BIOAVAILABILITY AND METABOLISM OF GRAPE TRANS-RESVERATROL ON CACO-2 CELLS

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1 Comm. Appl. Biol. Sci, 80/1, 2015 BIOAVAILABILITY AND METABOLISM OF GRAPE TRANS-RESVERATROL ON CACO-2 CELLS I. M. TOALDO*, C. GROOTAERT**, M. T. BORDIGNON-LUIZ*, J. VAN CAMP** *Laboratory for Food Biochemistry, Federal University of Santa Catarina, Admar Gonzaga 1346, Florianópolis, Brazil **Laboratory for Food Chemistry and Human Nutrition, Ghent University, Coupure Links 653, 9000 Gent, Belgium INTRODUCTION The consumption of polyphenol-rich diets is associated with a reduced risk of developing chronic diseases such as atherosclerosis, heart disease, cancer and diabetes (Manach et al., 2005). Grape derivatives, such as wine and grape juice are highly appreciated worldwide, and account for one of the most important sources of polyphenols in the human diet (Jackson, 2008). American varieties of V. labrusca L. are widely cultivated in North and South America and are a very relevant source of polyphenols in Western diets (Nixdorf and Hermosín-Gutiérrez, 2010). Many studies have reported that dietary intake of phenolic compounds is associated with health benefits related to cardiovascular function modulating various parameters such as, vascular and platelet function, blood pressure and the plasma lipid profile, as a result of improved resistance towards oxidative stress, inflammation, and endothelial dysfunction (Kemperman et al., 2013). The main phenolic compounds present in grape and grape juice are anthocyanins, mainly malvidin and cyanidin in their glucoside forms, flavonols (quercetin, myricetin, kaempferol) and flavan-3-ols (catechin and proanthocyanins), phenolic acids, trans-resveratrol and tannins (Jackson, 2008). The major grape polyphenols with clear impact on several cardiovascular markers are trans-resveratrol and quercetin. Several mechanistic studies with endothelial cells demonstrate the beneficial effect of trans-resveratrol and quercetin on angiogenesis, cell migration (Mojzis et al., 2008), markers for vasorelaxation and inflammatory factors (Duluc et al., 2013). trans-resveratrol (RV) (trans-3,4,5-trihydroxystilbene) is a natural polyphenol present in grapes, red wine and berries that has been reported to exhibit beneficial effects against cardiovascular diseases (Jackson, 2008). In the human organism, during the course of absorption, polyphenols undergo extensive modification. Dietary polyphenols are conjugated in the intestinal cells and later in the liver by methylation, sulfation and glucuronidation (Pandey and Rizvi, 2009). The Caco-2 cell-monolayer comprises a wellestablished in vitro model for intestinal absorption and can generate useful information in order to predict in vivo mechanisms. Indeed, polyphenols undergo excessive metabolization by the intestinal microbiota and the gut epithelial cells, resulting in metabolites that are released into the bloodstream with potential to elicit health benefits on target tissues (Storniolo and Moreno, 2012). In order to investigate the metabolism of trans-resveratrol and the bioactive potential of this grape polyphenol, the cell conversion and transport of trans-resveratrol through Caco-2 cells for the detection of metabolites was evaluated. In addition, to verify RV bioactivity on a different target tissue, cellular reactivity to RV was evaluated on the human endothelial cell line EA.hy926, using the MTT and SRB protocols.

2 MATERIAL AND METHODS Cell lines The experiments were performed using the continuous cell line originated from a human colon adenocarcinoma Caco 2 cell line (HTB 37, ATCC, Manassas, USA) and the human endothelial cell line EA.hy926 (CRL2922, ATCC). Cell toxicity assays trans-resveratrol (RV) (Sigma-Aldrich, St. Louis, MO, USA) cytotoxicity was evaluated by the MTT (mitochondrial activity) and SRB (intracellular protein content) assays. For both assays, Caco-2 and endothelial cells were cultivated in growth medium (DMEM with glutamax, high glucose 4.5 g/l, 10% fetal bovine serum, 1% non-essential amino acids) until 90% confluence, and subsequently seeded in 96-well plates at a concentration of cells per well. After 24h incubation (37 C, 10% CO2), cells were treated with exposure medium (DMEM, high glucose 4.5 g/l, 1% nonessential amino acids solution) spiked with RV at concentration range of µm. The MTT and SRB tests were performed after 1-day treatment for undifferentiated Caco-2 cells and after 3-days treatment for differentiated Caco-2 cells. For cytotoxicity experiments on endothelial cells, tests were performed after 2-days exposure to RV (1-100 µm). Results are expressed as % of optical density (570 nm for MTT, 490 nm for SRB) compared to untreated cells. All analyses were carried out in six replicates. Conversion and transport experiments on Caco-2 cells The metabolism of resveratrol by Caco-2 cells was assayed through conversion and transport experiments in spiked cell culture medium. For conversion experiments, differentiated Caco-2 cells were treated in exposure medium spiked with RV solution to final concentration of 100 µm. Cells cultivated in exposure medium without RV were taken as blank samples. The cells were incubated at 37 C at 10% CO2. For the transport experiments, differentiated Caco-2 cells were treated in exposure medium using the Transwell set-up. In the apical compartment, cells were treated with RV 100 µm and RV-free culture medium was applied in the basolateral compartment. The cells were incubated at 37 C at 10% CO2 and samples of culture medium were periodically collected and analysed for metabolites by HPLC-DAD. HPLC-DAD analysis for resveratrol and metabolites The determination of RV and metabolites were carried out on a Thermo HPLC-DAD chromatograph (Thermo Scientific, Waltham, MA, USA) using a reverse phase column C18 (150 x 4.6 mm, 3 µm) (Varian Inc., Palo Alto, CA, USA). Separation and detection of RV and metabolites were optimized using the mobile phase composed of ultrapure water 0.5% formic acid (solvent A) and methanol (solvent B) through a gradient elution program, as follows: 5% B over 7 min, 5 95% B over 8 min, 95% B over 5 min, returning to the initial condition in 5 min. The flow rate was set at 0.9 ml min -1 and detection was performed at 300 nm. Samples of cell culture medium were filtered through a 0.45 µm PTFE membrane and directly inject (25 µl) into the chromatograph.

