Coffee Modulates Transcription Factor Nrf2 and Highly Increases the

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 glutathione peroxidase increased 74.8, 59.4, and 135.2%, respectively, whereas the cytosolic level of Nrf2 increased 131.3%. At the 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

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INTRODUCTION

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 ECHassociated 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

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, considerable efforts have been made to identify substances that can © 2013 American Chemical Society

Received: Revised: Accepted: Published: 116

April 25, 2013 December 9, 2013 December 13, 2013 December 13, 2013 dx.doi.org/10.1021/jf401777m | J. Agric. Food Chem. 2014, 62, 116−122

Journal of Agricultural and Food Chemistry

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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 SBC18 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

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.

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MATERIALS AND METHODS

Chemicals. Caffeic, ferulic, and p-coumaric acids, 5-caffeoylquinic acid (5-CQA), 5-feruloylquinic acid (5-FQA), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-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 117

dx.doi.org/10.1021/jf401777m | J. Agric. Food Chem. 2014, 62, 116−122


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done at 323 nm for caffeic and ferulic acids and at 309 nm for pcoumaric acid. A previous standardization of this method was done (recovery, limit of detection, and limit of quantification),15 the 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 transcription of antioxidant enzymes, and it was quantified by HPLC as 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 Western 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 IU/L). 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 antibody against Nrf2, washed three times with Tris-HCl buffer, incubated with IgG secondary antibody coupled with horseradish peroxidase for 1 h at room temperature, and washed with Tris-HCl buffer; chemiluminescence was measured. The quantifications were adjusted for the corresponding β-actin level (loading control protein). Statistical Calculations. Statistical calculations (Kolmogorov− Smirnov and Student’s t test) were done using the SPSS 16.0 for

Windows software package, with a significance level of p < 0.05. All tests were done in triplicate, and the results are presented as means ± standard deviations.

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RESULTS AND DISCUSSION Bioactive Compounds in Coffee Brew. As the main purpose of this study was to evaluate the effect of the

Figure 1. Typical chromatograms obtained during the quantification of free phenolic acids (A) and chlorogenic acids (B) in coffee brew.

Figure 2. SOD (U/mg of protein), CAT (μmol/min/mg of protein), and GPx (U/mg of protein) average activities in the liver tissue of rats (n = 6) showing the differences between groups 1 (control) and 2 (coffee). (∗ indicates statistically significant difference from the control group at p < 0.05.)

administration of coffee brew on the activity of antioxidant enzymes, it was important to quantify the levels of the bioactive 118



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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 chlorogenic 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 Capacity. 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

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 119



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Figure 6. Liver tissues (group 1, left; group 2, right) showing normal architectures and no apparent damage.

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

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 AREresponsive 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 120



