Doxorubicin-Induced Oxidative Stress in Rats Is Efficiently

Article pubs.acs.org/JAFC

Doxorubicin-Induced Oxidative Stress in Rats Is Efficiently Counteracted by Dietary Anthocyanin Differently Enriched Strawberry (Fragaria × ananassa Duch.) Jacopo Diamanti,† Bruno Mezzetti,† Francesca Giampieri,† José M. Alvarez-Suarez,‡ José L. Quiles,§ Adrian Gonzalez-Alonso,§ Maria del Carmen Ramirez-Tortosa,⊗ Sergio Granados-Principal,⊗ Ana M. Gonzáles-Paramás,⊥ Celestino Santos-Buelga,⊥ and Maurizio Battino*,‡ †

Department of Agriculture, Food and Environmental Science, Marche Polytechnic University, 60121 Ancona, Italy Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche − Sez-Biochimica, Marche Polytechnic University, 60121 Ancona, Italy § Department of Physiology, Institute of Nutrition and Food Technology ‘‘José Mataix”, Biomedical Research Centre, University of Granada, 18071 Granada, Spain ⊗ Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology ‘‘José Mataix”, Biomedical Research Centre, University of Granada, 18071 Granada, Spain ⊥ Grupo de Investigación en Polifenoles (GIP-USAL), Universidad de Salamanca, 37008 Salamanca, Spain ‡

ABSTRACT: This study investigated the effects of two different strawberry cultivars, Adria and Sveva, against doxorubicin (DOX)-induced toxicity in rats. A controlled dietary intervention was conducted over 16 weeks with four groups: (i) normal diet; (ii) normal diet + DOX injection; (iii) Adria supplementation + DOX injection; and (iv) Sveva supplementation + DOX injection. Sveva presented higher total antioxidant capacity value and phenol and and vitamin C levels than Adria, which in turn presented higher anthocyanin contents. DOX drastically increased lymphocyte DNA damage, liver biomarkers of protein and lipid oxidation, and mitochondrial ROS content and markedly decreased plasma retinol level, liver antioxidant enzymes, and mitochondrial functionality. After 2 months of strawberry supplementation, rats presented a significant reduction of DNA damage and ROS concentration and a significant improvement of oxidative stress biomarkers, antioxidant enzyme activities, and mitochondrial performance. These results suggest that strawberry supplementation can counteract DOX toxicity, confirming the potential health benefit of strawberry in vivo against oxidative stress. KEYWORDS: strawberry, anthocyanins, doxorubicin, oxidative stress, DNA damage, mitochondrial functionality

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INTRODUCTION Doxorubicin (DOX) is an antibiotic anthracycline, clinically known as adryamicin, which has been employed for more than 30 years in the battle against cancer, especially in breast and esophageal carcinomas, solid tumors in childhood, osteosarcomas, and soft-tissue sarcomas.1 However, its use is limited by the acute and chronic toxic side effects it produces due to the fact that DOX acts at two fundamental levels: (i) altering DNA and (ii) producing free radicals and oxidative stress.1 To protect, or to prevent, the toxic effect of DOX, natural antioxidants in the diet can represent a valuable aid. In this context, fruits and vegetables possess vitamins, mineral salts, phenols, and other phytochemicals that act directly against free radicals, counteracting the biological oxidative status. In the past few years, a great number of studies have investigated the relationship between diet and physiological status,2,3 so that specific balanced diets are now widely recognized as healthy strategies even by UNESCO.4 Accordingly, the numerous beneficial effects attributed to phenolic compounds have given rise to a new interest in finding vegetal species with high phenolic content and relevant biological activity. Among them, berries constitute a rich dietary source of phenolic antioxidant and bioactive properties.5,6 Strawberry (Fragaria × ananassa © 2014 American Chemical Society

Duch.) fruits are very popular berries and are widely consumed in fresh forms and as food products such as preserves, jams, yogurts, and ice creams. Their health effects are attributed to high levels of antioxidant compounds, most of which are phenolic compounds such as anthocyanins, flavonols, flavanols, condensed tannins (proanthocyanidins, ellagitannins, and gallotannins), hydroxybenzoic and hydroxycinnamic acid derivatives, and hydrolyzable tannins.5,6 These compounds are reported to possess antioxidant, anticancer, anti-inflammatory, and antineurodegenerative biological properties.5 Various in vitro studies have been conducted using strawberry extracts to evaluate their antiangiogenic and chemopreventive properties on cancer cell lines in a dose-dependent manner7 and antiproliferative and anti-inflammatory activities, inducing apoptosis and cell cycle arrest against human stomach, prostate, intestine, and breast cancer cell lines.8 Anthocyanin compounds Special Issue: 2013 Berry Health Benefits Symposium Received: Revised: Accepted: Published: 3935

August 4, 2013 March 1, 2014 March 3, 2014 March 3, 2014 dx.doi.org/10.1021/jf405721d | J. Agric. Food Chem. 2014, 62, 3935−3943

Journal of Agricultural and Food Chemistry

Article

μm, Aqua C18 column (Phenomenex). The chromatographic conditions were similar to those previously described for anthocyanin determination.14 Double online detection was carried out by the combined use of a DAD and an API 3200 Qtrap MS equipped with an ESI source and a triple-quadrupole ion trap mass analyzer (Applied Biosystems, Darmstadt, Germany) controlled by Analyst 5.1 software and connected to the HPLC system via the DAD cell outlet. Mass spectrometry data were acquired in the negative mode for all phenolic compounds except anthocyanins, which were acquired in positive mode. Anthocyanins were quantified using the external standards of cyanidin-3-glucoside (Cy-3-glucoside) and pelargonidin-3-glucoside (Pg-3-glucoside) (at 520 nm), flavonols as quercetin-3-O-glucoside (at 360 nm), ellagic acid and ellagic acid glycosides as ellagic acid (at 360 nm), and ellagitannins as gallic acid (at 280 nm). Each freeze-dried strawberry sample was assayed in triplicate for each parameter analyzed. Experimental Design. Twenty-four male Wistar rats (Rattus norvegicus), initially weighing 110−130 g, were allocated in groups of three animals per box and maintained on a 12 h light/12 h darkness cycle, with free access to drinking water. Animals were match-fed for 16 weeks at 25 g/day on semisynthetic and isoenergetic diets according to the AIN93 criteria or an AIN93-modified diet supplemented with 10 g/100 g of strawberry lyophilized extract, as shown in Table 1, according to the method of Prior et al.15 For the

