The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 577-581

Hot Spices Influence Permeability of Human Intestinal Epithelial Monolayers¹

Erika Jensen-Jarolim^{*, 2}, Leszek Gajdzik^*, Ines Haberl, Dietrich Kraft^*, Otto Scheiner^*, and Jürg Graf^*

^* Department of General and Experimental Pathology and  Clinic of Surgery, University Hospital AKH, Logo, Vienna, Austria

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Indirect evidence suggests that hot spices may interact with epithelial cells of the gastrointestinal tract to modulate their transport properties. Using HCT-8 cells, a cell line from a human ileocoecal carcinoma, we studied the effects of spices on transepithelial electrical resistance (TER), permeability for fluorescein isothiocyanate (FITC)-labeled dextrans with graded molecular weight, and morphological alterations of tight junctions by immunofluorescence using an anti-ZO-1 antibody, a marker for tight junction integrity. Two different reactivity patterns were observed: paprika and cayenne pepper significantly decreased the TER and increased permeability for 10-, 20- and 40-kDa dextrans but not for -70 kDa dextrans. Simultaneously, tight junctions exhibited a discontinuous pattern. Applying extracts from black or green pepper, bay leaf or nutmeg increased the TER and macromolecular permeability remained low. Immunofluorescence ZO-1 staining was preserved. In accordance with the above findings, capsaicin transiently reduced resistance and piperine increased resistance, making them candidates for causing the effects seen with crude spice extracts. The observation that Solanaceae spices (paprika, cayenne pepper) increase permeability for ions and macromolecules might be of pathophysiological importance, particularly with respect to food allergy and intolerance.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Hot spices are used widely due to their taste and digestive properties associated with their pungent principles. These are predominantly piperine (6%) in black pepper (Piper nigrum) and capsaicinoidic compounds in paprika (Capsicum anuum: 0.01-0.22%), chili or cayenne pepper (C. frutescens: 0.3-1%). Although piperine may support digestion of food by a hydrocholagogue effect (Ganesh Bhat and Chandrasekhara 1987), the digestive properties of capsaicin (Govindarajan and Sathyanarayana 1991) may be attributed to enhancement of the activity of digestive enzymes (Platel and Srinivasan 1996) or to indirect effects on vascular endothelia, smooth muscles and mast cells, resulting in increase of vascular permeability and of mucosal blood flow (Gronbech and Lacy 1994, Reynier-Rebuffel et al. 1994).

Besides these neuronal and vascular effects, direct interaction of spices with gastrointestinal epithelia is likely to occur. Surprisingly, little is known about such direct interactions. Some studies suggest inhibitory effects on healing of gastric lesions by capsaicin (Kearney et al. 1989, Marotta and Floch 1991, Takeuchi et al. 1994), but no noxious effects were elicited by peppers (Graham et al. 1988). Our interest in studying possible effects of spices on gastrointestinal epithelial transport arises from the observations that spices can elicit food adverse reactions with heterogeneous organ manifestations (Bruijnzeel-Koomen et al. 1995, Hofer and Wüthrich 1985). Although in food allergy primary sensitization via air born allergens can be assumed (Valenta and Kraft 1996), it is conceivable that hot spices can promote antigen transfer through epithelia and thereby augment sensitization or allergic reactions.