3 Statistical analysis Statistical analysis was performed using the Statistica software package version 7.0 (StatSoft Inc., Tulsa, USA). Data were subjected to analysis of variance and the significance was assessed using the t-test. Confidence intervals and differences were regarded as significant at 95% and p<0.05, respectively. RESULTS AND DISCUSSION Cell toxicity of trans-resveratrol The bioactivity results for RV treatment on Caco-2 and endothelial cells are presented in in Fig.1 and Fig. 2, respectively. Fig. 1. MTT and SRB values for resveratrol bioactivity in Caco-2 cells. A) MTT; B) SRB. D1: 1-day treatment; D3: 3-days treatment. Error bars indicate standard deviations. *p<0.05 (t-test compare to untreated cells). Fig. 2. Bioactivity of resveratrol in endothelial cells. A) MTT; B) SRB. D2: 2-day treatment. Error bars indicate standard deviations. *p<0.05 (t-test compare to untreated cells).

4 In the MTT experiments with Caco-2 cells, the optical density (OD) showed increased values after 1-day treatment at low concentrations of RV. In comparison with untreated cells, the mitochondrial activity was significantly higher after the slight increase on RV concentration (10 and 20 µm). The MTT values showed non-significant difference with RV treatment at higher concentrations. In the SRB tests, the slight increases on OD values at lower concentrations of RV demonstrated a stress response in undifferentiated Caco-2 cells and cell reaction by production of proteins, possibly stimulating cell proliferation, thus increasing SRB values, as shown in Fig.1 (B). For differentiated Caco-2 cells, the MTT and SRB showed decreased values after exposure to increasing concentrations of RV. However, in comparison to untreated cells no significant difference was verified after RV exposure. Indeed, after 1-day and 3-days treatment, variations on OD values were inferior to 20% after treatment with RV at concentration range of 1 to 100 µm. These findings suggest that Caco-2 cells are responsive to RV compound in culture medium, without a toxicity effect upon 3-days exposure with RV at the analysed concentration range. For the endothelial cells, the increases on the optical density in MTT test verified at higher concentrations of RV ( µm) showed cellular reactivity upon exposure to RV in culture medium. In the SRB experiment, reduced OD values were observed after exposure to resveratrol. In comparison to the untreated cells, variations on OD values were inferior to 20% after 2-days exposure to RV at 1 to 100 µm. However, the SRB values were significantly decreased after treatment with RV at 100 µm. In fact, after cell exposure to the polyphenol compound, the decrease on SRB values can be related to reduced cell growth and secretion of proteins/enzymes. Moreover, the results demonstrated that for the endothelial cells treated with RV, increases on OD in MTT test associated with a significant decrease in SRB values are related to cell reactivity or stress. These findings suggest that the endothelial epithelium is responsive to this grape phenolic compound. Furthermore, the investigation of the effects of RV and its metabolism-products on specific endothelial functions is mandatory to predict beneficial biological mechanisms in cardiovascular diseases. Conversion and transport of trans-resveratrol by Caco-2 cells The HPLC-DAD chromatograms of the conversion and transport experiments in Caco-2 cells are presented in Fig. 3 and Fig. 4, respectively. Fig. 3. HPLC-DAD chromatograms of RV standard and cell culture medium for the conversion experiments on differentiated Caco-2 cells. Peaks: (1) and (2) RV isomers; (3) RV metabolite.

