Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H2O2 induced oxidative damage

Chemico-Biological Interactions 193 (2011) 232–239

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Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H2O2 induced oxidative damage Monica Deiana a,⇑, Alessandra Incani a, Antonella Rosa a, Angela Atzeri a, Debora Loru a, Barbara Cabboi a, M. Paola Melis a, Ricardo Lucas b, Juan C. Morales b, M. Assunta Dessì a a b

Dipartimento di Biologia Sperimentale, Sezione di Patologia Sperimentale, Università degli Studi di Cagliari, Cittadella Universitaria SS 554, 09042 Monserrato, Cagliari, Italy Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, 49 Americo Vespucio, 41092 Sevilla, Spain

a r t i c l e

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Article history: Received 16 May 2011 Received in revised form 4 July 2011 Accepted 10 July 2011 Available online 26 July 2011 Keywords: Hydrogen peroxide Hydroxytyrosol glucuronides Lipid peroxidation MAPK Oxidative stress

a b s t r a c t Hydroxytyrosol (2-(30 ,40 -dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 30 -O-b-D-glucuronide and 40 -O-b-D-glucuronide derivatives and 2-(30 ,40 -dihydroxyphenyl)ethanol-1-O-b-D-glucuronide, in comparison with the parent compound, to inhibit H2O2 induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H2O2 treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellularsignal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Hydroxytyrosol (2-(30 ,40 -dihydroxyphenyl)ethanol; HT) is the most potent antioxidant among the phenolics found in extravirgin olive oil. The biological activities of HT have been explored by several groups as recently reviewed by Granados-Principal et al. [1]. Two of the most relevant reported activities are antitumor and anti-inflammatory effects. The anticancer activity seems to be a result of its capacity to exert cytotoxic effects, such as induction of apoptosis, cell cycle arrest and antiproliferative effects. The antiinflammatory and antiplatelet aggregation action of HT, together with its antiatherogenic capacity and cardioprotective effects, are important in counteracting the development of cardiovascular diseases. HT biological effects stem mainly from its free radical scavenging and metal chelating properties, most probably due to its

Abbreviations: HT, hydroxytyrosol; HP, fatty acid hydroperoxides; UFA, unsaturated fatty acids; 7-keto, 7-ketocholesterol; Glu1, hydroxytyrosol 30 -O-b-Dglucuronide; Glu2, hydroxytyrosol 40 -O-b-D-glucuronide; Glu3, 2-(30 ,40 -dihydroxyphenyl)ethanol-1-O-b-D-glucuronide; ERK, extracellular-signal related kinase. ⇑ Corresponding author. Tel.: +39 070 6754126; fax: +39 070 6754032. E-mail address: [email protected] (M. Deiana). 0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.07.002

ortho-diphenolic structure, whose high antioxidant activity may be explained by the high electron donating effect of the second hydroxyl group. At the same time it also shows effects on cell signaling pathways and on gene expression [1]. A daily intake of 25–50 ml of extravirgin olive oil, typical of Mediterranean countries, may supply at least 1 mg of simple phenols, free HT and tyrosol, and 8 mg of their secoiridoids derivatives, mainly oleuropein and ligstroside-aglycones [2]. However, the majority of these complex polyphenols undergo gastro-intestinal biotransformation. HT and tyrosol conjugated forms, except oleuropein, are rapidly hydrolysed under gastric conditions, effectively increasing the relative amount of simple phenols, HT and tyrosol, entering the small and large intestine [3]. Oleuropein is not degraded under acidic conditions and is not absorbed in the parental form in the small intestine; however, once reached the large intestine, it may be subjected to rapid degradation by the colonic microflora, to yield HT [3]. The absorption of HT takes place in the small intestine and the colon [4] through a passive diffusion mechanism [5]. In the process of crossing enterocytes, HT is subjected to a classic phase I/II biotransformation, and then to an important first pass metabolism in the liver cells. This process leads to the formation of orthomethyl derivatives (homovanillic alcohol), glucuronide and sulfate