To study direct effects and to exclude the possible interaction between nerve endings and mast cells with the epithelium in the intact mucosa, we selected an intestinal epithelial cell line (HCT-8) derived from a human ileocecal adenocarcinoma as a model. These cells exhibit microvilli and polarized morphology when grown on permeable filters. HCT-8 cells express tight junctions and achieve a high transepithelial electrical resistance (TER)³ (>700 ohm·cm²) in confluent monolayers (Hunter et al. 1991). These features were prerequisites for examining the effects of spices on the permeability of monolayers and on their ability to promote the transfer of macromolecules across the epithelium.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Spice extracts.  Spices (black and green pepper grains, bay leaves, nutmeg, chili pepper, cayenne pepper and paprika powder) were purchased from a local retail shop. Spices were pulverized in a food processor and mixed 150 g/L with serum free Dulbecco's modified Eagle's Medium (DMEM) culture medium. Samples then were homogenized using micro glass beads and incubated in a water bath for 30 min at 37°C. Extracts then were centrifuged a 800 g for 10 min at room temperature. Supernatants were collected and again centrifuged at 1000 × g. Extracts were frozen and stored at 20°C until use of experiments. spice extract (50 µL), representing extract from 7.5 mg crude spice powder, was applied per 450 µL medium on 1 cm² epithelial monolayer in all experiments performed.

View larger version (29K): [in this window] [in a new window]   View larger version (30K): [in this window] [in a new window]   Fig 1. Evaluation of transepithelial electrical resistance (TER) of confluent intestinal epithelial HCT-8 cells before and after application of spice extracts. Resistances (·cm2) were measured every 8 s in an Ussing type chamber and calculated by a computer program. Mean values of two experiments (representing measurements on 2 cell wells) ± SD are shown. Paprika, cayenne pepper, chili pepper (A) and black pepper, green pepper, nutmeg and bay leaf (B) were extracted in DMEM. After stabilization of the resistance, 50 µL extract corresponding to extract from 7.5 mg crude spice was diluted 1:10 in DMEM and applied per cm2 HCT-8 monolayer (indicated by black arrows). As positive control, 12.5 mmol/L EDTA was applied (A).

Spice components.  We selected the most common spices, paprika and black pepper, for their heterogeneous TER reactivity patterns. Their major components, capsaicin and piperine, respectively, and further compounds present in spices (Coumarin, camphor, safrole, -pinene, xanthine) were applied apically on confluent HCT-8 monolayers (see further). Coumarin, camphor, safrole, -pinene, xanthine, capsaicin and piperine (Sigma, St. Louis, MO) were dissolved at 100 mmol/L in 50% ethanol/50% dimethyl sulfoxide (DMSO), and these stock solutions were further diluted in DMEM for measurement of TER.

Cell culture.  Intestinal epithelial cells derived from a human ileocecal adenocarcinoma (HCT-8; ATCC No. CCL-244) were cultured in DMEM 100 mL/L fetal calf serum (FCS), 10 g/L glutamine, 10 g/L N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] (HEPES), 1 × 10⁶ IU/L penicillin-streptomycin (Life Technologies, Gaithersburg, MD), in tissue culture flasks (Corning Costar, Cambridge, MA) until monolayers were confluent. After washing with PBS, cells were typsinized using a 25 g/L trypsin solution containing 1 mmol/L ethylene diamine tetraacetate (EDTA), 10 mmol/L glucose and 4 g/L phenol red. After washing with DMEM/10% FCS, cells were seeded onto cell culture inserts (Falcon, 12 mm diam, 0.4 µm pore size) at a density of 3 × 10⁵ cells per insert and cultured until monolayers were confluent. At d 9, resistance values ranging between 700 and 1500 ohm·cm² were achieved. Inserts were chosen randomly for a pair of stimulation experiments with a spice or spice component.

Measurement of TER.  Intestinal epithelial cells (HCT-8) were grown on permeable filter membranes until high TER (between 700 and 1500 ·cm²) indicated that the monolayers were confluent. Then filters were mounted in a specially designed Ussing-type chamber. Medium 1.5 mL was placed into the lower buffer chamber and 0.5 mL into the upper. Fluid levels were equally high inside and outside the insert wall. Current was passed across the monolayer; resulting voltages were recorded every 8 s and monitored by a computer program as described by Tschugguel et al. (1995). The data obtained were corrected for the series resistances of the medium and membrane support. Experiments were performed at 37°C and a gentle stream of humidified 5% CO₂-95% air was directed over the solutions during the experiments. All experiments with spice extracts and spice components were conducted on a single day, with the same batch of HCT-8 cells and with inserts chosen randomly.

