Hydroxytyrosyl acetate contributes to the protective effects against oxidative stress of virgin olive oil

Food Chemistry 131 (2012) 869–878

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Hydroxytyrosyl acetate contributes to the protective effects against oxidative stress of virgin olive oil G. Pereira-Caro a, R. Mateos b,⇑, B. Sarria b, R. Cert c, L. Goya b, L. Bravo b a

IFAPA Centro Venta del Llano, Bailén-Motril, Km 18.5, E23620 Mengíbar (Jaén), Spain CSIC, Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN), C/ José Antonio Novais, 10, Ciudad Universitaria, E-28040 Madrid, Spain c CSIC, Instituto de la Grasa, Avda. Padre García Tejero 4, E-41012 Sevilla, Spain b

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Article history: Received 19 February 2011 Received in revised form 5 July 2011 Accepted 19 September 2011 Available online 25 September 2011 Keywords: Hydroxytyrosol Hydroxytyrosyl acetate HepG2 cells Antioxidant defence capacity Oxidative stress biomarkers Olive oil phenols

a b s t r a c t The effects of virgin olive oil phenols, hydroxytyrosyl acetate (HTy-Ac) and hydroxytyrosol (HTy), on cell integrity and steady-state values of cellular redox status were assessed in HepG2 cells, as well as their potential protective effects against oxidative stress induced by tert-butyl hydroperoxide (t-BOOH). Direct treatment for 20 h with 0.5–10 lM HTy or HTy-Ac reduced ROS generation and increased glutathione peroxidase activity at the higher concentrations. Furthermore, after t-BOOH exposure, pretreatment with HTy-Ac and HTy for 2 or 20 h counteracted cell viability damage from 1 lM, counterbalanced reduced glutathione levels from 0.5 lM, protected against lipid peroxidation from 0.5 lM, decreased ROS generation from 1 lM as well as antioxidant enzyme activities from 1 lM. All these changes were statistically significant. HTy-Ac presents antioxidative stress protective effects at physiological concentrations similar to or even slightly higher than that of HTy, thus contributing to the protective role of virgin olive oil. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Numerous studies have shown an association between the consumption of virgin olive oil (VOO), the main source of fat in the Mediterranean diet, and reduced risk of cardiovascular diseases (Bendini, Cerretani, Carrasco-Pancorbo, & Gómez-Caravaca, 2007; Trípoli, Giammanco, Tabacchi, & Di Majo, 2005). Health effects of VOO intake are attributed to its high content of monounsaturated fatty acid (MUFA) but also to minor constituents with biological properties, such as phenolic compounds. In this sense, the EUROLIVE study (Covas, Nyyssonen, et al., 2006) provided clear evidence that VOO is more than just MUFA. This randomised, crossover intervention trial, held with 200 healthy male volunteers, demonstrated that the intake of three VOOs, with different phenolic contents, increased HDL-cholesterol and reduced lipid oxidative damage in a dose-dependent manner (Covas, de la Torre, et al., 2006). These results are in agreement with those previously reported by Marrugat et al. (2004) who observed that the intake of VOO with high-phenolic content decreased lipid oxidative damage. Furthermore, Visioli, Wolfram, Richard, Abdullah, and Crea (2009) recently showed that olive oil phenolic compounds obtained from ⇑ Corresponding author. Address: Department of Metabolism and Nutrition, Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN), CSIC, C/ José Antonio Novais, 10, Ciudad Universitaria, E-28040 Madrid, Spain. Tel.: +34 91 544 56 07/91 549 23 00; fax: +34 91 549 36 27. E-mail address: [email protected] (R. Mateos). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.09.068