5 The cellular uptake and conversion of resveratrol was investigated using the human Caco-2 cells. The chromatographic analysis of RV standard solutions showed the presence of an isomer form of RV, probably due to conversion of trans-resveratrol to cis-resveratrol. The photoinstability of resveratrol has been previously postulated. In fact, resveratrol undergoes a rapid isomerization from the more thermodynamically stable trans-isomer to the less stable cis- isomer upon UV light excitation (Figueiras et al., 2011). In the conversion experiments with cells treated with RV 100 µm, the presence of a metabolite peak was detected after 2 hours of incubation, with increased peak area up to 24h exposure. Fig. 4. HPLC-DAD chromatograms of apical and basolateral compartments after the transport experiments on Caco-2 cells. A) Apical; B) Basolateral. Peaks: (1) and (2) RV isomers; (3) RV metabolite. In the transport experiments, the analysis of culture medium showed the presence of resveratrol peaks (isomers forms) on both apical and basolateral compartments after 2h incubation, as shown in Fig. 4(A) and (B), respectively. The efficiency of RV transport through Caco-2 cells was 16.8% up to 24h of incubation. After RV treatment, the presence of a RV metabolite was detected in the apical medium after 7h incubation. As verified in both conversion and transport experiments on Caco-2 cells, the metabolite peak showed a reduced retention time in relation to resveratrol isomers peaks, demonstrating a hydrophilic characteristic. The transported metabolite is possibly a sulfate or glucuronated derivative of resveratrol, as previously reported (Storniolo and Moreno, 2012). This finding suggested that for absorption, RV is metabolized by intestinal cells and the metabolite-product is promptly secreted in its hydrophilic derivative form. Interestingly, we observed that also the intact polyphenol compound is secreted and transported through the Caco-2 cells monolayer. In fact, RV is absorbed in the large intestine being rapidly metabolized by enterocytes into sulfated and

6 glucuronidated forms within 15 min of entering the bloodstream (Saiko et al., 2008). Resveratrol crosses the apical membrane of the enterocyte by passive transport, although MRP2 (protein associated with drug resistance 2) is also associated with regulating transmembrane efflux (Storniolo and Moreno, 2012). In this study we highlight the high concentrations of this bioactive polyphenol that are transported through the intestinal barrier and disposed to reach the bloodstream leading to biological effects under in vivo conditions. Notwithstanding, the identification of trans-resveratrol metabolites, as well as the study of its bioactivity towards target tissues can represent valuable contribution leading to health implications. CONCLUSION trans-resveratrol promoted cellular reactivity in Caco-2 and endothelial cells. The results demonstrated that Caco-2 cells transport the intact polyphenol and cellular metabolism generates a hydrophilic metabolite of RV, which may improve its transport and bioactivity towards target tissues such as the endothelium, leading to health implications in cardiovascular diseases. ACKNOWLEDGEMENTS The authors acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil and the Research Group on Food Chemistry and Human Nutrition of Ghent University for financial support. REFERENCES Duluc, L., Jacques, C., Soleti, R., Iacobazzi, F., Simard, G., & Andriantsitohaina, R. (2013). Modulation of mitochondrial capacity and angiogenesis by red wine polyphenols via estrogen receptor, NADPH oxidase and nitric oxide synthase pathways. The International Journal of Biochemistry & Cell Biology 45: Figueiras, T. F., Neves-Petersen, M. T., & Petersen, S. B. (2011). Activation Energy of Light Induced Isomerization of Resveratrol. Journal of Fluorescence 21(5): Jackson, R. S Chemical Constituents of Grapes and Wine. In: Wine Science: Principles and Applications 3 ed. San Diego: Elsevier Inc.: Kemperman, R. A., Gross, G., Mondot, S., Possemiers, S., Marzorati, M., Wiele, T. V., Doré,J., & Vaughan, E. E. (2013). Impact of polyphenols from black tea and red wine/grape juice on a gut model microbiome. Food Research International 53: Manach, C., Williamson, G., Morand, C., Scalbert, A., & Rémésy, C. (2005). Biovailability and bioefficacy of polyphenols in humans. The American Journal of Clinical Nutrition 81: Mojzis, J, Varinska, L., Mojzisova, G., Kostova, I., & Mirossay, L. (2008). Antiangiogenic effects of flavonoids and chalcones. Pharmacological Research 57: Nixdorf, S. L., & Hermosín-Gutiérrez, I. (2010). Brazilian red wines made from the hybrid grape cultivar Isabel: phenolic composition and antioxidant capacity. Analytica Chimica Acta 659: Pandey, K.B., & Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Medicine and Cellular Longevity 2 (5): Saiko, P., Szakmary, A., Jaeger, W., & Szekeres, T. (2008). Resveratrol and its analogs: Defense against cancer, coronary disease and neurodegenerative maladies or just a fad? Mutation Research 658: Storniolo, C. E., & Moreno, J. J. (2012). Resveratrol metabolites have an antiproliferative effect on intestinal epithelial cancer cells Food Chemistry 134: