Procyanidins protect Caco-2 cells from bile acid- and oxidant-induced damage

Free Radical Biology & Medicine 41 (2006) 1247 – 1256 www.elsevier.com/locate/freeradbiomed

Original Contribution

Procyanidins protect Caco-2 cells from bile acid- and oxidant-induced damage Alejandra G. Erlejman a , Cesar G. Fraga b,c , Patricia I. Oteiza a,c,d,⁎ a

IQUIFIB-Department of Biological Chemistry (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina b Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina c Department of Nutrition, University of California, Davis, CA, USA d Department Environmental Toxicology, University of California, Davis, CA, USA Received 16 May 2006; revised 16 June 2006; accepted 6 July 2006 Available online 11 July 2006

Abstract Procyanidins can exert cytoprotective, anti-inflammatory, and anticarcinogenic actions in the gastrointestinal tract. Previous evidence has shown that procyanidins can interact with synthetic membranes and protect them from oxidation and disruption. Thus, in this study we investigated the capacity of a hexameric procyanidin fraction (Hex) isolated from cocoa to protect Caco-2 cells from deoxycholic (DOC)-induced cytotoxicity, cell oxidant increase, and loss of monolayer integrity. Hex interacted with the cell membranes without affecting their integrity, as evidenced by a Hex-mediated increase in the transepithelial electrical resistance, and inhibition of DOC-induced cytotoxicity. DOC induced an increase in cell oxidants, alterations in the paracellular transport, and redistribution of the protein ZO-1 from cell-cell contacts into the cytoplasm. Hex partially inhibited all these events at concentrations ranging from 2.5 to 20 μM. Similarly, Hex (5–10 μM) inhibited the increase in cell oxidants, and the loss of integrity of polarized Caco-2 cell monolayers induced by a lipophilic oxidant (2,2′-azobis (2,4-dimethylvaleronitrile). Results show that the assayed procyanidin fraction can interact with cell membranes and protect Caco-2 cells from DOC-induced cytotoxicity, oxidant generation, and loss of monolayer integrity. At the gastrointestinal tract, large procyanidins may exert beneficial effects in pathologies such us inflammatory diseases, alterations in intestinal barrier permeability, and cancer. © 2006 Elsevier Inc. All rights reserved. Keywords: Flavonoid; Flavanol; Polyphenol; Intestinal barrier permeability; Bile acids; Membrane interactions; Gastrointestinal tract; Cocoa

Introduction Flavonoids are found in a wide variety of vegetables and fruits, and numerous studies have associated flavonoid consumption with health benefits. A specific class of flavonoids known as procyanidins are oligomers of flavan3-ol subunits, i.e., (+)-catechin and (−)-epicatechin (EC),

Abbreviations: AMVN, 2,2′-azobis (2,4-dimethylvaleronitrile); DCDHF, 5(and-6)-carboxy-2′7′-dichlorodihydrofluorescein diacetate; DOC, deoxycholate; DPI, diphenyleneiodonium chloride; EC, (−)-epicatechin; FSA, fluorescein sulfonic acid; Hex, hexameric procyanidin fraction; LDH, lactate dehydrogenase; TEER, transepithelial electrical resistance. ⁎ Corresponding author. Department of Nutrition, University of California, Davis, One Shields Av., Davis, CA 95616, USA. Fax: +1 530 752 8966. E-mail address: [email protected] (P.I. Oteiza). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.07.002

which are present in high concentrations in cocoa, red wine, cranberries, and apples [1,2]. Procyanidins in cocoa are primarily composed of several units of EC linked by 4β→8 bonds (Fig. 1). While there is no conclusive information regarding the bioavailability and biotransformation of procyanidins, a study in humans demonstrated that cocoa procyanidins are not degraded in the stomach and can reach the intestine [3]. Of the procyanidins that reach the intestine, only dimers have been shown to be absorbed into the circulation [4,5]; higher molecular weight procyanidins are poorly absorbed in the intestinal lumen [6,7]. The human colonic microflora is capable of degrading procyanidins to aromatic acids [8], but this degradation decreases with the degree of procyanidin oligomerization [9]. In addition, procyanidins with more than 3 subunits are not transported across Caco-2 cell monolayers,

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and can remain in part adsorbed to the apical surface of cells [8]. The above evidence supports the concept that relevant amounts of procyanidins can reach the intestine and exert antiinflammatory, anticarcinogenic, or other beneficial effects within the gastrointestinal tract [10,11]. These effects could be relevant in diseases in which alterations in the permeability of the intestinal barrier are associated with gastrointestinal dysfunctions, such as Crohn's disease [12], celiac disease, alcoholic liver disease, fatty acid liver disease, food allergies, and acute pancreatitis (reviewed in [13]). Furthermore, the integrity of the apical plasma membrane and the conservation of intercellular tight junctions of the enterocyte are essential for maintaining the intestinal epithelial barrier, which is necessary for preventing the penetration of external toxic agents and microorganisms [14,15]. Although bile acids have relevant physiological functions in the intestinal lumen, they are also involved in the pathogenesis of several disorders affecting the intestine. Deoxycholic acid (DOC) can promote intestinal tumorogenesis [16,17] that may be associated with bile acid-mediated activation of several cell signals [18,19]. DOC, a hydrophobic bile acid, was recently shown to perturb the cell membrane, affecting its lipid distribution and physical properties. It was proposed that the observed alterations in cell signaling are induced by the effects of DOC at the level of membrane microdomains [19]. We have recently shown that different flavonoids can interact with synthetic membranes (liposomes), protecting the bilayer from both the disruption induced by a detergent and free radicalmediated lipid oxidation [20,21]. Procyanidins were particularly active in these protective actions and their effects consistently depended on the degree of oligomerization [22]. Here we hypothesize that procyanidins, through their interaction with cell membranes, could participate in the protection of the gastrointestinal tract from the deleterious effects of bile acids. To test this hypothesis, we used Caco-2 cells, a wellestablished model of intestinal epithelium [23], to investigate the capacity of a hexameric procyanidin fraction (Hex) to interact with and protect cell membranes from DOC-induced oxidant production and membrane damage. The effects of DOC were compared to those of a lipophilic oxidant, 2,2′-azobis(2,4dimethylvaleronitrile) (AMVN). We observed that Hex interacts with Caco-2 cells, protecting them from DOC-induced: (a) cytotoxicity, (b) increase in cell oxidants, (c) redistribution of tight junction proteins (ZO-1), and (d) monolayer permeabilization. Accordingly, Hex inhibited an AMVN-mediated increase in cell oxidants and the loss of monolayer integrity. Materials and methods Materials Procyanidins were purified [2,24,25] and supplied by Mars Incorporated (Hackettstown, NJ). The Hex fraction was composed of 76% hexamers, 4.5% monomers, 2.2% dimers, 1.0% trimers, < 1.0% tetramers, 11.5% pentamers, and 4.1% procyanidins larger than hexamers. Caco-2 cells were from the

Fig. 1. Chemical structure of B-type procyanidins. N is 4 for hexameric procyanidins.

American Type Culture Collection (Rockville, MA). Cell culture media and reagents were from InVitrogen Life Technologies (Carlsbad, CA). ZO-1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture transwell permeable supports were from Corning Incorporated (Corning, NY). The CellTiter-Glo Luminiscent Cell Viability assay was from Promega (Madison, WI). Lactate dehydrogenase (LDH) activity assay kit was from Wiener Lab, Rosario, Argentina. 5(and-6)-carboxy-2′7′-dichlorodihydrofluorescein diacetate (DCDHF), fluorescein sulfonic acid (FSA), and propidium iodide were from Molecular Probes (Eugene, OR). AMVN was from Wako Pure Chemical Ind. (Osaka, Japan). DOC, EC, apocynin, and diphenyleneiodonium chloride (DPI) were from Sigma Chem. Co. (St. Louis, MO). Cell culture and incubations Caco-2 cells were cultured at 37°C in a humidified atmosphere of CO2/air (5/95) in EMEM medium supplemented with 10% (v/v) fetal bovine serum and antibiotics (50 U/ml penicillin, and 50 μg/ml streptomycin). For the experiments we used both unpolarized (at confluence after 7–10 days in culture) and polarized cells. Polarized cells were obtained by seeding the Caco-2 cells on 0.4-μm-pore polyester membranes in transwell inserts (6- or 12-well plates) for 17–20 days until the formation of a cell monolayer. During this culture period the media in the apical and basolateral chambers were changed every 3 days. For the treatments, unpolarized or polarized cells were washed, and the media replaced by fetal bovine serum-free EMEM. Cells were then preincubated for 30 min in the presence of EC or procyanidins at the concentrations described for each experiment, after which 0.2 mM DOC or 10 mM AMVN was added to the cell culture. Depending on the experiment, EC and procyanidins remained or were removed from the cell culture medium during the subsequent incubation. After the corresponding incubation, the medium was collected and/or the cells