olive mill waste water, in which 2-(3,4-dihydroxyphenyl) ethanol (hydroxytyrosol, HTy) is the most bioactive component, increased total plasma glutathione levels when administered to 98 healthy volunteers. In addition, the consumption of phenolic-rich VOO could be responsible for the reduction of DNA damage in peripheral blood lymphocytes in postmenopausal women (Salvini et al., 2006). Accordingly, in the EUROLIVE substudy (Machowetz et al., 2007), the intake of VOO reduced urinary 8-oxodeoxyguanosine levels, regardless of the phenolic content. This outcome is relevant, as oxidative DNA and RNA alterations seem to play an essential role in the pathogenesis of major degenerative diseases. The main compounds that constitute the phenolic fraction of VOO are secoiridoid derivatives of HTy and of 2-(4-hydroxyphenyl) ethanol (tyrosol, Ty), and 2-(3,4-dihydroxyphenyl) ethyl acetate (hydroxytyrosyl acetate, HTy-Ac) (Mateos et al., 2001). HTy shows cardioprotective effects, preventing oxidative stress-induced endothelial dysfunction (Fitó, De la Torre, & Covas, 2007), reducing oxidation of low-density lipoprotein (De la Torre-Carbot et al., 2010; Rietjens, Bast, & Haenen, 2007), inhibiting lipid and protein oxidation in human plasma (Roche et al., 2009) and displays anti-inflammatory (Bitler, Viale, Damaj, & Crea, 2005) and antiplatelet aggregation activities (Dell’Agli et al., 2008). Moreover, HTy has shown a wide range of antitumor effects, inhibiting proliferation and promoting apoptosis in several human tumour-cell lines through several mechanisms (Corona et al., 2009; Fabiani, Fuccelli, Pieravanti, De Bartolomeo, & Morozzi, 2009; Han, Talorete, Yamada, & Isoda, 2009; Sirianini et al., 2010). HTy has also shown

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remarkable ability to protect against oxidative stress by scavenging most radical species in vivo, so that HTy completely counteracts CsA-induced oxidative stress, measured by the increase of GSH/ GSSG ratio and the decrease of lipid peroxidation levels in tubular cells in rat (Capasso et al., 2008). In vitro assays support HTy protective effects against H2O2-induced oxidative damage in renal tubular epithelial cells (Deiana et al., 2008; Incani et al., 2010), red blood cells (Paiva-Martins et al., 2010) and intestinal Caco-2 cells (Deiana et al., 2010). Finally, HTy has also shown chemoprotection against t-BOOH-induced oxidative stress in human hepatoma cells (Goya, Mateos, & Bravo, 2007), regulating the endogenous defence system by inducing antioxidant enzymes (Martín et al., 2010). HTy-Ac is another natural antioxidant present in VOO which has been much less studied than has HTy. HTy-Ac has shown protection effects against oxidative DNA damage in blood cells (Grasso, Siracusa, Spatafora, Renis, & Tringali, 2007) as well as iron-induced oxidative stress in human cervical cells (HeLa) (Bouallagui et al., 2010). However, its protective role against oxidative cell damage remains poorly understood. The aim of the present study was to comparatively study the effects of HTy-Ac and HTy on cell integrity and steady-state values of cellular redox status and to evaluate the potential protective effects of both compounds against oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) in HepG2 cells. Cell viability (Crystal Violet) and cell proliferation (BrdU) were used as cell integrity markers, and concentration of reduced glutathione (GSH), generation of reactive oxygen species (ROS) and activities of the antioxidant enzymes, glutathione peroxidase (GPx) and glutathione reductase (GR), as redox status markers and malondialdehyde (MDA) as a lipid peroxidation marker. 2. Materials and methods 2.1. Materials Culture media DMEM F-12 and foetal bovine serum (FBS) were from Biowhitaker Europe (Innogenetics, Madrid, Spain). Gentamicin, penicillin, streptomycin, tert-butyl hydroperoxide (t-BOOH), glutathione reductase (GR), reduced and oxidised glutathione, nicotine adenine dinucleotide phosphate reduced form (NADPH), dimethyl sulfoxide (DMSO), o-phthalaldehyde (OPT), dichlorofluorescein (DCFH), 2,4-dinitrophenyl hydrazine (DNPH), EDTA and mercaptoethanol were purchased from Sigma Chemicals (Madrid, Spain). Bradford reagent was from BioRad (Madrid, Spain). Crystal Violet indicator and dodecyl sulphate sodium salt (SDS) were acquired from Sigma Chemicals (Madrid, Spain). Cell proliferation ELISA 5-bromo-20 -deoxyuridine (BrdU) (colorimetric) assay kit was from Roche Diagnostics (Roche Molecular Biochemicals, Barcelona, Spain). Hydroxytyrosol (HTy) was recovered with 95% purity from olive oil wastewaters (Fernández-Bolaños et al., 2005), and further purified by column chromatography. Hydroxytyrosyl acetate (HTy-Ac) was obtained from HTy in ethyl acetate after incubation with p-toluene sulphonic acid and purification by column chromatography, following a patented procedure (Alcudia, Cert, Espartero, Mateos, & Trujillo, 2004). HTy and HTy-Ac had purities higher than 99.0%. All reagents were of analytical or chromatographic grade. 2.2. Cell culture Human HepG2 cells were grown in culture dishes (100 mm diameter) and maintained in DMEM-F12 supplemented with 2.5% foetal bovine serum (FBS) and 50 mg/l of each of the following antibiotics: gentamicin, penicillin and streptomycin, in a humidified atmosphere of 5% CO2 and 95% air at 37 °C.