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derivatives [6] and glutathionyl conjugates [3], which are detectable both in the plasma and in the urine [7]. Urinary recoveries as high as 80% of the ingested amounts of HT have been reported in humans [6,8]; over 90% of the urinary metabolites were conjugates, mainly glucuronidated metabolites [8–10]. 30 -O- and 40 -Ob-glucuronide of HT have been detected in human urine after ingestion of olive oil [11]. As unconjugated HT represents a very small proportion of circulating species, the testing of the biological activity of the conjugated metabolites is of great importance for the understanding of its in vivo properties. Few in vitro studies have been carried out on HT glucuronide conjugates and showed contrasting results: the 30 -O-b-glucuronide exerted a more potent radical scavenging activity than HT itself in a simple chemical system [7], while failed together with the 40 -O-b-glucuronide to protect LDL against oxidation [11]. The aim of this study was to examine the activity of the HT glucuronide metabolites in comparison with the parent compound against oxidative stress in vitro. H2O2 induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line that retains characteristics of the proximal tubular epithelium [12]. This cell line has been used to study the mechanisms of kidney toxicity [13,14]. The generation of H2O2 has been implicated in the pathogenesis of several forms of acute tubular cell injury, where the lipid peroxidation process plays a central role [15]. We have previously shown that H2O2 treatment in LLC-PK1 cells may induce a toxic effect both through a direct oxidative damage, initiating the membrane lipid peroxidation process [16], and inducing cell death, interacting selectively within signaling cascades that regulate cell survival following exposure to oxidative stress, as the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways [17]. As 30 -O- and 40 -O-b-glucuronide of HT have been detected in human urine, after ingestion of olive oil [11], three glucuronide metabolites of HT, 30 -O-b-D-glucuronide and 40 -O-b-D-glucuronide, as a mixture of isomers, and 2-(30 ,40 dihydroxyphenyl)ethanol-1-O-b-D-glucuronide (Fig. 1) have been synthesized and tested, in order to investigate the influence of the conjugation and its position within the phenolic structure. H2O2 induced toxicity was evaluated in terms of changes in the phosphorylation state of the kinases ERK 1/2 and Akt/PKB. H2O2 induced oxidative cell damage was also evaluated as malondialdehyde (MDA) production and through the more sensible and precise markers of the lipid peroxidation process, the modification of the profile of the major oxidizable membrane lipids, unsaturated fatty acids (UFA) and cholesterol. 2. Materials and methods 2.1. Chemicals All solvents used were HPLC grade (Merck, Darmstadt, Germany). Fatty acid standards, cholesterol, 5-cholesten-3b-ol-7-one (7-keto), H2O2, 2-thiobarbituric acid (TBA), trichloroacetic acid

Fig. 1. Structure of hydroxytyrosol glucuronides Glu1, Glu2 and Glu3 and their parent compound hydroxytyrosol (HT).

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(TCA), 1,1,3,3-tetraethoxypropane (TEP), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from Sigma Chemical (St. Louis, MO). Desferal (deferox amine methanesulfonate) was purchased from CIBA-Geigy (Basel, Switzerland). HT was purchased from Cayman Chemical Company (Ann Arbor, MI). Nitrocellulose membranes (Hybond-ECL), enhanced chemiluminescence reagent (ECL) and Hyperfilm-ECL were purchased from Amersham Biosciences (Buckinghamshire, UK). Phospho-Akt (ser473) antibody and Akt antibody, were obtained from Cell Signaling Technology, Inc. (Danvers, MA). Anti-phosphoMAP Kinase1/2 (Erk1/2) and anti-MAP Kinase 1/2 (Erk1/2) were obtained from Upstate cell signaling solution (Hampshire UK). Cell culture materials were purchased from Invitrogen (Milano, Italy).

2.2. HT glucuronides and partition coefficient measurement HT 30 -O-b-D-glucuronide (Glu1) and HT 40 -O-b-D-glucuronide (Glu2), as a 1.7:1 mixture of isomers, and 2-(30 ,40 -dihydroxyphenyl)ethanol-1-O-b-D-glucuronide (Glu3) have been synthesized as previously described [18]. HT glucuronides, whose solubility in water was greater than that of HT (5 g/100 ml = 0.32 M), were prepared in stock aqueous solutions (25 mM, pH 6.8). Partition coefficient (log P) values for Glu1,2 and Glu3 were calculated using the Crippen’s fragmentation [19] in the ChemBioDraw Ultra 11.0 software and compared with experimental log P values for HT, previously determined [20,21].