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were harvested at different time points. Cell viability was measured evaluating the cell ATP content (CellTiter-Glo Luminiscent Cell Viability) according to the manufacturer's protocol. LDH activity was determined in the supernatant obtained after centrifugation of the cell media at 800g for 10 min. Transepithelial electrical resistance Transepithelial electrical resistance (TEER) was determined in polarized Caco-2 cell monolayers, cultured in 6-well plates, using a Millicell-ERS Resistance System (Millipore, Bedford, MA) that includes a dual electrode volt-ohm-meter. TEER was calculated as TEER ¼ ðRm  Ri Þ  A; where Rm is transmembrane resistance; Ri, intrinsic resistance of a cell-free media; and A, the surface area of the membrane in cm2. For the evaluation of the effects of Hex on TEER, the incubation medium was removed from the apical and basolateral compartments, cells were rinsed, and fetal bovine serum-free EMEM was added to both compartments (1.5 and 2.5 ml to the apical and basolateral compartments, respectively). After 20 min of incubation, the initial TEER was determined (time 0), and cells were incubated without or with Hex (20 μM final concentration) added to the apical compartment. After 30 min of incubation, the media in a subset of the cells preincubated with Hex were replaced by media without Hex and all groups were incubated for a total period of 120 min. TEER determinations were performed every 15–30 min.

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10 mM NaN3, a metabolic inhibitor of benzoic acid transport, to confirm the validity of the experimental conditions [27]. Transport of FSA The paracellular transport through Caco-2 cell monolayers was determined by measuring the apical-to-basolateral clearance of FSA as previously described [28,29]. For these experiments, cells were preincubated for 30 min in the absence or the presence of 10 μM Hex. Subsequently, the media of a subset of cells preincubated with Hex were removed, cells were rinsed, and Hexfree media (+/−Hex) was added, while another subset was maintained in media containing Hex (+/+Hex). All groups were then added with 0.2 mM DOC or 10 mM AMVN and further incubated for 60 min. Afterward, the culture media was replaced in both compartments by Hank's balanced solution (0.3 ml in the apical compartment, and 1 ml in the basolateral compartment) and FSA was added to the apical compartment (0.42 mM final concentration). Immediately after FSA addition, 50-μl aliquots were taken from the apical compartment (time 0) and from the basolateral compartment, at time 0 and then every 10 min. After diluting the aliquots to 200 μl, the fluorescence was measured (λexc: 485 nm; λem: 530 nm). FSA clearance (CLFSA) was calculated from the equation CLFSA ¼ Fab =ðFFSA  AÞ; where Fab is flux of FSA (in fluorescence units/h); FFSA, the fluorescence of FSA in the apical compartment at zero time (in fluorescence units per nl); and A, the surface area of the membrane (1 cm2).

Transport of benzoic acid

Immunocytochemistry of ZO-1

The transcellular transport was determined by evaluating the passage of [7-14C]benzoic acid from the apical to the basolateral compartments through a monolayer of polarized Caco-2 cells cultured in 6-well plates. For these experiments, culture medium was replaced in both compartments by Hank's balanced solution at 37°C (2.5 ml, pH 7.4 in the basolateral compartment; 0.8 ml, pH 6 in the apical compartment). Hex (10 μM final concentration) was added to the apical compartment. After 30 min at 37°C, [7-14C]benzoic acid (22 mCi/ mmol) was added to the apical compartment to a final concentration of 10 μM concentration. Aliquots were taken from the basolateral compartment every 10 min, and the radioactivity (Q) was counted in a scintillation counter (1214 Rackbeta, Wallac-Perkin Elmer, Boston, MA). The transport was described by the apparent permeability coefficient (Papp) calculated as

Caco-2 cells cultured on coverslips were preincubated for 30 min in the absence or the presence of 10 μM Hex and subsequently in the absence or the presence of 0.2 mM DOC for a further 6 h. For immunocytochemistry, cells were rinsed twice with PBS, fixed with 4% (w/v) paraformaldehyde, 0.12 M sucrose in PBS for 1 h at room temperature, and subsequently incubated for 15 min with 1% (w/v) glycine in PBS. For the detection of ZO1, fixed cells were permeabilized by incubation with 0.01% (v/v) Triton X-100 in PBS for 15 min. Afterward, samples were blocked with 1% (w/v) BSA in PBS for 2 h and incubated overnight at 4°C with the primary antibody (1:100 dilution) against ZO-1. The coverslips were rinsed twice with PBS and incubated with a Cy2-conjugated secondary antibody (1:1000). Samples were mounted onto glass slides and microscopic observations were made using an Olympus BX50 epifluorescence microscope provided with a Cool-Snap digital camera.

Papp ¼ dQ=dt  1=C0  1=A;

Cell oxidant levels

where dQ/dt is the change of benzoic acid with respect to time in pmol/s; C0, the initial concentration of benzoic acid in the apical compartment (10 μM); and A, the surface of the membrane (4.7 cm2) [26]. Some cells were incubated with

Cell oxidant levels were evaluated using DCDHF. This probe crosses the cell membrane, and fluoresces when it is oxidized inside the cell. Caco-2 cells (1× 105) were grown in 12-well plates. For these experiments, cells were preincubated for 30 min in the absence or the presence of 2.5–10 μM Hex, and then divided into

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30 min of incubation at 37°C, the medium was removed; cells were rinsed with PBS and then incubated in 300 μl PBS containing 0.1% (v/v) Igepal. Then, cells were sonicated and the fluorescence of the mixture was determined (λexc: 475 nm; λem: 525 nm). To determine DNA content, samples were subsequently incubated with 50 μM propidium iodide for 20 min at room temperature, and the fluorescence (λexc: 538 nm, λem: 590 nm) was measured. Results are expressed as the ratio DCF fluorescence/propidium iodide fluorescence. Statistical analysis

Fig. 2. Effects of Hex on TEER in Caco-2 cell monolayers. Cells were incubated at 37°C for 120 min in the absence of additional treatments (■); in the presence of 20 μM Hex (●); or preincubated for 30 min in the presence of Hex and for a further 90 min in Hex-free media (○). Results are shown as mean ± SE of at least three independent experiments. *,#Significantly different from the control group at the corresponding time point.

+/−Hex and +/+Hex groups as previously described, treated with 0.2 mM DOC or 10 mM AMVN, and further incubated for 60 min. The medium was discarded, and cells were rinsed with PBS and incubated in 500 μl of EMEM containing 10 μM DCDHF. After

Data were analyzed by one-way analysis of variance (ANOVA) using Statview 5.0 (SAS Institute Inc., Cary, NC). Fisher least-significance difference test was used to examine differences between group means. A P value < 0.05 was considered statistically significant. Data are shown as means ± SE. Results Effects of Hex on TEER and transport in Caco-2 cell monolayers We previously demonstrated that procyanidins can interact with liposomes [22]. The observed decrease in membrane

Fig. 3. Effects of Hex on the transport of [7-14C]benzoic acid and FSA in Caco-2 cell monolayers. Cells were preincubated for 30 min in the absence or the presence of 10 μM Hex. At time 0, [7-14C]benzoic acid or FSA was added to the cells and transport was followed for 30 min; (■ and empty bar) cells not exposed to Hex; (● and light gray bars) cells preincubated with Hex, and then incubated in Hex-free media (+/−Hex); (○ and dark gray bars) cells preincubated and then incubated in the presence of Hex (+/+Hex). A subset of control cells was incubated with 10 mM NaN3 (▴ and full bar, A). Left panels: typical kinetics (0–30 min) of [7-14C]benzoic acid (A) or FSA (B) transport. Right panels: [7-14C]benzoic acid Papp (A) and FSA clearance (B). Values are shown as mean ± SE of three independent experiments. *Significantly different from the other groups.

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surface potential induced by procyanidin indicated that they adsorb to the polar head groups of phospholipids [22]. In the present experimental model, TEER values were measured to evaluate cell integrity and to assess the possible interactions of Hex with the cell plasma membrane. The average TEER value for the cell monolayers was 62 ± 58 Ω × cm2. TEER values higher than 200–350 Ω × cm2 are considered acceptable for an undamaged Caco-2 cell monolayer [30,31]. In cells incubated with Hex (20 μM) TEER values significantly increased between 30 and 120 min compared to cells incubated in the absence of Hex (Fig. 2). These higher values of TEER indicate the adsorption of Hex to the plasma membrane. Similar increases of TEER were observed when Caco-2 cells were exposed to an extract of mixed procyanidins [8]. We further observed that when Hex was removed from the media after the initial 30 min of incubation, TEER decreased to basal levels within approximately 60 min, indicating that Hex adsorption to the membrane is transient. Since the maximum value of TEER in the presence of Hex was observed after 30 min of incubation, this period of preincubation was subsequently used. To assess if Hex interactions with the plasma membrane could affect the functionality of Caco-2 monolayers, the transcellular transport of benzoic acid as an example of monocarboxylic acid transport and the paracellular transport of FSA were evaluated. The kinetics of [7-14C]benzoic acid transport for the different experimental conditions is shown in Fig. 3A (left panel). The calculated Papp was similar for cells incubated in the absence of Hex (control) compared to cells preincubated for 30 min with 10 μM Hex, and measured for further 30 min under +/−Hex and +/+Hex conditions (Fig. 3A, right panel). As expected, in the presence of NaN3 the transport was markedly inhibited. The kinetics of FSA paracellular transport (Fig. 3B, left panel) and the calculated FSA clearance (Fig. 3B, right panel) were similar under the different experimental conditions. These results indicate that, under the present experimental conditions, Hex per se does not affect transcellular and paracellular transport in Caco-2 monolayers. Effects of EC and procyanidins on DOC-induced cytotoxicity We next investigated if, similar to the capacity of procyanidins to protect liposomes from Triton-induced disruption [22], EC and procyanidins could protect Caco-2 cells from the cytotoxic effects of DOC. The release of the cytosolic enzyme LDH and cell viability were measured as indicators of cytotoxicity. DOC caused a dose- and time-dependent release of LDH from unpolarized Caco-2 cells (data not shown). The effects of EC and procyanidins were measured after cells were incubated for 6 h in the presence of 0.2 mM DOC. To determine the influence of the degree of procyanidin oligomerization on DOC-mediated LDH release, Caco-2 cells were incubated in the presence of equivalent amounts of monomer (EC) or procyanidins. While monomer, dimer, trimer, and tetramer had no significant effects, the pentameric and hexameric procyanidins inhibited DOC-induced LDH release (Fig. 4A). Hex prevented DOC-induced LDH release in a dosedependent manner (Fig. 4B), with the protective effect being