2.3. Olive oil phenol treatments Stocks solutions of HTy and HTy-Ac were dissolved in 1% DMSO in deionised water and diluted with free serum (DMEM-F12) to prepare test solutions (0.1% DMSO final concentration in cell culture media). First, the effect of HTy and HTy-Ac on the HepG2 cell viability and proliferation, after treatment with different concentrations (0.5, 1; 5; 10; 20; 50, 100; 200 and 500 lM) for 24 h, was evaluated, using Crystal Violet and BrdU assays, respectively. Afterwards, two different experiments were carried out. In experiment A, cells were exposed to different concentrations of both olive oil phenols (0.5, 1, 5, 10 lM), dissolved in serum-free culture medium, for 2 and 20 h to evaluate the direct effect of these phenols. In experiment B, cells were also exposed to different concentrations of HTy and HTy-Ac (0.5, 1, 5, 10 lM), dissolved in serum-free culture medium for 2 and 20 h, and then submitted to 400 lM t-BOOH for 3 h in order to determine the chemo-protective effect of both olive oil compounds. All cells were washed with PBS, collected by scraping, and treated as described below for each assay. In both experiments, cell viability, GSH and MDA levels, ROS generation, and GPx and GR activities were evaluated. Control cells were treated with 0.1% DMSO in free serum culture medium to maintain the same ratio of DMSO in treated cells. 2.4. Cell viability Cell viability was determined by using the Crystal Violet assay (Valdes et al., 2004). HepG2 cells were seeded at low density (104 cells per well) in 96-well plates and exposed to different treatment of the tested compounds. After the incubation period, cells were washed with PBS and Crystal Violet (0.2% in ethanol) was added to each well for 20 min. Then, plates were rinsed with tap water, three times, and drained (upside down) on paper towels. Afterwards, 100 ll of 1% sodium dodecyl sulphate (SDS) were added to estimate cell viability. The absorbance of each well at 570 nm was measured using a microplate reader (Bio-Tek, Winooski, VT, USA). Results are expressed as the percentage of cell viability referred to the absorbance measurement obtained with untreated cells. 2.5. Cell proliferation (BrdU) Cell proliferation was evaluated using a colorimetric immunoassay (ELISA), based on the measurement of BrdU incorporation into genomic DNA during its synthesis in proliferating cells. Briefly, HepG2 cells were seeded (104 cells per well) in 96-well plates and exposed to the indicated concentrations of tested compounds for 24 h. Afterwards, cells were labelled by adding BrdU for 4 h and subsequently anti-BrdU antibodies. The immune complexes were detected by the subsequent substrate (tetramethylbenzidine) reaction and quantified by measuring the absorbance at 620 nm using a scanning multi-well spectrophotometer (Bio-Tek, Winooski, VT, USA). Results were expressed as the percentage of cell growth referred to untreated cells. 2.6. GSH evaluation HepG2 cells were seeded in 60 mm diameter plates at a concentration of 1.5  106 per plate. The intracellular concentration of reduced glutathione was estimated by the fluorometric assay previously described (Alía et al., 2006; Goya et al., 2007). Briefly, GSH reacts with o-phthalaldehyde (OPT) at pH 8.0 and fluorescence is measured at an emission wavelength of 460 nm and an excitation wavelength of 340 nm, using a microplate reader

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(Bio-Tek). Results are expressed as percentage of GSH referred to GSH values obtained with untreated cells. 2.7. MDA determination HepG2 cells were seeded in 100 mm diameter plates at a concentration of 4.5  106 per plate. Cellular MDA was analysed by high-performance liquid chromatography (HPLC) as its 2,4dinitrophenylhydrazone (DNPH) derivative (Mateos, Goya, & Bravo, 2004). Values were expressed as the coefficient nmol of MDA/ mg protein. Protein concentration was measured using the Bradford method (Bradford, 1976). 2.8. ROS determination Cellular reactive oxygen species were quantified by the dichlorofluorescein (DCFH) assay, using a microplate reader (Bio-Tek) (Alía et al., 2006; Goya et al., 2007). After being oxidised with intracellular oxidants, DCFH turns into dichorofluorescein (DCF) and emits fluorescence. Results were expressed in fluorescence units. 2.9. GPx and GR determination The determination of the GPx activity is based on the oxidation of reduced glutathione by GPx, using t-BOOH as a substrate, coupled to the disappearance of NADPH by GR, which was monitored by following absorbance at 340 nm (Gunzler, Kremers, & Flohe, 1974). Alternatively, GR activity was determined by following the decrease in absorbance at 340 nm due to the oxidation of NADPH