2.3. Cell culture and experimental design The LLC-PK1 cells (a porcine renal epithelial cell line with proximal tubule epithelial characteristics) were obtained from European Collection of Cell Cultures (ECACC, Salisbury, UK). Frozen stocks were routinely thawed, grown in 75-cm2 tissue culture flasks in an incubator with a humidified atmosphere of 5% CO2/ 95% air at 37 °C, and passaged once a week. The maintenance culture medium was M199 supplemented with 10% fetal bovine serum, penicillin (100 U/ml)-streptomycin (100 lg/ml). Experiments were performed with cells from passages 44 to 60. For the experiments the cells were harvested and subcultured in the maintenance culture medium in 96-well plates by inoculating 104 cells in 200 ll/well, in 35-mm culture plates by inoculating 12.5  104 cells in 2.5 ml/dish and in 90-mm culture dishes by inoculating 5  105 cells in 10 ml/dish. After at least 24 h of growth the maintenance culture medium was replaced, after two washes, for the different experiments. The protein concentration in all experiments was determined by the Bradford protein assay (Sigma) [22].

2.4. Cytotoxic activity LLC-PK1 cells grown for 24 h in 96-well plates were exposed to various concentrations (5–500 lM) of HT and its glucuronide conjugates, in a volume of 200 ll/well, and the cytotoxicity was assessed by the MTT assay [23]. In order to assess the protective effect of HT and its glucuronide conjugates against H2O2 induced toxicity, cells were pre-treated with the tested compounds (5–10 lM; 24 h) prior to exposure to H2O2 (25 lM, 1 h). Cells were washed thoroughly with PBS following the removal of HT or metabolites and prior to the treatment with H2O2. After treatment the cells were washed with PBS prior to the addition of fresh medium and incubation for 24 h at 37 °C. After incubation the cytotoxic effect was assessed by the MTT assay [23].

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2.5. Immunoblotting All experiments were performed on 70% confluent cells in 6well plate. Three sets of cell treatments were performed: (a) cells exposed to H2O2 (25 lM) in PBS for 1 h; (b) cells pre-treated with HT or its glucuronide metabolites (5–10 lM, 24 h) before the treatment with H2O2 (25 lM, 1 h); (c) control cells. Following treatment, the cells were washed with ice-cold PBS prior to the addition of 150 ll lysis buffer for protein extraction [50 mM Tris base, Triton X-100 (1:100 v/v), 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, 0.5 mM PMSF, 1 mM sodium ortho-vanadate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, and mammalian protease inhibitor cocktail (1:100 v/v)]. Cells were scraped on ice and lysates were incubated for 45 min on ice before centrifugation at 2000g at 4 °C for 5 min. The samples were boiled at 98 °C for 3 min in boiling buffer (62.5 mM Tris, pH 6.8 containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.0025% bromophenol blue). The boiled samples were run on 8% SDS–polyacrylamide gels (20 lg/lane), and the proteins were transferred to nitrocellulose membranes (Hybond-ECL) by semi-dry electroblotting (1.5 mA/ cm2). The nitrocellulose membrane was then incubated in a blocking buffer [TBS supplemented with 0.05% (v/v) Tween 20 (TTBS)] containing 4% (w/v) skimmed milk powder for 1 h at room temperature, followed by three 5 min washes in TTBS. The blots were then incubated with either anti-Akt (1:1000 dilution), anti-phospho-Akt (Ser473) (1:1000 dilution), anti-p42/44 MAPK (1:1000 dilution), anti-phospho-p42/44 MAPK (Thr202/Tyr204) (1:1000 dilution) in TTBS containing 1% (w/v) skimmed milk powder (antibody buffer) overnight at room temperature on a three-dimensional rocking table. The blots were washed twice for 10 min in TTBS and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2000 dilution) in antibody buffer for 1 h. Finally, the blots were washed twice for 10 min in TTBS and exposed to ECLÒ reagent for 1–2 min as described in the manufacturer’s protocol (Amersham Biosciences). The blots were exposed to HyperfilmECL for 2–5 min in an auto-radiographic cassette and developed. The molecular weights of the bands were calculated from comparison with pre-stained molecular weight markers that were run in parallel with the samples (molecular weight, 27,000–180,000 and 6000–45,000; Sigma). Protein bands were quantified using Quantity One software (Bio-Rad, Hertfordshire, UK). 2.6. Uptake of HT and glucuronides Cells were incubated with 250 lM of HT or glucuronide conjugates for 90 min at 37 °C. Following exposure, cells were washed with ice-cold PBS and rapidly lysed on ice using aqueous methanol (50%, v/v) containing HCl (0.1%). Lysed cells were scraped and left on ice to solubilize for 45 min and then centrifuged at 2000g for 5 min at 4 °C to remove unbroken cell debris and nuclei. The supernatants were recovered and analyzed by an Agilent Technologies (Palo Alto, CA) 1100 liquid chromatograph equipped with a diode array detector, HPLC-DAD, using a Agilent Technologies column, Eclipse XDB – C8, 150  4.6 mm, with a mobile phase of H2O/ H3PO4(99/1):MeOH/CH3CN(50/50) 97:3, v:v at a flow rate of 0.5 ml/min. Detection of the peaks was carried out at 280 nm. For each experiment a calibration curve was performed using standard compounds (0–1 mM). 2.7. Antioxidant activity Three sets of cell treatments were performed: (a) cells exposed to H2O2 (100 lM) in PBS for 1 h; (b) cells pre-treated with HT or glucuronide conjugates (5–10 lM) 30 min before H2O2 treatment; (c) control cells. After H2O2 treatment, the cells were scraped on ice and centrifuged at 2000g at 4 °C for 5 min. After centrifugation