found in berry fruit play a fundamental role in the antioxidant, chemopreventive, and anti-inflammatory activities,5 but in addition to anthocyanins, other phenolic compounds, such as ellagitannins and flavonols, seem to exert a protective role in carcinogenesis by reducing the bioavailability of carcinogens.9 Many studies have also confirmed the influence of genotype on the phytochemical composition of strawberry fruit antioxidant capacity, so that it is possible to find new genotypes and cultivars producing fruit with higher contents of bioactive compounds, explore new genetic resources, or develop tailored breeding programs.10−12 The aim of this study was to analyze the potential antioxidant effects of freeze-dried strawberry powder, added to a semisynthetic and isoenergetic diet, on doxorubicin-induced oxidative stress in rats. The effect of freeze-dried strawberry powder was tested by comparing two strawberry cultivars originated by the strawberry breeding program of the university: Sveva and Adria. These two genotypes were selected after previous studies10−12 that allowed the differentiation of their fruits according to the total antioxidant capacity (TAC) and the vitamin C and polyphenolics they contained: Sveva presents higher total antioxidant capacity, associated with a higher value of total polyphenol and vitamin C, whereas Adria shows higher values of anthocyanin contents but lower TAC.

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Table 1. Composition of Control Diet and Contribution of Strawberry Daily Dose

MATERIALS AND METHODS

Chemicals. Doxorubicin was obtained as doxorubicin hydrochloride (2 mg/mL) from Sigma-Aldrich (St. Louis, MO, USA), as well as 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) diammonium salt, potassium persulfate, Trolox ≥98.0%, ascorbic acid, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, Folin−Ciocalteu reagent, sodium carbonate, gallic acid, potassium chloride (KCl), sodium acetate (NaAc), and hydrochloric acid (HCl). Ethanol of HPLC grade, phosphate buffer solution 10×, 2thiobarbituric acid 0.8%, acetic acid 99.9%, 2′,7′-dichlorofluorescein (DCF) ≥90%, sucrose, Tris 10 mM, EDTA-Na, bovine serum albumin (BSA), succinic acid disodium salt hezahydrate, 5,5-dithiobis(2nitrobenzoic acid) (DTNB), dinitrophenylhydrazine (DNPH), 1chloro-2,4-dinitrobenzene (CDNB), heparin, carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP), tetramethylphenylenediamine (TMPD), cyanidin-3-glucoside, pelargonidin-3-glucoside, quercetin-3-O-glucoside, and ellagic acid were also purchased from Sigma-Aldrich. Methanol and ethanol of ACS grade were purchased from Carlo Erba Reagenti, Milano, Italy, whereas DCFH2DA, cellpermeant indicator for reactive oxygen species, was acquired from Molecular Probes, Invitrogen (Carlsbad, CA, USA). High-purity water (Milli-Q Water System, Millipore Corp., Bedford, MA, USA) was employed throughout. Fruits and Fruit Analysis. Ripe fruits of Adria and Sveva cultivars were harvested from plants grown in an experimental open field in the Aso valley area (Italy), frozen at −20 °C, lyophilized by Labconco Freezone 12L model 78670 (Kansas City, MO, USA), and then kept under vacuum condition until milled with a Mixer B-400 (Büchi Labortechnik, Flawil, Switzerland). Samples of strawberry fruit powder were analyzed to determine their nutritional composition; each freezedried strawberry sample was assayed in triplicate for each parameter analyzed. The strawberry powder was kept under vacuum, in darkness, at a temperature of 4 °C until meal preparation. Fruit analysis was carried out as illustrated in detail in our previous publications.11,13 Briefly, TAC and total phenol (TPH) content of the strawberry fruits were measured spectrophotometrically, whereas vitamin C was quantified by reversed-phase HPLC.12,13 Anthocyanin extraction and HPLC-MS/MS analysis were performed as previously described.14 The HPLC-DAD/ESI-MS-driven analysis for the identification and quantification of phenolic compounds was performed through a Hewlett-Packard 1200 series liquid chromatography. The separation was achieved using a 150 mm × 4.6 mm i.d., 5

cornstarch casein dextrinized cornstarch sucrose soybean oil fiber mineral mix vitamin mix L-cystine choline bitartrate strawberry powderb

AIN-93M (groups C and C-DOX) (g/kg diet)

AIN-93M + strawberry (groups A-DOX and S-DOX)a (g/kg diet)

465.7 140.0 155.0 100.0 40.0 50.0 35.0 10.0 1.8 2.5 0.0

465.6 133.0 155.0 30.0 34.0 33.1 35.0 10.0 1.8 2.5 100.0

a

Strawberry extract was made from the Adria variety for the A-DOX group and from the Sveva variety for the S-DOX group. b100 g of strawberry powder contains 7 g of protein, 6 g of fat, 70 g of carbohydrates, and 27.1 g of fiber. This was considered when in the formulation of the AIN-93M + strawberry diet to make both diets isocaloric. formulation of the diet supplemented with strawberry extract, the nutritional composition of the extract was considered according to the USDA database. The remaining energetic nutrients were recalculated to make both diets isocaloric. The rats were randomly assigned to four experimental groups of six animals as follows: two groups fed AIN93 diet, a group fed AIN93 diet supplemented with Adria strawberry powder, and a group fed AIN93 diet supplemented with Sveva strawberry powder. Food intake was indirectly monitored through the weekly body weight control and daily spillage monitoring. At the end of the 16 weeks, rats were subjected to two intraperitoneal injections of 10 mg/kg/day DOX or saline solution with a 24 h gap between each injection, according to the method of Quiles et al.16 Thus, experimental groups were organized as follows: Cgroup, animals fed AIN93 diet for 16 weeks and subjected to two intraperitoneal injections of saline solution 48 h before death; C-DOX group, animals fed AIN93 diet for 16 weeks and subjected to two intraperitoneal injections of DOX 48 h before death; A-DOX group, animals fed AIN93 freeze-dried Adria strawberry powder diet for 16 weeks and subjected to two intraperitoneal injections of DOX 48 h 3936

dx.doi.org/10.1021/jf405721d | J. Agric. Food Chem. 2014, 62, 3935−3943


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Table 2. Fruit Nutritional Contents for Processed Fruit of Adria and Sveva Cultivarsa

a

cultivar

TAC (mmol Trolox equiv/kg FW)

