accumulative effect already mentioned was observed as a single
dose of coffee brew promoted only a small and not statistically
significant increase of ORAC (p = 0.403).18
On the basis of these results it was possible to conclude that
a small and repetitive dose of coffee brew was sufficient to
modulate the protein Nrf2, increasing the activity of antioxidant
enzymes and improving the defensive status against oxidative
stress. By comparison of the two studies involving a single dose
and repetitive doses of coffee, it was possible to verify an
accumulative effect that can be considered a beneficial
consequence for coffee drinkers.
Evolution of Body Weight and Changes in Liver
Tissue. Next, it was important to verify the occurrence of any
damage to the health of the animals. The evolution of body
weight is a parameter that could indicate eventual toxicity of
coffee. At the beginning of the study, group 1 (control) showed
an average weight equal to 203.86 ± 7.00 g, whereas group 2
(coffee) showed an average weight equal to 201.80 ± 6.83 g.
These results were compared, and no statistically significant
difference was obtained (p = 0.617). After the 28 day period,
group 1 showed an average value equal to 349.10 ± 10.97 g,
whereas group 2 showed 356.66 ± 19.55 g, indicating no
statistically significant difference between them (p = 0.428).
This result suggests that coffee did not present systemic toxicity
and did not affect the appetite of the animals or the growing
curve.15
The levels of the hepatic biomarkers AST and ALT were
determined for group 1, showing average results equal to 74.5
± 5.3 IU/L for AST and 30.2 ± 2.9 IU/L for ALT, compatible
with the figures reported in other publications.1,30,39 Group 2
was evaluated using the same methodology, showing average
values equal to 71.5 ± 3.8 IU/L for AST and 27.9 ± 4.0 IU/L
for ALT (Figure 5), not statistically different from group 1
according to the Student t test (p = 0.290 for AST; p = 0.284
for ALT). This finding was an excellent indication that no
injury was caused to the liver by a continued exposure to coffee.
To strengthen the previous conclusion, a histopathological
analysis of the liver tissue was done. After the preparation of the
samples, it was observed that the hepatic histoarchitecture of
both groups showed no alterations, presenting normal
structures without inflammatory infiltration or necrosis when
observed under the microscope (Figure 6). In addition, no fat
accumulation in the liver was observed after sacrifice, and the
health indicators for both groups (increase of weight,
appearance of eyes and skin, physical activity, and response
to external stimulus) were identical.
It was possible to conclude that the administration of 2 mL
of coffee brew per day to rats greatly increased the cytosolic
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Journal of Agricultural and Food Chemistry
levels of Nrf2 and the activity of antioxidant enzymes,
improving the defensive status against oxidative stress with
no apparent damage to the liver tissue. As a comparison, a
human of 70 kg BW would have to consume three to four
medium-size cups per day of coffee brew, which means
approximately 550 mg of phenolic acids and 500 mg of caffeine.
■
AUTHOR INFORMATION
Corresponding Author
*(S.J.V.V.) Mailing address: R. Oswaldo Cruz 266, Santos/SP,
Brazil 11045-907. E-mail: laq@unisanta.br. Phone: +55 13
3202.7100. Fax: +55 13 3234.5297.
Funding
This work was financially supported by the State of São Paulo
Research Foundation, FAPESP, and the National Council for
Scientific and Technological Development, CNPq.
Notes
The authors declare no competing financial interest.
■ ABBREVIATIONS USED
SOD, superoxide dismutase; CAT, catalase; GPx, glutathione
peroxidase; Nrf2, nuclear factor E2-related factor; Keap1,
Kelch-like ECH-associated protein-1; Tris-HCl buffer, tris-
(hydroxymethyl)aminomethane hydrochloride; ORAC, oxygen
radical absorbance capacity; TE, Trolox equivalent; O2−,
superoxide radical; O22−, peroxide anion; BW, body weight;
AST, aspartate transaminase; ALT, alanine transaminase; ROS,
reactive oxygen species; ARE, antioxidant response element;
HPLC, high-performance liquid chromatography; CQA,
caffeoylquinic acid; FQA, feruloylquinic acid
■
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