pellets were separated from supernatants: the pellet was used for lipid analyses and for the evaluation of the protein concentration by the Bradford protein assay [22], while MDA quantitation was performed in the supernatant. 2.7.1. Determination of MDA MDA concentration was determined as MDA-TBA adduct by HPLC [24]. 0.4 ml of the supernatant were added with 0.1 ml of TCA (10%) and 0.2 ml of TBA (0.6%) and incubated at 90 °C for 45 min. After centrifugation aliquots of the supernatant were injected into the HPLC-DAD system. Separation of MDA-TBA adduct was carried out using a Spherisorb column, Inertsil 5 ODS-2, 250  4.6 mm, with a mobile phase of 450 mM KH2PO/MeOH (65/35, v/v) at a flow rate of 1 ml/min. Detection of the adduct peak was carried out at 532 nm. For each experiment a calibration curve was performed using standards (0–10 lM) of TEP. 2.7.2. Determination of fatty acids, cholesterol and oxidized products Total lipids were extracted from the cell pellet dissolved in 12 ml of CHCl3/MeOH (2/1, v/v) solution as indicated by the Folch et al. procedure [25]. Separation of cholesterol and free fatty acids was obtained by mild saponification [26] as follows: 6 ml of the CHCl3 fraction, containing the lipids, from each sample was dried down and dissolved in 5 ml of ethanol; 100 ll of Desferal solution (25 mg/ml of H2O), 1 ml of a water solution of ascorbic acid 25% (w/v), and 0.5 ml of 10 N KOH were added. The mixtures were left in the dark at room temperature overnight. After addition of 10 ml of n-hexane and 7 ml of H2O, samples were centrifuged for 1 h at 900g. The hexane phase with cholesterol was collected, the dried residue was dissolved in 0.25 ml of MeOH and aliquots of the samples were injected into the HPLC system. After addition of further 10 ml of n-hexane to the mixtures, samples were acidified with 37% HCl to pH 3–4 and then centrifuged for 1 h at 900g. The hexane phase with free fatty acid was collected, a part was evaporated and the residue was dissolved in 0.25 ml of CH3CN with 0.14% (v/v) CH3COOH. Aliquots of the samples were injected into the HPLC system. The recovery of fatty acids and cholesterol was calculated by using an external standard mixture. All solvents evaporation was performed under vacuum. Separation of UFA and cholesterol was carried out with a HPLC-DAD system. Cholesterol, detected at 203 nm, and 7-ketocholesterol (7-keto), detected at 245 nm, were measured using a Varian column (Middelburg, The Netherlands), Inertsil 5 ODS-3, 150  3 mm, and MeOH as mobile phase, at a flow rate of 0.4 ml/min. UFA, detected at 200 nm, and conjugated fatty acids hydroperoxides (HP), detected at 234 nm, were measured using a Varian column, Inertsil 5 ODS-2, 150  4.6 mm, with a mobile phase of CH3CN/H2O (70/30, v/v) containing CH3COOH 0.12% at a flow rate of 1.5 ml/min. The identification of the peaks was made using standard compounds and the second derivative as well as conventional UV spectra, generated using the Agilent Chemstation A.10.02. software, as detailed in a previous paper [16]. 2.8. Statistical analysis GraphPad INSTAT software (GraphPad software, San Diego, CA) was used to calculate the means and standard deviations of three independent experiments (n = 9 for each sample/condition). Evaluation of the statistical significance of differences was performed using one-way analysis of variation (ANOVA) and the Bonferroni post test. 3. Results HT glucuronides, that did not exert any cytotoxic effect in the concentration range 5–500 lM, were examined in comparison