After recording the stable baseline resistance (3 min), 50 µL extracts from black or green pepper, paprika, chili pepper, cayenne pepper, nutmeg or bay leaf were applied to the medium of the upper buffer chamber, achieving a 1:10 dilution in 450 µL medium. Thus extract obtained from 7.5 mg crude spice per square centimeter of epithelial surface was applied apically in duplicate experiments. The resistance was monitored for 7 or 9 min after application of substances. Alternatively the 100 mmol/L spice components solutions, and as control solvent solution alone, were diluted in DMEM of the upper buffer chamber. Increasing concentrations of 0.03, 0.3 and 3 mmol/L (plus 5 mmol/L for capsaicin) were tested to determine the minimal effective dose. As controls, 50 µL of 125 mmol/L EDTA, known to increase epithelial permeability, or medium were applied apically to 450 µL medium.

View larger version (33K): [in this window] [in a new window]   Fig 2. Evaluation of transepithelial electrical resistance (TER) of confluent intestinal epithelial HCT-8 cells before and after application of spice components capsaicin, piperine, -pinene and xanthin. Resistances (·cm2) were measured every 8 s in an Ussing type chamber and calculated by a computer program. Mean values of two experiments ± SD are shown. Spice components were diluted 0.3-5 mmol/L in DMEM. After stabilization of the resistance, 0.3 mmol/L of piperine, -pinene and xanthine, or 5 mmol/L capsaicin was applied per cm2 HCT-8 monolayer (indicated by black arrows).

Determination of permeability using FITC dextrans.  FITC-conjugated Dextrans of graduated molecular weight (10, 20, 40 and 70 kDa; Sigma, St. Louis, MO) in 10 g/L Tris buffered saline (TBS), were dialyzed against TBS for 24 h and against DMEM for 24 h. HCT-8 cells were cultured on inserts as described above. When cells were confluent, medium of the upper chamber compartment was exchanged with medium containing different FITC dextrans. Simultaneously spice extracts, EDTA or medium in same concentrations per square centimeter as described above were applied in duplicate. After 0, 15, 30 and 120 min, 100-µL samples were collected from the lower chamber compartment, transferred to a Nunclon 96 plate (Nunc, Roskilde, Denmark) and analyzed using a cytofluor (Millipore, Bedford, U.K.) (excitation 485 nm; emission 530 nm). Permeability was calculated from differences between values obtained at 15, 30 and 120 min: flux (pmol.·cm²·s¹)/upper concentration (pmol·cm³) and is given in µm·s¹.

Immunofluorescence.  HCT-8 cells were seeded on 16-well Lab-Tek tissue chamber slides (Nunc, Roskilde, Denmark) at a density of 1 × 10⁵ per well. After 24 h culture, large monolayered cell clusters were visible. Medium was exchanged with fresh medium, and then cells were exposed to corresponding amounts of spice extracts per square centimeter epithelia as in the experiments above, 12.5 mmol/L EDTA or medium alone. As the most dramatic changes of TER occurred already during the first minute, supernatants were discarded after 3 min exposure and cells washed two times with PBS++ (PBS containing 1 mmol/L MgCl₂ and 0.1 mmol/L CaCl₂), 5 g/L bovine serum albumin (BSA) and one time with PBS++ without BSA. Cells then were fixed with 50 µl ice cold aceton for 2 min on ice (Anderson et al. 1989). Then cells were washed with PBS, and nonspecific binding sites were blocked with PBS containing 5 g/L BSA 30 min at 37°C. After three times washing with PBS, 20 µL of rat anti-Zonula occludens (ZO-1) antibody (kindly provided by Dr. Jim Anderson, Yale) diluted 1:50 in PBS, 5 g/L BSA was applied and incubated at 37°C for 30 min. Bound immunoglobulin was detected with FITC-conjugated rabbit anti-rat IgG (Serotec, Oxford, U.K.) by incubating 30 min at 37°C. For control, only the secondary antibody was used. Finally all preparations were washed three times in PBS and one time in water, and were mounted using Moviol. Fluorescence staining was viewed using an AH2 Olympus microscope.