TPH (mg GAE/kg FW)

vitamin C (g ascorbic acid/kg FW)

Adria Sveva

18.08 ± 0.33b 19.67 ± 0.30a

1749.47 ± 48.31b 2594.67 ± 54.59a

0.45 ± 0.02b 0.90 ± 0.08a

Mean values followed by different letters are significantly different. Test SNK, p ≤ 0.05. Determination of Liver Antioxidant Enzyme Activity. Catalase activity (Cat) was measured following the method described by Aebi,23 by monitoring H2O2 decomposition at 240 nm as a consequence of the catalytic activity of catalase. Superoxide dismutase (SOD) was determined according to the method of Kakkar,24 on the basis of the inhibition of the formation of NADH−phenazine methosulfate−nitroblue tetrazolium formazan by SOD, measured by spectrophotometry at 540 nm. For glutathione peroxidase (GPx) measurement the method used25 was based on the ability of GPx to remove H2O2 by coupling its reduction to H2O with oxidation of reduced glutathione. Glutathione reductase (GRx) activity was assayed by using the modified method of Carlberg and Mannervik,26 based on the capacity of GRx to reduce oxidized glutathione (GSSG) back to reduced glutathione (GSH). Glutathione transferase activity (GST) was determined according to the modified method described by Habig,27 which measures the conjugation of CDNB with GSH, producing a dinitrophenyl thioether detected at 340 nm spectrophotometrically. Each measurement was repeated in duplicate, with two readings for each replicate. Evaluation of Reactive Oxygen Species (ROS) in Liver Mitochondria. Liver mitochondria were isolated by differential centrifugation as previously described.16 Mitochondria protein was measured according to the Lowry procedure17 using BSA as standard. ROS production by mitochondrial preparations was measured in fluorescence plate readers.28,29 The results obtained are expressed as arbitrary fluorescence units (AFU) and are converted into nanomoles of ROS by linear regression method, using the DCF dilutions. Each measurement was repeated in duplicate, with two readings for each replicate. Liver Mithocondrial Respiratory Efficiency. The oxygen consumption rate (OCR) in liver mitochondria was measured with an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica MA, USA), as previously described.27 The mitochondrial assay solution (MAS) consists of 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EDTA, and 0.2% (w/v) fatty acid-free BSA, pH 7.2 at 37 °C. To minimize variability between wells, mitochondrial suspension were first diluted 10 times in cold MAS buffer to which was added 10 mM pyruvate, 2 mM malate, and 4 μM FCCP to achieve the final concentration of 10 μg of mitochondria in 50 μL of MAS solution. Then, 50 μL of the mitochondria diluted solution was delivered to each well (except for background correction wells) while the plate was on ice. The plate was then transferred to a centrifuge equipped with a swinging bucket microplate adaptor, and spun at 4089 rpm for 20 min at 4 °C. After centrifugation, 450 μL of MAS plus substrate was added to each well. The mitochondria were then placed at 37 °C for 10 min in the XF Prep Station incubator (Seahorse Bioscience). The plate was transferred to the XF24 instrument and the experiment initiated. Changes to the concentrations of dissolved oxygen in the media were measured for each 2 s interval by solid state sensor probes. After determination of the basal mitochondrial respiration, rotenone (2 μM), succinate (10 mM), antimycin A (4 μM), and ascorbate plus 1 mM TMPD (10 mM and 100 μM, respectively) were sequentially added to each well. Data are presented as the average of three replicates per well ± standard error. Results are expressed as OCR in picomoles of consumed oxygen per minute and were adjusted for protein concentration (pmol O2/min/mg protein). Statistical Analysis. Results are presented as the mean ± standard error (SE). One-way variance analysis was used to test the differences between genotypes, groups, and treatments. Statistically significant differences (p ≤ 0.05) were determined with the SNK test. Pearson correlation was evaluated for fruit nutritional parameters and biological

before death; S-DOX group, animals fed AIN93 freeze-dried Sveva strawberry powder diet for 16 weeks and subjected to two intraperitoneal injections of DOX 48 h before death. At the end of the experimental period (24 h after the second intraperitoneal injection), animals were killed by cervical dislocation followed by decapitation at the same time of day to avoid any circadian fluctuation. After decapitation, blood was collected by decantation onto glass tubes coated with lithium-heparin; isolated plasma and the “buffy coat”, enriched in white cells, were removed, diluted 1:1 with RPMI1640 medium, and layered onto an equivalent volume of Histopaque to obtain peripheral blood lymphocytes. Weights of the heart, liver, and right kidney were taken to evaluate the relative weight of organs (%) calculated as grams per 100 g of body weight. A part of the livers was homogenized on ice at 4 °C in 5 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA, in a ratio (10%) of 1 g of wet tissue to 10 mL of buffer, using an IKA-Werk (Janke Kunkel, UE) homogenizer. After centrifugation at 9143 rpm for 20 min at 4 °C, the supernatant was kept; proteins were measured according to the Lowry procedure17 using BSA as standard and then stored at −80 °C until analysis. Animals were handled according to the guidelines of the Spanish Society for Laboratory Animals, and the investigation was approved by the Ethical Committees of the Ministry of Science and Technology and the University of Granada. Evaluation of Rat Plasma Lipophilic Antioxidant Contents. Plasma levels of retinol and α-tocopherol were determined by reversed-phase HPLC as previously described16 using a Spherisorb S5 ODS1 (Merck, Darmnstadt, Germany) column and ethanol/ purified water 97:3 (v/v) as mobile phase. The HPLC system was a Beckman in-line diode array detector, model 168 (Fullerton, CA, USA), connected to a Waters (Milford, MA, USA) 717 plus autosampler. Determinations were performed at 325 nm for retinol and at 292 nm for α-tocopherol. Antioxidant molecules were identified by predetermining the retention times of individual standards and quantified through two calibration curves, daily prepared with known concentrations of retinol (0.1−1.7 μg/mL) and α-tocopherol (1.7− 30.2 μg/mL) standards. Each measurement was repeated in duplicate, with two readings for each replicate. DNA Double-Strand Break (Comet) Analysis. The comet assay was used to measure DNA double-strand breaks in peripheral blood lymphocytes as previously described.18 Analysis was performed using a CCD camera and Komet 3.0 image analysis program (Kinetic Imaging Ltd., Liverpool, UK). The percent of DNA in the tail (mean of 100 comets per gel) is taken as a measure of DNA break frequency. The comet-like DNA formations were placed into five arbitrary classes (0− 4) according to the extent of DNA damage represented by a cometlike “tail”. Each comet was assigned a value according to its class, and the overall score for 100 comets ranged from 0 (100% of comets being class 0) to 400 (100% of comets in class 4). Each lymphocyte sample was assayed in duplicate, and 50 comets (i.e.,100 comets/sample) on two different slides were evaluated for every sample in each experiment. Evaluation of Liver Biomarkers of Oxidative Stress. Protein carbonyl content was determined in liver homogenates by the DNPH method as described by Levine,19 whereas the GSH content was determined through the DTNB assay as carried out by Ellman.20 Liver lipid peroxidation was measured by the assay of thiobarbituric acidreactive substances (TBARS) according to a standardized method,21 whereas the oxide−xylenol orange (FOX2) method was used to determine hydroperoxides according to a method reported by Jiang.22 Each measurement was repeated in duplicate, with two readings for each replicate. 3937



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of flavonols in sample of about 2.10 ± 0.03 and 7.49 ± 2.0 mg/ kg for Adria and Sveva, respectively. Quercetin-3-glucuronide was, in accordance with previous studies,30 the most abundant flavonol in the two varieties, approximately 1 ± 0.0 and 3.5 ± 0.7 mg/kg in Adria and Sveva, respectively. An ellagic acid pentoside, an ellagic acid deoxyhexoside, and ellagic acid/ellagic acetyl rhamnoside were also detected and quantified in both strawberries (Table 4) with a total of ellagic acid conjugates of about 6.5 ± 0.1 and 18.7 ± 0.9 mg/kg in Adria and Sveva varieties, respectively. Finally, as previously reported,31 a major ellagitannin pigment was identified as agrimoniin, with concentrations of 21.4 ± 3.3 mg/kg in Adria and 39.0 ± 3.0 mg/kg in Sveva (Table 4). Although proanthocyanidins have been previously identified and quantified in strawberries,30 in our samples several minor peaks corresponding to these polyphenols were detected but not quantified because of their low concentration. Hematological and Clinical Analysis. Data on body weight and organ ratios showed slight differences among rat groups but no significant variations were found, thus indicating that the strawberry diet did not interfere with animal development (Table 5) while sacrifice after doxorubicin injection occurred before any possible modification could be identified. Evaluation of Rat Plasma Retinol and α-Tocopherol Content. Concerning plasma levels of retinol (Figure 1), the highest value were found in C group while the lowest in CDOX group; freeze-dried strawberry supplementation buffered the decrease of retinol in A-DOX group and in S-DOX group (p < 0.05) compared to C-DOX group. Plasma α-tocopherol did not show differences among all groups, although higher values were obtained in rats fed with freeze-dried strawberries, both Adria and Sveva (Figure 1). DNA Double-Strand Break Assays. The comet assay for the evaluation of DNA damage was performed on peripheral blood lymphocytes collected before and after DOX injection to evaluate DNA damage due to endogenous oxidative DOXmediated stress and the efficacy of long-term freeze-dried strawberry diet in preventing such oxidative-induced damage. Before the DOX injection, all groups showed similar base values of DNA damage, whereas after the DOX injection an increase of DNA damage was observed in all groups, but with different rates: indeed, as shown in Figure 2, comparing the variation of DNA damage values detected before and after the DOX injection, the lower increase of DNA damage was found in the C group, as expected for rats not submitted to DOX injection, whereas greater variation was observed in C-DOX and S-DOX groups. Freeze-dried Adria supplementation buffered the increase in DNA damage, as demonstrated by the lower increase (p < 0.05) in the A-DOX group compared to the C-DOX group. Evaluation of Liver Biomarkers of Oxidative Stress. Table 6 shows the changes in liver biomarkers of oxidative stress. Treatment with DOX resulted in a significant increase in protein carbonyl levels (3.2-fold, p < 0.05) as well as a dramatic decrease in GSH levels (1.7-fold, p < 0.05) in the C-DOX group compared to the C group, whereas the supplementation with freeze-dried strawberry restored the condition of the C group in A-DOX and S-DOX groups, especially for protein carbonyl levels. Similar trends were observed in markers of lipid oxidation. A significant increase in TBARS concentration was in fact observed in the liver (2.0-fold, p < 0.05) of C-DOX rats, whereas in A-DOX and S-DOX groups, treatment with freezedried strawberry powders resulted in values similar to those of

assays. Statistical processing was carried out using STATISTICA software (Statsoft, Tulsa, OK, USA).

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RESULTS Strawberry Fruit Nutritional Composition. Fruit nutritional composition was analyzed on freeze-dried fruit (Table 2). Sveva freeze-dried powder presented a higher TAC value than Adria (19.67 vs 18.08 mmol Trolox equiv/kg FW, respectively) as well as higher phenols and vitamin C levels (Table 2), whereas Adria freeze-dried powder showed higher total anthocyanin contents (Table 3). The use of optimized HPLC Table 3. Identification and Quantification of Anthocyanin Pigments in Adria and Sveva Freeze-Dried Fruits peak 1 2 3 4 5 6 7

identification

Adria cultivar (mg/kg)

Sveva cultivar (mg/kg)

3.798 ± 0.4

1.754 ± 0.1

(epi)afzelechin-(4→8)Pg3-glucoside Cy-3-glucoside Pg 3,5-diglucoside Pg 3-glucoside Pg 3-rutinoside Pg 3-malonylglucoside Pg 3-acetylglucoside

32.232 0.588 1157.376 97.848 32.562 7.114

total anthocyanin content

1331.518 ± 4.6

± ± ± ± ± ±

2.1 0.0 12.0 4.0 3.0 4.0

25.974 0 790.234 26.176 45.62 3.284

± ± ± ± ± ±

2.2 0.0 7.0 2.0 2.2 1.0

893.042 ± 2.3

conditions coupled to diode array and mass detection allowed detection of seven different anthocyanin pigments in freezedried strawberry fruits (Table 3). For both varieties, up to five pigments could be assigned to Pg derivates, and the major peak in the HPLC chromatograms corresponded to Pg-3-glucoside; one pigment could be identified as (epi)afzelechin-(4→8)-Pg 3glucoside and the last one as a Cy derivative. Individual concentrations of the anthocyanin identified in the freeze-dried cultivars are shown in Table 3 with total concentrations of anthocyanin in samples of approximately 1331.52 ± 3.6 and 893.04 ± 2.3 mg/kg for Adria and Sveva, respectively. With regard to flavonols, two compounds were identified and quantified as quercetin derivates and three as kaempferol derivates in both cultivars (Table 4), with total concentrations Table 4. Identification and Quantification of Total Flavonols, Total Ellagic Acid Conjugates, and Ellagitanins in Adria and Sveva Freeze-Dried Fruits identification flavonols quercetin-3-hexose quercetin-3-glucuronide kaempferol-3-glucoside kaempferol-3-glucuronide kaempferol-3-malonylglucoside total ellagic acid conjugates ellagic acid pentoside ellagic acid deoxyhexoside ellagic acid + ellagic acetyl rhamnoside total ellagitannins agrimoniin

Adria cultivar (mg/kg) 0.48 1.005 0.21 0.413

± ± ± ±

0.0 0.0 0.0 0.0

Sveva cultivar (mg/kg) ± ± ± ± ± ±

2.10 ± 0.03

1.2 3.5 1.2 0.89 0.74 7.49

0.3 0.7 0.3 0.3 0.2 2.0

0.55 ± 0.02 1.8 ± 0.3 4.1 ± 0.5

0.96 ± 0.1 6.9 ± 0.03 10.9 ± 1.8

6.5 ± 0.1

18.7 ± 0.9

21.4 ± 3.3

39.0 ± 3.0 3938



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Table 5. Body Weight and Organ Body Ratio of the Rat Groups Fed the Four Dietsa groupb C C-DOX A-DOX S-DOX

body wt (g) 401.4 397.6 421.4 424.4

± ± ± ±

19.9ns 15.8ns 7.1ns 15.1ns

heart ratio (% of body wt) 0.29 0.26 0.26 0.27

± ± ± ±

0.02ns 0.01ns 0.01ns 0.02ns

liver ratio (% of body wt) 2.51 2.76 2.58 2.58

± ± ± ±

0.15ns 0.05ns 0.07ns 0.05ns

Rx kidney ratio 0.31 0.31 0.27 0.27

± ± ± ±

0.02ns 0.02ns 0.01ns 0.01ns

Values are expressed as group mean values and SE; mean values followed by different letters are significantly different. Test SNK p ≤ 0.05. ns, nonsignificant. bC group, AIN93 diet; C-DOX group, AIN93 diet + DOX; A-DOX group, AIN93 diet + 10% Adria strawberry fruit + DOX; S-DOX group, AIN93 diet + 10% Sveva strawberry fruit + DOX. a

Figure 2. Percentage of DNA damage detected before and after DOX injection. Columns labeled with different letters are significantly different (p < 0.05). C group, AIN93 diet; C-DOX group, AIN93 diet + DOX; A-DOX group, AIN93 diet + 10% Adria strawberry fruit + DOX; S-DOX group, AIN93 diet + 10% Sveva strawberry fruit + DOX.

significantly reduced (p < 0.05) in the C-DOX group when compared with control groups. Freeze-dried strawberry supplementation significantly (p < 0.05) increased the enzyme activities with respect to the C-DOX group, with values close to those of the C group, especially for the A-DOX group. Liver Mitochondria Respiration Efficiency and ROS Contents. To examine the protection of strawberries on mitochondrial function against DOX-induced stress, we measured the OCR of isolated rat liver mitochondria. This experiment began assessing mitochondrial basal respiration in an uncoupled state (pyruvate, malate, and FCCP). After the measurement of basal OCR, the following compounds were sequentially added at four different fixed times: rotenone, succinate, antimycin A, and ascorbate plus TMPD. Injection of rotenone inhibited complex I and, as expected, respiration fell to a minimum in all groups (Figure 3a). Injection of succinate allowed the mitochondria to respire via complex II, bypassing complex I, so that OCR values increased. Indeed, A-DOX and S-DOX groups reached the maximal respiration rate (78.53 ± 0.9 and 81.50 ± 1.0 pmol O2/min/protein, respectively) with a significant increase (p < 0.05) compared to the C group, whereas for the C-DOX group a significant decrease was found (40.39 ± 1.1 pmol O2/min/protein). Electron flow was then inhibited at complex III level by the addition of antimycin A and, in fact, respiration stopped again as expected in all groups. Finally, addition of ascorbate and TMPD, which act as electron donors to complex IV, elicited a moderate increase in the OCR, especially in the A-DOX and S-DOX groups, although without any significant difference from the C-group except for C-DOX. To assess if improved mitochondrial functionality observed in A-DOX and S-DOX groups could be due to lower mitochondrial ROS levels, we analyzed the ROS concentration

Figure 1. Effect of strawberry and DOX treatment on the plasma content of retinol and α-tocopherol. Results are expressed as the mean ± SE. Columns labeled with different letters are significantly different (p < 0.05). C group, AIN93 diet; C-DOX group, AIN93 diet + DOX; A-DOX group, AIN93 diet + 10% Adria strawberry fruit + DOX; SDOX group, AIN93 diet + 10% Sveva strawberry fruit + DOX.

the C group. In the C-DOX group a significant increase (1.8fold, p < 0.05) was outlined also for hydroperoxide levels, whereas supplementation with freeze-dried strawberry restored the condition of the C group only in the A-DOX group but not in the S-DOX group. With regard to the activity of the antioxidant enzymes, a positive effect of freeze-dried strawberry-enriched diet on liver antioxidant enzymes was found (Table 7). GPx, GR, and GST activities were significantly decreased (p < 0.05) in the C-DOX group compared to the C group, whereas after xx months, freeze-dried strawberry supplementation significantly increased (p < 0.05) GPx, GR, and GST levels in the A-DOX and S-DOX groups compared to those of the C-DOX group, leading to values similar to those of control group. The same trends were found also for catalase and SOD activities (Table 7): they were 3939