Fig. 4. Effects of EC and procyanidins (dimer through hexamer) on DOCinduced LDH release and Caco-2 cell viability. (A) Cells were preincubated for 30 min at 37°C in the absence or the presence of 30 μM monomer, 15 μM dimer, 10 μM trimer, 7.5 μM tetramer, 6 μM pentamer, or 5 μM hexamer. Subsequently, 0.2 mM DOC was added, and the cells were incubated for further 6 h, and LDH was determined in the media. (B, C) Cells were preincubated for 30 min in the presence of 2.5–20 μM Hex. Subsequently, cells (+/+Hex or +/−Hex conditions) were incubated with 0.2 mM DOC for a further 6 h. LDH activity was determined in the incubation media. Results are shown as means ± SE of six independent experiments. In A *significantly different from the other groups; in B and C, values having different letters are significantly different.

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significant across the range of concentrations tested (2.5– 20 μM). The extent of the protection by Hex toward DOCmediated release of LDH was similar for unpolarized and polarized Caco-2 cells (47 ± 9 and 49 ± 8% of LDH released, respectively, for 10 μM Hex). These percentages were calculated by considering as 100%, the LDH released by cells incubated in the absence of EC or procyanidins. The incubation of cells in the presence of 0.2 mM DOC caused a 13% decrease in cell viability. Hex (10–20 μM) prevented this DOC-mediated decrease in cell viability in a dose-dependent manner when Hex was present in the media during the exposure to DOC (+/+Hex conditions) (Fig. 4C). Under +/−Hex conditions, Hex partially protected Caco-2 cells from DOC-mediated LDH release, but did not prevent the loss of cell viability induced by DOC (Figs. 4B and C). Effects of Hex on DOC-induced Caco-2 monolayer permeabilization, ZO-1 redistribution, and oxidant increase It was recently reported that bile acids affect the TEER of Caco-2 monolayers and the distribution of tight junction proteins [18]. Thus, we next investigated the capacity of Hex to protect Caco-2 cell monolayers from DOC-induced redistribution of the tight junction protein ZO-1 and from the associated alterations in the paracellular transport of FSA. In the absence of all treatments, the distribution of ZO-1 in Caco-2

cells, as assessed by inmunocytochemistry, showed the location of the protein mainly in cell boundaries (Fig. 5A). Treatment of cells with 0.2 mM DOC for 6 h led to a decrease of ZO-1 in the areas of contact among cells and to an increase in the cytosolic distribution of ZO-1 (Fig. 5B). Cells treated with Hex throughout the incubation with DOC showed an important prevention of DOC-induced ZO-1 redistribution (Fig. 5C). Accordingly, DOC caused a significant increase in the paracellular transport of FSA that was partially prevented by coincubating cells with 10 μM Hex (Fig. 5D). An inhibitor of NADPH-oxidase, apocynin, prevented the DOC-induced increase in FSA transport, suggesting the involvement of NADPH oxidase-mediated oxidant production in the DOCmediated alteration of monolayer permeability. Bile acids have been reported to increase cell oxidants. Treatment of isolated crypt epithelium with 5 mM DOC [32] and Caco-2 cells with cholic acid (50–200 μM) increased oxidant production [18]. Similarly, under the current experimental conditions, the incubation of Caco-2 cells in the presence of 0.2 mM DOC for 60 min led to a 40% increase of cell oxidant levels as measured with the probe DCDCHF (Fig.6). The preincubation of cells with Hex for 30 min and the subsequent incubation of cells with DOC (+/+Hex and +/−Hex conditions) caused a partial prevention of the DOC-induced increase in cell oxidants (Fig. 6). The increase in oxidants was also prevented by the NADPH-oxidase inhibitors DPI and