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with HT for their protective effects at 5–10 lM against oxidative stress-induced cell damage in LLC-PK1 cells. We first tested the effect of HT metabolites on the phosphorylation states of ERK 1/2 and Akt/PBK since we have previously shown that incubation of kidney cells with H2O2 resulted in a decrease of cell viability that also correlated with a change in the phosphorylation states of these kinases [17]. Exposure to H2O2 (25 lM for 1 h) resulted in a 55.4 ± 10.0% decrease in the ability of the cells to reduce MTT (Fig. 2). Pre-treatment with the glucuronide metabolites, Glu1,2 and Glu3, did not exert any protection while a significant protection against H2O2-induced cellular injury was observed when cells were pre-treated with HT (viability of 73.7 ± 17.5% at 5 lM and 84.5 ± 17.1% at 10 lM) (Fig. 2). H2O2 treatment (25 lM for 1 h) also resulted in a significant increase of the phosphorylation of ERK1 and ERK2 above basal level (Fig. 3A) together with a decrease of Akt phosphorylation (Fig. 3B). No changes were observed in the total content of the kinases. Both Glu1,2 and Glu3 were unable to induce any change in the phosphorylation states of ERK1/2 and AKT (Fig. 3). Pre-treatment with HT instead showed a strong inhibition of ERK 1/2 activation (Fig. 3A) at both concentrations tested (5–10 lM) as indicated by the statistically significant reduction in the ERK1/2 phosphorylation state. These changes in the ERK activation state were paralleled by a significant increase in the phosphorylation of Akt, indicating a protective action against H2O2-induced inhibition of the kinase activity (Fig. 3B). Depending on the concentration used and the time of exposure, H2O2 treatment of LLC-PK1 cells may result in the induction of a lipid peroxidation process, indicated by the detection of various oxidation products [16,27]. In order to induce a detectable lipid peroxidation process but not cell death, LLC-PK1 cells were treated with H2O2 (100 lM for 1 h). Membrane oxidative injury was assessed by measuring MDA production and the modification of the more oxidizable membrane lipids, UFA and cholesterol. H2O2 (100 lM) exposure for 1 h resulted in a significant increase of MDA level in the treated samples compared with controls (Fig. 4). Pre-treatment with both glucuronide metabolites and HT significantly inhibited MDA production at all tested concentrations although HT was more efficient than the corresponding metabolites. The lipid fraction of the LLC-PK1 cells was extracted and the levels of the major UFA (docosahexaenoic acid (22:6 n-3), eicosapentaenoic acid (20:5 n-3), arachidonic acid (20:4), eicosatrienoic acid (20:3 n-9), linoleic acid (18:2), oleic acid (18:1 n-9)) and cholesterol were assessed together with their oxidation products (conjugated fatty acid hydroperoxides (HP) and the 7-ketocholesterol (7-keto)). Although the levels of the measured UFA, expressed as total UFA (Fig. 5A) and the levels of cholesterol (Fig. 5B) were not significantly modified by H2O2 treatment, a highly significant increase of HP (181.3 ± 5.0% of the control) (Fig. 5A) and 7-keto (167.3 ± 0.8% of the control) (Fig. 5B) was

Fig. 2. Cell viability (MTT assay), expressed as % of the control, in LLC-PK1 cultures pre-treated for 24 h with HT and HT glucuronides (Glu1,2 and Glu3), 5 and 10 lM, and treated with H2O2, 25 lM, for 1 h. ⁄⁄⁄p < 0.001 respect to the control; ap < 0.001, c p < 0.05 respect to the H2O2 treated cells.

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observed, indicating an ongoing oxidative process. A significant protective effect was observed in the samples pre-treated with HT glucuronides at the highest concentration tested, comparable to that of HT, maintaining HP values at the level of the control sample (Fig. 5A). Similar efficacy was exerted in protecting cholesterol from oxidation, as the concentration of 7-keto was significantly lower in all pre-treated samples with respect to the oxidized samples (Fig. 5B). To exclude the hydrolytic/enzymatic cleavage of the glucuronide metabolites to release free HT, at 90 min incubation in the LLC-PK1 cultures, the analyses of HT and glucuronide metabolites in the medium and in the lysed cells were performed. As shown in Fig. 6A, the quantity of HT added was mainly found in the medium and around 7.5% was measured in the lysed cells. There was no detectable uptake of glucuronide metabolites into kidney cells at 90 min exposure; the amount of Glu1,2 and Glu3 added was entirely detected by comparison with standard compounds in the medium (Fig. 6B and C), whereas the peak of HT was not present, indicating that no degradation process had undergone. The calculated partition coefficients (log P) of Glu1,2 and Glu3, 0.92 and 0.82, respectively, indicated that these metabolites are much more polar than HT, which experimental log P ranged from 0.08 to 0.09 [20,21].

4. Discussion It is well established that HT is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites determined in human plasma and urine samples following olive oil intake [4,6,8–11,28]. These metabolites should be taken into consideration to provide in vivo relevance for any activity. If the conjugates also show beneficial activity at realistic physiological concentrations, the connection between the biological activity and the benefits of HT in the diet becomes more robust. We have previously shown that the main O-methylated metabolite of HT, homovanillic alcohol, is able to protect both enterocytes [29] and renal tubular epithelial cells [16,17] against oxidative damage. Its efficacy was close to that of HT, acting both as scavenger of oxidizing species and modulating the MAP kinase and PI3 kinase signaling pathways that regulate cell survival following exposure to H2O2 oxidative action. Conjugation of xenobiotics with glucuronic acid is a common detoxification pathway that leads to an increased polarity/hydrophilicity of the molecule. It has generally been assumed that its role is the rapid excretion of the parent compound and its biological inactivation. The increased polarity of the glucuronide limits its access to cell what is consistent with our data that did not show any detectable uptake of HT glucuronides in LLC-PK1 cells. None of the tested glucuronides exhibited protection against H2O2-induced loss in renal cells viability and also failed to prevent H2O2-induced changes in the phosphorylation states of ERK 1/2 and Akt/PKB. HT was able to enter LLC-PK1 cells and to prevent cell death and related deactivation of the pro-survival Akt/PKB signaling pathway and activation of the pro-death ERK 1/2 signals, in agreement with data obtained in previous studies [17]. These results strengthen the hypothesis that protection against H2O2 toxic action by HT is exerted intracellularly rather than extracellularly on membrane receptors. Many reports show evidence for non-receptor-mediated activation of ERK in different H2O2 treated cell lines [30–32]; H2O2 permeates the cell membrane by passive diffusion and alters the cellular redox status, eventually converting to radicals such as hydroxyl radicals in the presence of transition metals [33] and directly oxidizing the sulfhydryl groups of cysteine residues to disulfide bonds, modifying the function of proteins [34]. HT has been reported to react directly with intracellular H2O2 and/or related radicals. However, HT protection may also be

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Fig. 3. Modulation of H2O2-induced ERK phosphorylation (A) and Akt de-phosphorylation (B) by HT and HT glucuronides (Glu1,2 and Glu3) in LLC-PK1 cells, treated with HT and HT glucuronides, 5 and 10 lM, 24 h prior to the addition of H2O2, 25 lM, for 1 h. ⁄⁄⁄p < 0.001 respect to the control; ap < 0.001, bp < 0.01, cp < 0.05 respect to the H2O2 treated cells.