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Table 6. Liver Changes in Protein and Lipid Markers of Oxidative Stressa groupb C C-DOX A-DOX S-DOX

GSH (nmol/mg protein) 164.39 96.13 139.85 132.67

± ± ± ±

protein carbonyl content (nmol/mg protein)

2.54a 1.27c 1.04b 0.34b

7.65 24.33 8.74 5.71

± ± ± ±

TBARS (nmol/mg protein)

0.23b 2.54a 0.93b 1.15b

0.46 0.90 0.37 0.42

± ± ± ±

hydroperoxides (nmol/mg protein)

0.16b 0.15a 0.06b 0.01b

11.42 20.4 12.22 12.96

± ± ± ±

0.04c 0.28a 0.37bc 0.05b

Data are presented as means ± SE. Means belonging to the same set of data with different letters are significantly different (p < 0.05). ns, nonsignificant. bC, AIN93 diet; C-DOX group, AIN93 diet + DOX; A-DOX group, AIN93 diet + 10% Adria strawberry fruit + DOX; S-DOX group, AIN93 diet + 10% Sveva strawberry fruit + DOX.

a

Table 7. Changes in Liver Antioxidant Enzyme Activitya groupb C C-DOX A-DOX S-DOX

GST (nmol/min/mg protein) 430.01 221.42 425.26 424.02

± ± ± ±

3.95a 27.49b 33.43a 4.37a

GPx (nmol/min/mg protein) 0.55 0.31 0.53 0.52

± ± ± ±

GR (nmol/min/mg protein)

0.04a 0.03b 0.07a 0.03a

170.96 83.81 167.73 164.13

± ± ± ±

5.75a 4.61b 6.02a 3.41a

SOD (IU/mg protein) 123.33 45.66 106.75 89.18

± ± ± ±

1.81a 0.05c 2.68ab 2.54b

catalase (IU/mg protein) 18.98 9.73 19.90 19.40

± ± ± ±

1.52a 0.78b 1.78a 1.01a

Data are presented as means ± SE. Means belonging to the same set of data with different letters are significantly different (p < 0.05). bC group, AIN93 diet; C-DOX group, AIN93 diet + DOX; A-DOX group, AIN93 diet + 10% Adria strawberry fruit + DOX; S-DOX group, AIN93 diet + 10% Sveva strawberry fruit + DOX.

a

Figure 4. Effect of strawberry and DOX treatment on mitochondrial ROS concentration. Results are expressed as the mean ± SE. Columns labeled with different letters are significantly different (p < 0.05). C group, AIN93 diet; C-DOX group, AIN93 diet + DOX; A-DOX group, AIN93 diet + 10% Adria strawberry fruit + DOX; S-DOX group, AIN93 diet + 10% Sveva strawberry fruit + DOX.

values were found for groups fed Adria and Sveva fruits (1.74 ± 0.2 and 1.86 ± 0.1 nmol DCF/mg protein, respectively), highlighting that the lower the ROS mitochondrial levels are, the higher is the mitochondrial respiration.

■

DISCUSSION This study evaluates the effect of a long-term diet supplemented with freeze-dried strawberry powder against DOX-induced oxidative stress in rats. AIN93 diet was supplemented with diverse freeze-dried strawberry powders of two cultivars with marked differences regarding fruit phenol and anthocyanin contents. In fact, fruits of Sveva cultivar possessed higher contents of phenols, vitamin C, and TAC than the fruits of Adria cultivar (Table 2), which instead possessed higher anthocyanin contents but lower vitamin C and TAC (Tables 2 and 3). The strawberry fruit quantity was related to a human diet, which considers a calorie intake of 2000 kcal/day: this means that it was equivalent to a substitution of 200 calories with strawberry fruit, calculated to correspond approximately to a quantity of 60 g of strawberry freeze-dried powder or, if expressed in anthocyanidins present in the

Figure 3. Effects of strawberry and DOX treatment on oxygen consumption in liver mitochondria of rats. (a) Mitochondria oxygen consumption was monitored with sequential injection of rotenone, succinate, antimycin A, and ascorbate/TMPD at the indicated time points into each well, after baseline rate measurement. (b) Average changes in mitochondrial OCR after succinate injection. Data are presented as means ± SEM (pmol/min). Columns labeled with different letters are significantly different (p < 0.05). C group, AIN93 diet; C-DOX group, AIN93 diet + DOX; A-DOX group, AIN93 diet + 10% Adria strawberry fruit + DOX; S-DOX group, AIN93 diet + 10% Sveva strawberry fruit + DOX.

in liver mitochondria (Figure 4). We found the highest values of ROS in mitochondria of the C-DOX group, although not significantly different from the C group (2.56 ± 0.2 and 2.50 ± 0.2 nmol DCF/mg protein, respectively), whereas the lowest 3940



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supplementation in healthy female volunteers.43 Some authors attribute this protection against DNA damage to different mechanisms that could involve pathways such as DNA repair and alkylation or formation of anthocyanin−DNA complexes and antioxidant enzymes.44 Moreover, it can be considered that an anthocyanin-rich diet45 indirectly acts on DNA protection through up-regulation expression of different genes involved in detoxification molecules,46 even if this aspect seems to depend on the time of exposure to dietary components as mentioned previously.47 In the present work, the antioxidant capacity provided by the strawberry diet resulted also in enhanced protection of liver components such as proteins and lipids against oxidative stress. Liver protein carbonyls and TBARS/hydroperoxide levels were significantly increased (p < 0.05) by DOX injection in the CDOX group, due to the ROS-induced production, demonstrating that the natural antioxidant defense mechanism to scavenge excessive free radicals has been compromised. The freeze-dried strawberry diet prevented this increase in A-DOX and S-DOX rats, because they showed values similar to those of the C group. The ability of strawberries to modulate the activity of hepatic antioxidant enzymes has been demonstrated in our study, too. We found that DOX administration resulted in a significant decrease in GSH concentration and in the activities of antioxidant enzymes GR, GPx, GST, SOD, and catalase in the C-DOX group compared to the C-group. The effects of DOX were partially reduced by freeze-dried strawberry diet, suggesting that the increase of GSH and endogenous antioxidant enzymes might be one of the important mechanisms of the strawberry against oxidative stress damage, as previously reported.48 Mitochondria are key targets in DOX toxicity, especially their inner membrane.1 DOX can be metabolized into a lipophilic aglycone able to diffuse throughout the mitochondrial membrane, accumulating within it; this aglycone is the starting point for several reactions that release electrons, producing ROS and disturbing the functional integrity of membranes and of the respiratory chain.1 In the present study, the treatment of rats with two doses of 10 mg/kg doxorubicin showed negative effects in liver mitochondria: in fact, the drug increased the total mitochondrial ROS concentration in C-DOX rats, whereas in A-DOX and S-DOX rats, supplementation with strawberry resulted in a decrease of ROS levels induced by DOX injection. To assess if the decrease of ROS, due to freeze-dried strawberry supplementation, could affect mitochondrial functionality, we performed the electron flow experiment to follow and interrogate each complex of the electron transport chain. As expected, respiration stopped with all of the inhibitor (rotenone and antimycin A) injection in all groups, whereas it rose with the injection of the electron donors, especially with succinate (Figure 3b) in S-DOX and A-DOX, demonstrating an improved functionality of the electron transport chain in rats fed strawberry. Interestingly, the extent of the increase was more relevant in the same groups that also presented lower ROS levels. These results could be explained by the possible protection mechanisms of the strawberry: first of all, the well-known antioxidant capacity of the fruits to counteract, both in vitro and in vivo, oxidative stress33,48 and then the interaction between polyphenols and membranes.5 Indeed, from the results obtained in studies conducted to assess the potential of polyphenols to access the inner mitochondrial membrane,49 it seems that mitochondria are able to absorb significant

powder, 80 mg per day for the Adria-fed group and 54 mg per day for the Sveva-fed one (calculated on the results for nutritional compounds of freeze-dried strawberry of cv. Adria and Sveva in Table 3). First, initial and final body weights of the rats were measured. There were no differences in final body weight (p = 0.38) between the treatment groups. It is known that increased liver weight is one symptom of toxicity; however, there were no statistically significant differences in mean liver weight per 100 g of body weight between treatment groups (p = 0.38), suggesting that strawberry supplementation did not affect the liver, as previously reported.32 For plasma analysis, we observed a higher concentration of retinol in A-DOX and S-DOX groups than in the C-DOX group, suggesting that strawberry supplementation could increase the levels of this vitamin and could contribute to strengthen the antioxidant status of rat plasma. However, we did not observe any difference in αtocopherol concentration among groups, but this result is consistent with the very low concentration of this vitamin in strawberries with respect to retinol.5 Retinol and α-tocopherol have a high antioxidant capacity and play a key biological role, especially in protecting cells and tissues from oxidative damage, as well as membrane lipids and lipoproteins from peroxidation.33 Some studies found that retinol significantly reduces lipid oxidation, providing good results in the heart, brain membranes, liver microsomes, and kidney,32 and that it also possesses a protective dose-dependent effect against the chromosomal aberrations induced by doxorubicin in rat bone marrow cells.34 Similarly, other studies showed that intraperitoneal vitamin E (100 mg/kg/day) for 8 days inhibits the general toxic and hepatotoxic effects of DOX and hepatotoxicity in rats, by decreasing malondialdehyde, glutathione peroxidase, and catalase activities.35 The freeze-dried strawberry supplementation seems to protect DNA from DOX-induced stress. Indeed, the evaluation of delta values of percentage damage before and after DOX injection showed that rats fed freeze-dried Adria cultivar, characterized by high anthocyanin contents, presented lower values of DNA damage among all other rat groups subjected to DOX injection, suggesting that a diet rich in anthocyanin is able to protect DNA against in vivo induced oxidative stress. In recent years many in vitro and in vivo studies have focused on the capacity of anthocyanins in protecting against DNA damage. For example, blueberry anthocyanin extracts were found to decrease ROS and DNA damage by tail moment of comet assay as well as to reduce gene and protein expression of p53, phospho-p53, and p21 in UV-irradiated HepG2 cells.36 Similarly, an anthocyanin-rich strawberry extract was able to increase cell viability and decrease DNA damage in fibroblasts exposed to UV-A irradiation,37 whereas cyanidin-3-glucoside (C3G) was found to prevent DNA fragmentation and procaspase-3 cleavage in UV-induced skin damage, very likely by blocking cellular oxidative stress-related events, in particular inflammation and apoptosis.38 However, several in vivo studies have documented the presence of conflicting evidence. In studies with cranberry juice or black currant juice, no increase in protection from oxidative DNA damage was found,39,40 nor was any damage found in a study involving women consuming a polyphenol-rich blueberry.41 On the contrary, in another intervention study, consumption of a high-polyphenol juice determined a decrease in oxidative DNA damage,42 whereas the intake of a mixture of blueberry and apple juices reduced the levels of H2O2-induced DNA damage after 4 weeks of 3941