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(9) Giudice, A.; Montella, M. Activation of the Nrf2-ARE signaling pathway: a promising strategy in cancer prevention. BioEssays 2006, 28, 169−181. (10) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mzur, M.; Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 2007, 39, 44− 84. (11) Yeh, C. T.; Yen, G. C. Induction of hepatic antioxidant enzymes by phenolic acids in rats is accompanied by increased levels of multidrug resistance-associated protein 3 mRNA expression. J. Nutr. 2006, 136, 11−15. (12) Yeh, C. T.; Ching, L. C.; Yen, G. C. Inducing gene expression of cardiac antioxidant enzymes bydietary phenolic acids in rats. J. Nutr. Biochem. 2009, 20, 163−171. (13) Nardini, M.; Cirillo, E.; Natella, F.; Scaccini, C. Absorption of phenolic acids in humans after coffee consumption. J. Agric. Food Chem. 2002, 50, 5735−5741. (14) Mattila, P.; Hellström, J.; Törrönen, R. Phenolic acids in berries, fruits and beverages. J. Agric. Food Chem. 2006, 54, 7193−7199. (15) Vicente, S. J. V. Antioxidant characterization of coffee (Cof fea arabica, L) and effects of its oral administration to rats. Thesis, University of São Paulo, São Paulo, Brazil, 2009; available at www. teses.usp.br. (16) Toci, A.; Farah, A.; Trugo, L. C. Efeito do processo de descafeinaçaõ com diclorometano sobre a composiçaõ quı ́mica dos cafés Arabica e Robusta antes e após a torrefaçaõ . Quim. Nova 2006, 29, 965−971. (17) Associaçaõ Brasileira da Indústria do Café (ABIC); http://www. abic.com.br/scafe_dicas.hmtl (accessed Jan 2013). (18) Vicente, S. J. V.; Ishimoto, E. Y.; Cruz, R. J.; Pereira, C. D. S.; Torres, E. A. F. S. Increase of the activity of phase II antioxidant enzymes in rats after a single dose of coffee. J. Agric. Food Chem. 2011, 59, 10887−10892. (19) Corrêa, T. A. F. Comparison of the antioxidant capacity of coffee roasts and their effects on cardiovascular risks factors in healthy subjects. Thesis, University of São Paulo, São Paulo, Brazil, 2012; available at www.teses.usp.br. (20) Nardini, M.; Cirillo, E.; Natella, F.; Mencarelli, D.; Comisso, A.; Scaccini, C. Detection of bond phenolic acids: prevention by ascorbic acid and ethylenediaminetetraacetic acid of degradation of phenolic acids during alkaline hydrolysis. Food Chem. 2002, 79, 119−124. (21) Kowalski, R.; Wolski, T. Evaluation of phenolic acid content in Silphium perfoliatum L. leaves, inflorescences and rhizomes. Electr. J. Polish Agric. Univ. 2003, 6, 9−17. (22) Vitorino, M. D.; França, A. S.; Oliveira, L. S.; Borges, M. L. A. Metodologia de obtençaõ de extrato de café visando a dosagem de compostos não voláteis. Rev. Bras. Armaz. 2001, 26, 17−24. (23) Woolliams, J. A.; Wiener, G.; Anderson, P. H.; McMurray, C. H. Variation in the activities of glutathione peroxidase and superoxide dismutase and in the concentration of copper in the blood of various breed crosses of sheep. Res. Vet. Sci. 1983, 34, 253−256. (24) Aebi, H. Catalase in vitro. In Methods in Enzymology; Packer, L., Ed.; Academic Press: Orlando, FL, USA, 1984; Vol. 105, pp 121−126. (25) Paglia, D. E.; Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 1967, 70, 158−169. (26) Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (27) Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem. 2001, 49, 4619−4626. (28) Prior, R. L.; Hoang, H.; Gu, L.; Wu, X.; Bacchiocca, M.; Howard, L.; et al. Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORACFL)) of plasma and other biological and food samples. J. Agric. Food Chem. 2003, 51, 3273−3279.

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.

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AUTHOR INFORMATION

Corresponding Author

*(S.J.V.V.) Mailing address: R. Oswaldo Cruz 266, Santos/SP, Brazil 11045-907. E-mail: [email protected]. 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.

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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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REFERENCES