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attributed to the rapid generation of the arylating catechol quinone as an oxidation product [35]. Hydrophilic catechols, such as HT, are readily oxidized in vivo to catechol quinone electrophiles, thereby possessing properties similar to those of lipophilic arylating

tocopherols quinones, including an ability to undergo Michael additions with cellular thiols such as cysteinyl containing proteins; their hydrophilicity allows the catechol quinone electrophile to react with available thiol nucleophilic groups in enzymes and signaling molecules within the hydrophilic environment of the cell [35]. Glucuronide metabolites of HT did not exert the same efficacy of the parent compound in protecting renal cells against H2O2-induced death, however, they were able to exert an antioxidant effect. This is in agreement with reported data for other phenolic antioxidants in the literature. It has been shown that some flavonoids after glucuronidation may still act as antioxidants in biological fluids [36–38], for example inhibiting xanthine oxidase and lipoxygenase [39], retaining a reduced cytoprotective capacity in vitro against H2O2 induced oxidative stress [40], and displaying a slightly decreased ability to scavenge radicals (TEAC test) [41]. Even more relevant is the fact that quercetin and its glucuronide metabolites, at physiological concentrations, can inhibit the expression of key molecules involved in monocyte recruitment during the early stages of atherosclerosis [42]. The antioxidant activity of HT glucuronides is controversial; the 30 -O-b-glucuronide conjugate showed a more efficient radical scavenging potency (DPPH test) than HT itself [7]. However Khymenets et al. reported that 30 -and 40 -O-b-glucuronide metabolites, although slightly

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Fig. 5. Values of the total UFA together with fatty acid hydroperoxides (HP) (A) and cholesterol and 7-ketocholesterol (7-keto) (B) measured in LLC-PK1 cells after 1 h incubation with H2O2 and treated with HT or HT glucuronides (Glu1,2 and Glu3), 5 and 10 lM, expressed as % of the control values (total UFA as sum of 22:6 n-3, 11.3 ± 1.7 lg/mg; 20:5 n-3, 8.1 ± 0.9 lg/mg; 20:4, 40.9 ± 6.2 lg/mg; 20:3 n-9, 13.5 ± 1.1 lg/mg; 18:2, 19.6 ± 2.1 lg/mg; 18:1 n-9, 145.2 ± 10.2 lg/mg; HP, 0.2 ± 0.1 nmol/mg; cholesterol, 78.3 ± 5.2 lg/mg; 7-keto, 0.2 ± 0.1 lg/mg protein). ⁄⁄⁄p < 0.001 respect to the control, ap < 0.001 respect to the H2O2 treated cells.

C B

A

mAU medium

140 120 100 80 60 40 20 medium

500 400 300 200 100

mAU 280 nm 0

5

10

15

20

min

mAU 120 280 nm 0

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5

7.5

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cell lysate

cell lysate

15

20

7.5

10

12.5 15

17.5 20 min

cell lysate

5

10

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25 min

40 280 nm 0

280 nm 10

5

140 120 100 80 60 40 20 0

20 HT 5

2.5

280 nm

80 60

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mAU 600 500 400 300 200 100

medium

140 120 100 80 60 40 20

Glu1,2

HT

mAU

Glu 3

mAU

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10

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25 min

Fig. 6. Chromatograms showing HT (A) and its glucuronide metabolites Glu1,2 (B) and Glu3 (C) detected in the medium and cell lysate after 90 min of incubation of LLC-PK1 cells with 250 lM of each compound.

active, did not display the antiradical activity of the parent compound and appeared to maintain only some residual activity in protecting LDL from Cu-mediated oxidation [11]. Our study assesses for the first time the ability of the glucuronide metabolites of HT to protect cells against the lipid peroxida-

tion process. We have shown that both a mixture of HT 30 -O-b-Dglucuronide and 40 -O-b-D-glucuronide, and 2-(30 ,40 -dihydroxyphenyl)ethanol-1-O-b-D-glucuronide exhibited a significant protection against H2O2 induced membrane oxidative injury in renal cells comparable to that of the parent compound. Cell pre-treat-

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ment with the glucuronide metabolites significantly inhibited the formation of MDA and fatty acid and cholesterol major oxidation products, HP and 7-keto. In fact, exposure of LLC-PK1 cells to H2O2 may also result in the disruption of the membrane structure, due to the lipid peroxidation process initiated by the oxidative attack from the aqueous phase [16]. The antioxidant action of a phenolic compound in a membrane system depends both on the radical scavenging ability of aqueous radicals at the surface of the membrane and on the formation of lipid peroxyl radicals within the membrane. HT has been shown to react directly with H2O2 [42,43], to act as oxygen-centered radical scavenger [44] in the reaction medium and near the membrane surface [45] and as scavenger of chain propagating lipid peroxyl radicals generated from membrane UFA and cholesterol [29]. HT metabolites tested are highly polar compounds, as indicated by the calculated log P and the high solubility in water, which makes it difficult to pass the cell membranes. Therefore, their protective action is more likely to be due to the ability of scavenging the initiating aqueous radicals originated from reaction with H2O2 both in the reaction medium and near the membrane surface. In our experimental conditions, the scavenging ability of the HT glucuronides does not seem to be influenced by the substitution of the catechol structure. In fact, Glu1,2, where glucuronic acid is conjugated to one of the hydroxyl groups of the catechol, protected cells as efficiently as Glu3, where the conjugation affects the aliphatic chain and leaves intact the ortho-diphenolic structure. In contrast, the activity of the glucuronide metabolites of several flavonoids were reported to be highly dependent on the conjugation position [39]. Our results have shown that HT glucuronide metabolites are able to protect renal cells against H2O2 induced membrane oxidative damage, more likely through a direct antioxidant action. Moreover, Glu1,2 and Glu3 revealed no effect on cell death probably due to their incapacity to interact with intracellular signaling pathways. These findings are interesting in light of the fact that, although the glucuronide metabolites were less efficacious in our renal cell model, it should be noted that in vivo they would be present at greater concentrations than the unmetabolized parent compound. Although further researches either in animal experimentations or ex vivo models for renal oxidative damage are needed, these results suggest that HT glucuronide metabolites when concentrated in the renal compartment [46] may significantly contribute, together with HT and other of its metabolites, to the protective action against H2O2 mediated renal diseases where the lipid peroxidation process plays a central role. Conflict of interest statement The authors declare no conflict of interest. References [1] S. Granados-Principal, J.L. Quiles, C.L. Ramirez-Tortosa, P. Sanchez-Rovira, M.C. Ramirez-Tortosa, Hydroxytyrosol: from laboratory investigations to future clinical trials, Nutr. Rev. 68 (2010) 191–206. [2] R. de la Torre, Bioavailability of olive oil phenolic compounds in humans, Inflammopharmacology 16 (2008) 245–247. [3] G. Corona, X. Tzounis, M.A. Dessi, M. Deiana, E.S. Debnam, F. Visioli, J.P.E. Spencer, The fate of olive oil polyphenols in the gastrointestinal tract: implications of gastric and colonic microflora-dependent biotransformation, Free Radic. Res. 40 (2006) 647–658. [4] M.N. Vissers, P.L. Zock, A.J. Roodenburg, R. Leenen, M.B. Katan, Olive oil phenols are absorbed in humans, J. Nutr. 132 (2002) 409–417. [5] C. Manna, P. Galletti, G. Maisto, V. Cucciolla, S. D’Angelo, V. Zappia, Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells, FEBS Lett. 470 (2000) 341–344. [6] E. Miro-Casas, M.I. Covas, M. Farre, M. Fito, J. Ortuno, T. Weinbrenner, P. Roset, R. de la Torre, Hydroxytyrosol disposition in humans, Clin. Chem. 49 (2003) 945–952.

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