utilised in the reduction of oxidised glutathione (Goldberg & Spooner, 1987). For both determinations, HepG2 cells were seeded in 100 mm diameter plates at a concentration of 4.5  106 per plate. Results were expressed as mU or lU/mg protein for GPx or GR, respectively. Protein concentration was measured using the Bradford method (Bradford, 1976). 2.10. Statistical analysis Prior to statistical analysis, data were tested for homogeneity of variances by the test of Levene. Multiple comparisons were carried out using one-way ANOVA, followed by Bonferroni tests, when variances were homogeneous, or by the Tamhane test when variances were not. The level of significance was established at p < 0.05. The statistical package SPSS (version 17.0) was used. 3. Results 3.1. Cytotoxic evaluation of olive oil phenols on HepG2 cells The effects of 0.5 to 500 lM HTy and HTy-Ac on HepG2 cell viability, using the Crystal Violet assay, are shown in Fig. 1A. There were no significant differences in cell viability after a 24 h exposure to HTy or HTy-Ac up to 100 lM. Regarding the antiproliferative effects of these polyphenols, evaluated using the BrdU assay, HTy at 50 lM showed a high capacity to inhibit cell proliferation (Fig. 1B) whereas a higher concentration of HTy-Ac, 200 lM, was required to significantly inhibit the percentage of cell proliferation

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Fig. 2. Effects of HTy and HTy-Ac on intracellular concentration of reduced glutathione and malondialdehyde. HepG2 cells were treated with 0.5–10 lM concentrations of HTy and HTy-Ac for 2 and 20 h. (A) Results of fluorescence analysis for GSH are expressed as a percentage of control values ± SD of four different samples per condition. (B) MDA levels in cytoplasm contents measured by HPLC are expressed as nmol MDA/mg protein. Values represent means ± SD of four determinations. Different letters denote statistically significant differences (p < 0.05).

compared to untreated cells. Taking these results together, no cytotoxic effects were observed at 0.5, 1, 5 or 10 lM HTy and HTy-Ac, and therefore these concentrations were selected for experiments A and B. 3.2. Experiment A: direct effect of olive oil phenols on HepG2 cells 3.2.1. Reduced glutathione and malondialdehyde levels HepG2 cells were incubated with increasing concentrations of HTy or HTy-Ac during 2 and 20 h to evaluate the direct effect of these phenol compounds on GSH content and MDA levels (Fig. 2). Fig. 2A shows that treatment of HepG2 cells with 0.5– 10 lM HTy did not induce statistically significant differences in GSH concentrations compared with control cells, although slight variations in GSH contents between cells, treated at the same concentration and different incubation times, were observed, being more relevant at 0.5 and 5 lM. Regarding HTy-Ac, no differences were observed between treated and untreated cells (Fig. 2A). Finally, HepG2 cells, treated with increasing concentrations of HTy or HTy-Ac, did not show changes in MDA levels compared with untreated cells (Fig. 2B) at either incubation period. Random differences were detected between different treatments not showing a relationship with the incubation time or concentration tested. 3.2.2. ROS generation After a 2 h exposure of HepG2 cells with test concentrations of HTy or HTy-Ac, a significant dose-dependent decrease in ROS generation was observed over time (from 0 to 120 min) compared with control cells (Fig. 3). A significant decrease in ROS levels, below that of untreated cells, was observed after 20 h of incubation with both phenols, not following a dose–response at any of the four concentrations assayed. It is noteworthy that a greater de-

crease in ROS production was observed when cells were incubated with phenols for 2 h than for 20 h. 3.2.3. Activity of antioxidant enzymes (GPx and GR) Glutathione peroxidase and glutathione reductase activities were determined as indices of intracellular antioxidant enzymatic activity. As shown in Fig. 4, treatment with 0.5–10 lM HTy or HTyAc for 2 h did not change GPx activity. However, treatment of HepG2 cells for 20 h with both compounds enhanced GPx activity starting at 1 lM up to 10 lM. Maximal activity of this enzyme was obtained at 10 lM. Regarding GR activity, treatment with HTy or HTy-Ac during 2 h did not induce significant alterations at any of the phenolic concentrations tested. However, after 20 h of incubation, 10 lM HTy significantly increased GR activity in contrast to HTy-Ac that did not induce significant changes at any of the tested concentrations (Fig. 4). 3.3. Experiment B: protective effect of olive oil phenols on HepG2 cells against oxidative damage induced by t-BOOH 3.3.1. Cell viability When HepG2 cells were treated with 400 lM t-BOOH during 3 h, a dramatic decrease in cell viability (50–60%) was observed (Fig. 5A). This cell damage was partially counterbalanced with the pre-treatment with 10 lM concentrations of either HTy or HTy-Ac for 2 h. The treatment with 1 and 5 lM HTy for 20 h partially prevented the induced cell damage while the highest concentration assayed, 10 lM, largely protected against the oxidative insult. A similar response was observed with HTy-Ac treatment, that protected HepG2 cells in a dose-dependent manner from 1 lM.