(1) Kim, H. J.; Yu, M. H.; Lee, I. S. Inhibitory effects of methanol extract of plum (Prunus salicina L., cv ‘Soldam’) fruits against benzo(α)pyrene-induced toxicity in mice. Food Chem. Toxicol. 2008, 46, 3407−3413. (2) Bichler, J.; Cavin, C.; Simic, T.; Chakraborty, A.; Ferk, F.; Hoelzl, C.; et al. Coffee consumption protects human lymphocytes against oxidative and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (TrpP-2) induced DNA-damage: results of an experimental study with human volunteers. Food Chem. Toxicol. 2007, 45, 1428−1436. (3) Tao, K. S.; Wang, W.; Wang, L.; Cao, D. Y.; Li, Y. Q.; Wu, S. X.; Dou, K. F. The multifaceted mechanisms for coffee’s anti-tumorigenic effect on liver. Med. Hypotheses 2008, 71, 730−736. (4) Higgins, L. G.; Cavin, C.; Itoh, K.; Yamamoto, M.; Hayes, J. D. Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein. Toxicol. Appl. Pharmacol. 2008, 226, 328−337. (5) Boettler, U.; Sommerfeld, K.; Volz, N.; Pahlke, G.; Teller, N.; Somoza, V.; Lang, R.; Hofmann, T.; Marko, D. Coffee constituents as modulators of Nrf2 nuclear translocation and ARE (EpRE)-dependent gene expression. J. Nutr. Biochem. 2011, 22, 426−440. (6) Muriel, P.; Arauz, J. Coffee and liver diseases. Fitoterapia 2010, 81, 297−305. (7) Volz, N.; Boettler, U.; Winkler, S.; Teller, N.; Schwarz, C.; Bakuradze, T.; Eisenbrand, G.; Haupt, L.; Griffiths, L. R.; Stiebitz, H.; Bytof, G.; Lantz, I.; Lang, R.; Hofmann, T.; Somoza, V.; Marko, D. Effect of coffee combining green coffee bean constituents with typical roasting products on the Nrf2/ARE pathway in vitro and in vivo. J. Agric. Food Chem. 2012, 60, 9631−9641. (8) Kwak, M. K.; Wakabayashi, N.; Kensler, T. W. Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme inducers. Mutat. Res. 2004, 555, 133−148. 121



Article

(29) Reitman, S.; Frankel, S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 1957, 28, 56−63. (30) Pari, L.; Prasath, A. Efficacy of caffeic acid in preventing nickel induced oxidative damage in liver of rats. Chem. Biol. Interact. 2008, 173, 77−83. (31) Farah, A.; Donangelo, C. M. Phenolic compounds in coffee. Braz. J. Plant Physiol. 2006, 18, 26−36. (32) Lafay, S.; Gil-Izquierdo, A.; Manach, C.; Morand, C.; Besson, C.; Scalbert, A. Chlorogenic acid is absorbed in its intact form in the stomach of rats. J. Nutr. 2006, 136, 1192−1197. (33) Monteiro, M.; Farah, A.; Perrone, D.; Trugo, L. C.; Donangelo, C. Chlorogenic acid compounds from coffee are differentially absorbed and metabolized in humans. J. Nutr. 2007, 137, 2196−2201. (34) Lafay, S.; Morand, C.; Manach, C.; Besson, C.; Scalbert, A. Absorption and metabolism of caffeic acid and chlorogenic acid in the small intestine of rats. Br. J. Nutr. 2006, 96, 39−46. (35) Suri, R.; Crozier, A. Free phenolic acids in human urine after drinking coffee rich in chlorogenic acids. J. Trop. Agric. Food Sci. 2012, 40, 221−232. (36) Redeuil, K.; Smarrito-Menozzi, C.; Guy, P.; Rezzi, S.; Dionisi, F.; Williamson, G.; Nagy, K.; Renouf, M. Identification of novel circulating coffee metabolites in human plasma by liquid chromatography−mass spectrometry. J. Chromatogr., A 2011, 1218, 4678−4688. (37) Natella, F.; Nardini, M.; Gianetti, I.; Dattilo, C.; Scaccini, C. Coffee dirnking influences plasma antioxidant capacity in humans. J. Agric. Food Chem. 2002, 50, 6211−6216. (38) Kakarla, P.; Vadluri, G.; Kesireddy, S. R. Response of hepatic antioxidant system to exercise training in aging female rat. J. Exp. Zool. 2005, 303, 203−208. (39) Shanmugarajan, T. S.; Krishnakumar, E.; Somasundaram, I.; Sivaraman, D.; Arunsundar, M.; Balaji, R.; Sivakumar, S. M. Salutary effect of ferulic acid against D-galactosamine challenged liver damage. J. Biol. Sci. 2008, 8, 1271−1279.

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