Fig. 5. Effects of Hex on DOC-induced alterations in ZO-1 distribution and FSA transport in Caco-2 cell monolayers. Cells were preincubated for 30 min in the absence (A, B), or the presence (C) of 10 μM Hex, and subsequently incubated for a further 6 h with 0.2 mM DOC (B, C). The cellular distribution of ZO-1 was characterized by immunocytochemistry. (D) Caco-2 cell monolayers were incubated in the absence of additional treatments (control); preincubated for 30 min in the absence of additional treatment, and subsequently incubated for 6 h with 0.2 mM DOC (DOC); with 0.2 mM DOC and 0.3 mM apocynin (apocynin); or preincubated for 30 min in the presence of 10 μM Hex and subsequently incubated for 6 h with 0.2 mM DOC (+/+Hex conditions). FSA clearance values are shown as mean ± SE of three independent experiments. Values having different symbols are significantly different.

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Fig. 6. Effects of Hex on DOC-induced increase of Caco-2 cells oxidants. Cells were incubated in the absence of additional treatment (control); preincubated in the absence of additional treatment and subsequently incubated for 60 min with 0.2 mM DOC (DOC); preincubated for 30 min in the presence of 2.5–10 μM Hex, and subsequently incubated for 60 min with 0.2 mM DOC (+/+Hex or +/−Hex conditions); or incubated for 60 min with 0.2 mM DOC and 10 μM DPI (DPI) or 0.3 mM apocynin (Apocynin). DCF fluorescence is expressed as relative fluorescence (RF) per DNA content (propidium iodide fluorescence). Results are shown as mean ± SE of six independent experiments. Values having different letters are significantly different.

apocynin. The above results indicate that Hex can partially prevent the DOC-mediated increase in oxidants and the subsequent alterations in barrier integrity. Effects of Hex on AMVN-induced Caco-2 monolayer permeabilization and oxidant increase We have previously shown that flavonoids and procyanidins, by interacting with liposome membranes, can protect membrane lipids from AMVN-induced oxidation [20]. Thus, we investi-

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gated if Hex could protect Caco-2 cell monolayers from AMVN-induced alterations in permeability and oxidation. The treatment of Caco-2 cell monolayers with AMVN for 60 min caused a 100% increase in the DCF fluorescence, and this increase was prevented by Hex (Fig. 7). When Hex was present in the media during both the preincubation (30 min) and the incubation with AMVN (+/+Hex conditions), the inhibition of DCDHF oxidation was significant at all the concentrations tested (2.5–10 μM). When Hex was removed from the media after the 30 min preincubation (+/−Hex conditions) a significant inhibition (47%) was observed at the highest Hex concentration tested (10 μM) (Fig. 7A). The capacity of Hex (10 μM) to protect the integrity of Caco2 cell monolayers exposed to AMVN was evaluated by measuring the paracellular transport of FSA (Fig. 7B). The incubation of the monolayer in the presence of 10 mM AMVN caused a significant increase (88%, P < 0.05) in the apical-tobasolateral flux of FSA that was partially prevented when cells were incubated in the presence of both AMVN and Hex (10 μM). Even when Hex (10 μM) was removed from the media after the 30 min preincubation (+/−Hex conditions), a significant protection against AMVN-induced permeabilization of the Caco-2 monolayer was observed (Fig. 7B). These results further support the participation of oxidants in affecting the permeability of the intestinal barrier. Discussion This work demonstrates that procyanidins can interact with cell plasma membranes, protecting Caco-2 cells from bile acidinduced cytotoxicity, increases in cell oxidants, and alterations in tight junction protein distribution and barrier integrity. Supported by their relatively high concentration in a number of edible plants [33] and their limited intestinal absorption [6], procyanidins may exert beneficial health effects along the whole

Fig. 7. Effects of Hex on AMVN-induced Caco-2 cell oxidants and FSA transport. Cells were incubated in the absence of additional treatment (control); preincubated in the absence of additional treatment and subsequently, incubated for 60 min with 10 mM AMVN (AMVN); or preincubated for 30 min in the presence of 2.5–10 μM Hex and subsequently incubated for 60 min with 10 mM AMVN (+/+Hex or +/−Hex conditions). (A) DCF fluorescence; and (B) FSA clearance measured in cells treated as described above, but only the 10 μM Hex concentration was tested. Results are shown as mean ± SE of four independent experiments. Values having different letters are significantly different.

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gastrointestinal tract. In the present experiments, we tested the hypothesis that large procyanidins can protect the intestinal epithelium from bile acid-induced damage in part through their interactions with the cell plasma membrane. As a control, we proved that, under the current experimental conditions, the interactions of Hex with cell membranes do not affect important physiological processes, i.e., membrane integrity (TEER) and transcellular and paracellular transport of Caco-2 monolayers. Bile acids have a relevant role in the intestinal tract facilitating the absorption and digestion of dietary fats and of other fat-soluble nutrients. However, bile acids can also exert deleterious effects on the gastrointestinal mucosa. Through different mechanisms DOC has been proposed to be involved in colon tumorogenesis, e.g., DOC can promote sloughing of the intestinal epithelium and induce cell proliferation [32]. Interestingly, intestinal epithelium is directly exposed to fecal water, where DOC can reach 100–200 μM concentrations [34]. In cultured Caco-2 cells, and depending on its concentration, DOC can induce both cell proliferation or apoptotic death [35]. We observed that in Caco-2 cell monolayers, DOC induced cytotoxicity, oxidant production, and alterations of the barrier integrity. In a previous study [22], Hex was the tested procyanidin with the highest capacity to protect liposomes from Triton X-100 disruption to micelles. Accordingly, when evaluating the protective actions of EC and procyanidins against DOC-induced cytotoxicity, we found that the larger procyanidins tested (pentamer and hexamer) inhibited DOC-induced LDH release. The protective effects of Hex were in general observed across the range of concentrations tested (2.5–20 μM). The limited effects on LDH release observed for lower molecular weight procyanidins, i.e., monomer-tetramer, stress the relevance of the membrane-related effects of larger procyanidins. These differential actions could be explained as a compromise between the incorporation of the compounds into the cells that decrease as increases procyanidin oligomerization and the adsorption to the cell surface that increases as procyanidins oligomerization increase. To assess the physiological relevance of the tested concentrations it is worth noting that the ingestion of 20 g of a procyanidin containing chocolate would generate an intestinal concentration of approximately 18 μM procyanidins of 4– 10 EC units, considering an intestinal volume of 600 ml [1]. It was previously calculated that the ingestion of one glass of wine would lead to a concentration of total procyanidins close to 300 μM [8]. Thus, the concentrations of Hex shown to be effective in the current experiments could be reached in the intestine from dietary sources. We observed that oxidants generated by DOC or AMVN lead to an increased permeabilization of Caco-2 cell monolayers. Accordingly, several studies have shown that oxidantmediated damage of the mucosa can cause alterations in the integrity of the intestinal barrier, a mechanism that can be involved in the pathogenesis of several intestinal diseases [36– 38]. This is supported by recent findings in Drosophila, indicating that the regulation of the redox balance is crucial to the host's defense and survival from continuous gastrointestinal infection [39]. Both nitrogen [37,38] and oxygen [36,38,40]