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endothelial activation in the multi-ethnic study of atherosclerosis (MESA). Am. J. Clin. Nutr. 2006, 83, 1369−1379. (3) Helmersson, J.; Arnlov, J.; Larsson, A.; Basu, S. Low dietary intake of β-carotene, α-tocopherol and ascorbic acid is associated with increased inflammatory and oxidative stress status in a Swedish cohort. Br. J. Nutr. 2009, 101, 1775−1782. (4) Bach-Faig, A.; Berry, E. M.; Lairon, D.; Reguant, J.; Trichopoulou, A.; Dernini, S.; Medina, F. X.; Battino, M.; Belahsen, R.; Miranda, G.; Serra-Majem, L. Mediterranean diet foundation expert group. Mediterranean diet pyramid today. Science and cultural updates. Public Health Nutr. 2011, 14, 2274−2284. (5) Giampieri, F.; Tulipani, S.; Alvarez-Suarez, J. M.; Quiles, J. L.; Mezzetti, B.; Battino, M. The strawberry: composition, nutritional quality, and impact on human health. Nutrition 2012, 28, 9−19. (6) Giampieri, F.; Alvarez-Suarez, J. M.; Mazzoni, L.; Romandini, S.; Bompadre, S.; Diamanti, J.; Capocasa, F.; Mezzetti, B.; Quiles, J. L.; Ferreiro, M. S.; Tulipani, S.; Battino, M. The potential impact of strawberry on human health. Nat. Prod. Res. 2013, 27, 448−455. (7) Seeram, N. P.; Heber, D. Impact of berry phytochemicals on human health: effects beyond antioxidation. In Antioxidant Measurement and Applications; ACS Symposium Series 956; American Chemical Society: Washington, DC, USA, 2006; Chapter 21, pp 326−336. (8) Boivin, D.; Blanchette, M.; Barrette, S.; Moghrabi, A.; Beliveau, R. Inhibition of cancer cell proliferation and suppression of TNF induced activation of NFκB by edible berry juice. Anticancer Res. 2007, 27, 937−948. (9) Starvic, B.; Matula, T. I.; Klassen, R.; Downie, R. H.; Wood, R. J. Effect of flavonoids on mutagenicity and bioavailability of xenobiotics in food. . In Phenolic Compounds in Food and Their Effects on Health II. Antioxidants & Cancer Prevention; ACS Symposium Series 507; American Chemical Society: Washington, DC, USA, 1992; Chapter 17, pp 239−249. (10) Diamanti, J.; Capocasa, F.; Battino, M.; Mezzetti, B. Evaluation of F. × ananassa intra-specific and inter-specific back-crosses to generate new genetic material with increased fruit nutritional quality. J. Berry Res. 2010, 1, 103−114. (11) Diamanti, J.; Capocasa, F.; Balducci, F.; Battino, M.; Hancock, J.; Mezzetti, B. Increasing strawberry fruit sensorial and nutritional quality using wild and cultivated germplasm. PLoS One 2012, 7, e46470. (12) Diamanti, J.; Capocasa, F.; Denoyes, B.; Petit, A.; Chartier, P.; Faedi, W.; Maltoni, M. L.; Battino, M.; Mezzetti, B. Standardized method for evaluation of strawberry (Fragaria × ananassa Duch.) germplasm collections as a genetic resource for fruit nutritional compounds. J. Food Compos. Anal. 2012, 28, 170−178. (13) Tulipani, S.; Mezzetti, B.; Capocasa, F.; Bompadre, S.; Beekwilder, J.; de Vos, C. H.; Capanoglu, E.; Bovy, A.; Battino, M. Antioxidants, phenolic compounds, and nutritional quality of different strawberry genotypes. J. Agric. Food Chem. 2008, 56, 696−704. (14) Lopes da Silva, F.; Escribano-Bailon, M. T.; Perez-Alonso, J. J.; Rivas-Gonzalo, J.; Santos-Buelga, C. Anthocyanin pigments in strawberry. LWT−Food Sci. Technol. 2007, 40, 374−382. (15) Prior, R. L.; Wu, X.; Gu, L.; Hager, T. J.; Hager, A.; Howard, L. R. Whole berries versus berry anthocyanins: interactions with dietary fat levels in the C57BL/6J mouse model of obesity. J. Agric. Food Chem. 2008, 56, 647−653. (16) Quiles, J. L.; Ramirez-Tortosa, M. C.; Huertas, J. R.; Ibañez, S.; Gomez, J. A.; Battino, M.; Mataix, J. Olive oil supplemented with vitamin E affects mitochondrial coenzyme Q levels in liver of rats after an oxidative stress induced by adriamycin. Biofactors 1999, 9, 331− 336. (17) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265−275. (18) Quiles, J. L.; Ochoa, J. J.; Battino, M.; Gutierrez-Rios, P.; Nepomuceno, E. A.; Frías, M. L.; Huertas, J. R.; Mataix, J. Life-long supplementation with a low dosage of coenzyme Q10 in the rat: effects on antioxidant status and DNA damage. Biofactors 2005, 25, 73−86.

concentrations of polyphenols. Once compartmentalized into the cell membranes, these substances seem to enhance membrane integrity and to protect the functional integrity of the mitochondrial respiratory chain.50 Another possible mechanism of strawberries is the activation of the endogenous defense systems, mainly by the modulation of the expression of some antioxidant enzymes. Even if literature data report controversial results that can differ among the several types of cells or pathological conditions studied, it is reasonable to hypothesize that coordination of the endogenous and exogenous antioxidant response is achieved, at least in part, through antioxidant responsive elements (AREs), which are found in the promoters of many of the genes that are inducible by oxidative and chemical stress. For instance, an increasing number of studies indicate that the dietary polyphenols are able to stimulate the transcription of gene coding for antioxidant and phase II detoxifying enzymes, through selective activation of AREs sequences localized in specific gene promoters.45 In conclusion, our in vivo feeding trial on rats submitted to long-term diet supplemented with freeze-dried strawberry powders confirms the potential health benefits of strawberry fruit against oxidative stress; in fact, a reduction of physiological oxidative damage in rats was observed, and this protective effect was strictly linked to the bioactive composition of strawberry fruit. More studies have evidenced that the genotype plays a fundamental role in the phytochemical composition of strawberry fruit, and this variability among fruit genotype is also reflected in the bioactive action developed against oxidative stress.11−13 For this reason it is important to characterize the bioactive composition of strawberries to better understand which compounds are able to improve human health benefits. Additional studies are necessary to characterize the bioactive compounds that play a fundamental role against oxidative stress; these efforts could possibly elucidate by which mechanisms strawberry fruit and their phytochemicals (anthocyanins and polyphenols) can improve antioxidant defenses and would add to the understanding of their potential role in protecting against DNA damage, both in vitro and in vivo.

■

AUTHOR INFORMATION

Corresponding Author

*(M.B.) Phone +39 071 2204646. Fax: +39 071 2204123. Email: [email protected]. Funding

The research was partially granted by EUBerry Project EU FP7 No. 265942. The GIP-USAL is financially supported by the Spanish government through Projects BFU2012-35228 and CSD2007-00063 (Consolider-Ingenio 2010 Programme). Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank Monica Glebocki for extensive editing of the manuscript. REFERENCES

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