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Fig. 3. Effects of HTy and HTy-Ac on intracellular ROS production. HepG2 cells were treated with different concentrations of HTy (1) and HTy-Ac (2) for 2 or 20 h. Results are expressed in fluorescence units ± SD of 7–8 different samples per condition. SD values are not shown due to bars overlapping. Different letters indicate statistically significant differences (p < 0.05) compared to time-matched controls.

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Fig. 4. Effects of HTy and HTy-Ac on the activities of GPx and GR. HepG2 cells were treated with different concentration of HTy and HTy-Ac for 2 or 20 h. Values represent means ± SD of four determinations. Different letters indicate statistically significant differences (p < 0.05).

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Fig. 5. Protective effects of HTy and HTy-Ac against oxidative stress on cell viability, intracellular concentration of reduced glutathione and malondialdehyde. HepG2 cells were treated with 0.5–10 lM concentrations of HTy and HTy-Ac for 2 and 20 h, washed with PBS, and 400 lM t-BOOH was added to all cultures, except controls, for 3 h. (A) Cell viability is expressed as a percentage of control values. Values represent means ± SD of eight determinations. (B) Results of GSH fluorescence analysis are expressed as a percentage of control values. Values represent means ± SD of four different samples per condition. (C) MDA levels in cytoplasmic contents, measured by HPLC, are expressed as nmol MDA/mg protein as means ± SD of four determinations. Different letters denote statistically significant differences (p < 0.05).

3.3.2. Reduced glutathione concentration The treatment of HepG2 cells with 400 lM t-BOOH induced a remarkable decrease (70%) in the concentration of GSH compared with untreated cells (Fig. 5B). GSH decrease was partly prevented with the pre-treatment with 0.5–10 lM HTy or HTy-Ac for 20 h, whereas the 2 h pre-treatment required 10 lM concentrations of HTy or HTy-Ac, to significantly attenuate the decrease of GSH levels induced by t-BOOH. 3.3.3. Malondialdehyde levels The treatment with 400 lM t-BOOH for 3 h significantly increased cytosolic concentration of MDA (Fig. 5C). This cell lipid oxidative damage is partially or totally reversed with the pretreatment at 0.5–10 lM HTy and HTy-Ac, after 2 and 20 h. Similarly, cells treated with 0.5–5 lM HTy for 2 h were partially protected from the increase of MDA induced by t-BOOH, reaching

control values at 10 lM. Complete inhibition of lipid peroxidation induced with t-BOOH was observed in cells pre-treated with HTy for 20 h. When HepG2 cells were pretreated with different concentrations of HTy-Ac for 2 and 20 h and then submitted to t-BOOH, MDA increase was completely counteracted (Fig. 5C).

3.3.4. ROS generation HepG2 cells treated with t-BOOH showed a significant 2-fold increase in ROS generation after 2 h as compared with control cells (Fig. 6). However, when cells were pretreated with HTy and HTyAc for 2 h, ROS production was partially prevented at doses of 1 and 0.5 lM, respectively. The most significant reduction of ROS generation was observed when cells were pre-treated for 20 h with either phenol, although control ROS levels were not reached, even at the highest concentrations.

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Fig. 6. Protective effects of HTy and HTy-Ac against t-BOOH-induced intracellular ROS generation. HepG2 cells were treated with 0.5–10 lM concentrations of HTy and HTyAc for 2 and 20 h, washed with PBS, and 400 lM t-BOOH was added to all cultures, except controls, for 120 min. Values are expressed in fluorescence units and represent means ± SD of 7–8 different samples per condition. SD values are not included due to bars overlapping. Different letters indicate statistically significant differences (p < 0.05) compared to time-matched controls.

3.3.5. GPx and GR activities The treatment of HepG2 cells with 400 lM t-BOOH, for 3 h, induced a significant increase (about 2.5–3-fold) in both GPx and GR activities (Fig. 7). When cells were pretreated with 1–10 lM HTy for 2 and 20 h, the t-BOOH-induced increase in GPx activity was partially counteracted in a dose- and time-dependent manner. Interestingly, concentrations of HTy-Ac lower than those of HTy were required to induce significant differences in comparison with t-BOOH-treated cells. Accordingly, cells pretreated with all the tested concentrations of HTy for 2 and 20 h showed a decrease in GR activity, reaching control levels at 0.5 lM for 2 h. Similar results were observed in cells pretreated with HTy-Ac for 2 and 20 h.

4. Discussion Virgin olive oil polyphenols present a strong antioxidant activity in vitro (Capasso et al., 2008; Deiana et al., 2010; Goya et al., 2007; Incani et al., 2010) and in vivo (Covas, de la Torre, et al., 2006; Marrugat et al., 2004). Due to their powerful antioxidant activity, these compounds have attracted much attention in light of their possible role in dietary prevention of pathologies in which aetiology and progression have been related to ROS-mediated tissue injury. In this sense, although the biological activities of phenolic compounds have been commonly related to their free radicalscavenging role, recent evidence strongly supports the idea that

natural biophenols may also offer indirect protection by increasing the endogenous defence systems (Martín et al., 2010). High bioavailability of olive oil phenols has been demonstrated in human studies (Caruso, Visioli, Patelli, Galli, & Galli, 2001; Visioli et al., 2000; Vissers, Zock, & Katan, 2004; Vissers, Zock, Roodenburg, Leenen, & Katan, 2002). Olive oil phenols, after intestinal absorption, are transported to the liver where they are metabolised and produce their biological effects. HepG2 cells constitute a validated model of human liver that has been well characterised and is widely used in biochemical and nutritional studies (Alía et al., 2006; Goya et al., 2007; Martín et al., 2010). Therefore, this model has been used to compare HTy-Ac to HTy effects on the antioxidant endogenous properties, with and without external hepatic injury induced with t-BOOH. While HTy has been extensively studied as a disease preventive agent, little attention has been paid to HTyAc. We have previously reported a higher intestinal absorption of HTy-Ac than of HTy (Mateos et al., 2011) and both compounds have displayed extensive uptake and metabolism in HepG2 cells (Mateos, Goya, & Bravo, 2005). However, elevated doses of these dietary compounds can be toxic and mutagenic in cell culture systems and, if consumed in excess, they may cause adverse metabolic reactions in mammals (Rodgers & Grant, 1998). Cell viability results, using the Crystal Violet assay, demonstrated that concentrations of HTy and HTyAc up to 100 lM can be safely used to test potential protective effects against a condition of oxidative stress. Interestingly, up to 200 lM concentrations of HTy-Ac did not affect cell proliferation

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Fig. 7. Protective effects of HTy and HTy-Ac against oxidative stress on the activities of GPx and GR. HepG2 cells were treated with 0.5–10 lM HTy and HTy-Ac for 2 and 20 h. Values represent means ± SD of four determinations. Different letters indicate statistically significant differences (p < 0.05).

whereas 50 lM HTy significantly altered cell proliferation according to BrdU assay results. The concentrations used in the present study (0.5–10 lM), are in the range of plasma concentrations of individuals who consume virgin olive oil in Mediterranean countries. Daily consumption of 25–50 ml of virgin olive oil, which contains about 180 and 300 mg/kg of olive oil phenols, results in an estimated intake of about 9 mg of olive oil phenols per day which is equivalent to 58 lmol of hydroxytyrosol-equivalents per day, of which 30–90% could be absorbed (Vissers et al., 2002), leading to plasma concentrations of 5–10 lM. In this respect a recent study has shown that a single ingestion of 40 ml of olive oil with high content of phenolic compounds (366 mg/kg) leads to a plasma hydroxytyrosol concentration of around 10–30 lM (Covas, de la Torre, et al., 2006). In experiment A, cell toxicity and cellular redox status were determined in cells treated for short and long periods with different micromolar concentrations of HTy and HTy-Ac. The results obtained were compared with those from experiment B, where human hepatoma HepG2 cells were pretreated with HTy and HTy-Ac for 2 and 20 h before being submitted to oxidative stress induced by t-BOOH. Complete protection against t-BOOH was observed when cells were pretreated for 20 h with either compound, showing similar activities at the highest concentration. GSH is the main nonenzymatic antioxidant defence. Within the cell, GSH plays an important role in hepatocyte defence against oxidative stress, acting as a substrate in GPx-catalysed detoxification of organic peroxides, reacting with free radicals and repairing free radical-induced damage through electron-transfer reactions (Scharf, Prustomersky, Knasmuller, Schulte-Hermann, & Huber, 2003). Severe GSH depletion reflects intracellular oxidation, whereas an increase in GSH concentration could be expected to

protect the cell against a potential oxidative stress (Alía et al., 2006; Cuello et al., 2007; Scharf et al., 2003). The studied olive oil phenols did not significantly affect GSH concentration in nonstress situations, whereas pre-treatment with micromolar concentrations of HTy and HTy-Ac for 20 h partially prevented the decrease in GSH concentration induced by t-BOOH. Lower protection effects were obtained after 2 h of pretreatment without obtaining significant statistical differences, except at the highest concentration with both compounds. No differences were observed between the activities of HTy and HTy-Ac. These findings are in accordance with that previously reported with higher doses of HTy (10–40 lM) (Goya et al., 2007) where 10 lM of HTy partially avoided the notable decrease in the concentration of GSH induced by t-BOOH. Moreover, Zhang, Cao, Jiang, Geng, and Zhong (2009) reported that 12.5–50 lM HTy attenuated GSH depletion in HepG2 cells treated with 10 mM acrylamide in a dose-dependent manner. A relevant step in cell membrane degradation is the reaction of ROS with double bonds of polyunsaturated fatty acids (PUFAs), yielding lipid hydroperoxides. Breakdown of such hydroperoxides results in the formation of a great variety of aldehydes. MDA, a three carbon compound formed by scission of peroxidised PUFAs, is one of the main lipid peroxidation products (Pilz, Meineke, & Gleiter, 2000). Since MDA is elevated in several diseases related to free radical damage, it has been widely used as an index of lipoperoxidation in biological and medical sciences. Taking this into account, our group has developed a sensitive method for determining MDA levels in cell cultures (Mateos et al., 2004). Treatment of HepG2 cells with 400 lM t-BOOH induced a significant increase in MDA intracellular concentration which was 2.5-fold higher than that obtained in untreated cells. MDA levels did not change in non-stress situations. Both HTy and HTy-Ac were able to protect

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cell culture against lipid peroxidation induced by t-BOOH, showing partial protection after 2 h of pretreatment and almost complete protection after 20 h of incubation. HTy-Ac was able to preserve (more efficiently) the integrity of the biological membranes in comparison with HTy, suggesting a better accessibility of HTy-Ac to the cell membrane, thus reaching the radicals formed due to its stronger lipophilic nature. These results are in agreement with that previously reported by Paiva-Martins et al. (2010) that indicate that one of the main phenolic compounds found in olive oil, 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde, might play a much more important protective role against ROS-induced oxidative injury in human red blood cells (RBC) than HTy, probably due to its stronger lipophilic nature. Accumulation of ROS in several cellular components is a major cause of molecular injury, leading to cell ageing and to age-related degenerative diseases, such as cancer, brain dysfunction and coronary heart disease (Valko, Rhodes, Moncol, Izakovic, & Mazur, 2006). Direct evaluation of ROS by intracellular DCF fluorescence can be used as an index to quantify overall oxidative stress in cells (Lebel, Ishiropoulos, & Bondy, 1992). ROS generation in cultured HepG2 cells was significantly reduced in the presence of any concentration of HTy and HTy-Ac in both the short and long treatments, HTy-Ac being significantly more active than HTy except at 1 lM after 2 h of incubation. These results support the idea that both olive oil compounds strongly decrease the steady-state generation of ROS by HepG2 cells in culture. Additionally, a significant increase of ROS generation in HepG2 cells treated with 400 lM t-BOOH for 120 min was partially prevented in cultured cells pretreated with HTy and HTy-Ac for 2 and 20 h, resulting in reduced cell oxidative damage, this being more efficient after long term treatment at higher doses (5 and 10 lM). In this sense, similar effects were observed with both olive oil phenols. These findings are in agreement with previous studies which have been carried out in the same cell line with HTy (Goya et al., 2007) and with other natural dietary antioxidants, such as the flavonoid quercetin (Alía et al., 2006). Similarly, olive oil phenolic compounds have been shown to scavenge ROS under natural and chemically simulated oxidative stress conditions (Paiva-Martins et al., 2009). Cell antioxidant enzymes (glutathione reductase and glutathione peroxidase) play an important key in the defence against oxidative stress. GPx is involved in eliminating peroxides, providing an important cellular defence mechanism (Hayes, Flanagan, & Jowsey, 2005; Lei, Cheng, & McClung, 2007), whereas GR is responsible for the regeneration of oxidised glutathione (Argyrou & Blanchard, 2004). Enhancement of glutathione-related enzymes helps to prepare cells to prevent ROS generation in the presence of stressors and thus to suppress oxidative-stress damage (Martín et al., 2010). In agreement, our results show that long-term treatment of HepG2 cells with HTy and HTy-Ac increase the activity of these protective enzymes which are involved in peroxide detoxification, acting as a barrier against hydroperoxide attack and thus preventing ROS cytotoxicity. This outcome indicates that these olive oil phenols are able to confront basal or increased stress-induced ROS generation. However, the treatment of HepG2 with 10 lM HTy significantly increased GR activity, whereas HTy-Ac did not. The significant increase in the activity of GPx and GR observed after 3 h of treatment with t-BOOH, indicates a positive response of the cell defence system against acute oxidative stress. However, the rapid return of the antioxidant enzyme activity to basal values would place the cell in a favourable condition to deal with a new insult. Pretreatment of HepG2 cells for 20 h with any concentration of HTy and HTy-Ac prevents the increase in the activity of GPx and GR induced by t-BOOH more efficiently than do 2 h incubation periods. HTy-Ac was more effective than was HTy, as substantial recuperation of basal conditions was observed at the lowest assayed concentration, 0.5 lM. These findings sug-

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gest that the pretreatment with HTy or HTy-Ac fortified inherent cellular defence capacity by returning the cell to a steady-state activity and preparing cells to counteract ROS generation in the presence of stressors. These data are in agreement with a recent study that shows that HTy may enhance endogenous defence capacity, inducing higher expression and activity of antioxidant/ detoxifying enzymes through a mechanism involving nuclear translocation of Nrf2 and the activation of PI3K/AKT and ERKs pathways in HepG2 cells (Martín et al., 2010). Accordingly, HTy induced significant protection against hydrogen peroxide-induced renal epithelial injury, linked to their interactions within the MAP kinase and PI3 kinase signalling cascades (Incani et al., 2010). Further research is necessary to elucidate the mechanism of action of HTy-Ac.

5. Conclusion Although no relevant changes in endogenous antioxidant defence were observed when cells were directly treated with HTy and HTy-Ac, both phenolic antioxidants reduced generation of ROS, maintained MDA and GSH concentration and slightly enhanced the activity of GPx and GR enzymes in HepG2 cells, preventing conditions which favour oxidative stress. Furthermore, both HTy-Ac and HTy have the ability to protect human HepG2 cells against oxidative damage by reducing free radical activity and preparing the antioxidant defence system by activating intracellular mechanisms involved in signalling processes. These findings support the idea that HTy presents chemoprotective effects and give further evidence that HTy-Ac contributes to the protective role of virgin olive oil, showing equal or slightly higher protection capacity against oxidative stress than HTy.

Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by projects AGL2007-64042, AGL200766373-C03/ALI and CSD2007-00063 from the Consolider-Ingenio Program of the Spanish Ministry of Science and Innovation (CICYT), a grant (RTA2007-000036-00-00) from the Research National Institute of Food, Agriculture and Technology (INIA), a contract (110105090014) with CSIC-IFAPA. G.P.-C. is a predoctoral fellow of the National Research Institute of Food, Agriculture and Technology (INIA). References Alcudia, F., Cert, A., Espartero, J. L., Mateos, R., & Trujillo, M. (2004). Method of preparing hydroxytyrosol esters, esters thus obtained and use of same. PCT WO 2004/005237. Alía, M., Mateos, R., Ramos, S., Lecumberri, E., Bravo, L., & Goya, L. (2006). Influence of quercetin and rutin on growth and the antioxidant defense system in a human hepatoma cell line (HepG2). European Journal of Nutrition, 45, 19–28. Argyrou, A., & Blanchard, J. S. (2004). Flavoprotein disulfide reductases: Advances in chemistry and function. Progress in Nucleic Acid Research and Molecular Biology, 78, 89–142. Bendini, A., Cerretani, L., Carrasco-Pancorbo, A., & Gómez-Caravaca, A. M. (2007). Phenolic molecules in virgin olive oils: A survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules, 12, 1679–1719. Bitler, C. M., Viale, T. M., Damaj, B., & Crea, R. (2005). Hydrolyzed olive vegetation water in mice has anti-inflammatory activity. Journal of Nutrition, 135, 1475–1479. Bouallagui, Z., Bouaziz, M., Lassoued, S., Engasser, J. M., Ghoul, M., & Sayadi, S. (2010). Hydroxytyrosol acyl esters: Biosynthesis and activities. Applied Biochemistry and Biotechnology. doi:10.1007/s12010-010-9065-2.

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