active species have been found to be associated with intestinal barrier damage, both in vitro and in several pathologies such us inflammatory bowel disease, including ulcerative colitis and Crohn's disease [36–38]. In Caco-2 cells, cholic acid increased oxidants, altered the distribution of tight junction proteins, and led to alterations in TEER [18]. NADH-dehydrogenase and xanthine oxidase were proposed to be involved in the high oxidant production by cholic acid [18]. We observed that the DOC-mediated increase in cell oxidants occurs in part through a mechanism involving NADPH-oxidase. In addition, it has been recently demonstrated that NADPH-oxidase participates in bile acid-induced apoptosis [41]. However, the partial prevention of oxidant production following NADPH-oxidase inhibition, suggests the coexistence of other mechanisms underlying DOC-induced oxidant production, for which procyanidins will also afford antioxidant protection. Hex partially prevented the DOC-mediated increase in cell oxidants, the mobilization of the tight junction protein ZO-1 from the intercellular space to the cytosol, and the increase in paracellular permeability. The finding that NADPH-oxidase inhibitors prevented both the DOC-induced increase in cell oxidants and the altered FSA paracellular transport indicates the involvement of oxidants in DOC-induced perturbations of the monolayer integrity. Due to the hydrophobic nature of DOC and to its capacity to increase cell oxidants, we investigated if Hex could protect cells from another lipophilic prooxidant. We observed that AMVN also induced a rise in cell oxidants, and the permeabilization of Caco-2 monolayers, events that were inhibited by Hex. These results reinforce the role of oxidants in affecting the barrier integrity and the potential capacity of large procyanidins to protect the intestinal epithelium from oxidative damage-mediated dysfunction. Hex inhibited AMVN- and DOC-induced increases in cell oxidants at concentrations as low as 2.5–5 μM when the procyanidin was present during the incubation with AMVN or DOC. When cells were exposed to Hex for 30 min, and Hex was subsequently removed from the media, most of the protective effects were observed, but at higher concentrations. This is in agreement with the results obtained for the TEER measurements that indicated that Hex binds to the cell membrane surface and, although transiently, extends its effect beyond the period of direct contact of the procyanidin with the monolayer. These results also support the previous findings that, in synthetic membranes, procyanidins can interact with membranes and exert long-term effects [20–22]. In support of this interaction, it was previously described that erythrocytes isolated from rats given a flavanol- and procyanidin-rich meal were less susceptible to in vitro oxidant-mediated hemolysis [42]. Furthermore, the possibility that these compounds can accumulate in the cell plasma membrane and exert action over extended periods of time constitutes a relevant factor to consider when defining their local concentrations and the physiological properties of these procyanidins. The effects of the intestinal mucus on the binding of procyanidins to the epithelium must be considered when evaluating the physiological actions of procyanidins at the gastrointestinal tract. The availability of large procyanidins to

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epithelial cells would be in part conditioned, among other factors, by the interactions of flavonoids with the different mucus components and with the characteristics of mucus that can vary depending on the diet [43], the nutritional status [44], and with the presence of certain drugs [45]. Furthermore, several intestinal pathologies are associated with alterations in the thickness and composition of the mucus [46], resulting in increased exposure of the epithelial cells to toxins and pathogens. Due to the limited absorption of procyanidins, the physiological action of dietary highly oligomerized procyanidins would be restricted to the oral cavity and the gastrointestinal tract. Accordingly, a lower risk of cancer in the upper aerodigestive tract was found in individuals consuming flavonoid-rich diets [47]. Furthermore, the consumption of polyphenols from black tea protected rat colon mucosa from 1,2-dimethylhydrazine-induced DNA damage [48]. The present study demonstrates that a procyanidin fraction enriched in hexameric oligomers can interact with the cell membrane and protect Caco-2 cells from bile acid-induced cytotoxicity, oxidant production, and barrier permeabilization. At the oral cavity and digestive tract, large procyanidins could protect the epithelium and prevent or ameliorate the outcome of different pathologies associated with increased oxidant production and alterations of the barrier integrity, such as inflammatory diseases, alterations in the intestinal barrier permeability, and cancer. The final assessment of the health relevance of the present findings will arise from further studies in animal models and in human populations. Acknowledgments This work was supported by grants from the University of Buenos Aires (B054), ANPCyT (01-08951), and CONICET (PIP 02120), Argentina; and from Mars Inc., USA. References [1] Gu, L.; Kelm, M. A.; Hammerstone, J. F.; Beecher, G.; Holden, J.; Haytowitz, D.; Gebhardt, S.; Prior, R. L. Concentrations of proanthocyanidins in common foods and estimations of normal consumption. J. Nutr. 134:613–617; 2004. [2] Hammerstone, J. F.; Lazarus, S. A.; Schmitz, H. H. Procyanidin content and variation in some commonly consumed foods. J. Nutr. 130: 2086–2092; 2000. [3] Rios, L. Y.; Bennett, R. N.; Lazarus, S. A.; Remesy, C.; Scalbert, A.; Williamson, G. Cocoa procyanidins are stable during gastric transit in humans. Am. J. Clin. Nutr. 76:1106–1110; 2002. [4] Holt, R. R.; Lazarus, S. A.; Sullards, M. C.; Zhu, Q. Y.; Schramm, D. D.; Hammerstone, J. F.; Fraga, C. G.; Schmitz, H. H.; Keen, C. L. Procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr. 76:798–804; 2002. [5] Sano, A.; Yamakoshi, J.; Tokutake, S.; Tobe, K.; Kubota, Y.; Kikuchi, M. Procyanidin B1 is detected in human serum after intake of proanthocyanidin-rich grape seed extract. Biosci. Biotechnol. Biochem. 67:1140–1143; 2003. [6] Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C. Bioavailability and bioefficacy of polyphenols in humans: I. Review of 97 bioavailability studies. Am. J. Clin. Nutr. 81:230–242; 2005.

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