Statistical analysis.  Statistical analysis was performed using Student's t test; paired tests for changes of TER, and unpaired tests for anlaysis of permeability with FITC-dextrans.

	RESULTS

Abstract Introduction Methods Results Discussion References

Effects of spice extracts on TER of HCT-8 monolayers.  Two general types of reactions were observed, an immediate and significant decrease or increase of the TER. A decrease of TER occurred when using cayenne pepper (P < 0.005), chili pepper (P < 0.001) or paprika (P < 0.001), reflecting a dramatic increase of ion permeability (Fig. 1A). For chili pepper, this reduction of resistance was pronounced but transient and was followed by an increase to values above baseline values (P < 0.005). Application of EDTA used as control, reduced resistance to only ~60% during the experiment (P < 0.005); this might be due to the apical and not basolateral application (Collares-Buzato et al. 1994). In contrast, significant increases of TER were observed after application of extracts from black pepper (P < 0.01), green pepper (P < 0.005), nutmeg (P < 0.001) or bay leaf (P < 0.0005 (Fig. 1B).

Effects of spice components on TER of HCT-8 monolayers.  A concentration of piperine of 0.3 mmol/L increased TER (P < 0.05), resembling the effect produced by pepper extracts. Assuming a 6% content of piperine in black pepper, this amount (0.3 mmol/L) corresponds approximately to the quantity present in crude spice extract applied in the experiment shown in Fig. 1B. Application of capsaicin reduced TER immediately (P < 0.05) but only at a concentration of 5 mmol/L. This was ~100 times higher than the concentration present in native extracts from cayenne pepper and 1000 times above that in paprika. Using 0.03, 0.3 or 3.0 mmol/L coumarin, camphor, safrole, -pinene, xanthine or a respective volume of solvent control, or significant changes of TER were noticed. Two examples are shown in Fig. 2. Thus piperine, and to a certain extent also capsaicin, can be considered as candidates for causing effects on epithelial TER observed with crude spices.

Spices affect the permeability of HCT-8 monolayers for macromolecules.  In controls, the permeability of HCT-8 decreased with increasing molecular size (Fig. 3). The application of pepper extract did not affect permeability for macromolecules of all sizes tested, but a significant increase of permeability was observed for the 10, 20 and 40 kDa dextrans (P < 0.05) after application of paprika extract (Fig. 3). The passage of 10-kDa macromolecules was favored compared with the 20- and 40-kDa molecules. A significant effect of paprika on the transfer of 70 kDa dextrans was not observed. The sustained decrease of TER in our model directly correlated with increased permeability for macromolecules of sizes 40 kDa.

View larger version (16K): [in this window] [in a new window]   Fig 3. Changes of permeability of confluent HCT-8 monolayers after apical paprika and pepper application or without treatment are shown. FITC dextrans (10, 20, 40 and 70 kDa) served as marker molecules for paracellular transfer of macromolecules. Mean values of the duplicate 30 and 120 min measurements ± SEM are shown. Note increase of permeability by paprika for molecules < 70 kDa.

Morphological changes of HCT-8 epithelia in immunofluorescence.  To examine whether the observed TER changes were accompanied by morphological alterations, particularly of the tight junctions, immunofluorescence experiments were performed.

We examined the localization of ZO-1, a cytoplasmic protein associated to tight junctions (Fig. 4). A fine continuous linear web outlined all cell contacts in untreated control HCT-8 cells (panel A) and after pepper treatment (not shown). After paprika treatment, the ZO-1 staining appeared fragmented (panel B).

View larger version (92K): [in this window] [in a new window]   Fig 4. ZO-1 staining of cultured HCT-8 intestinal epithelial cells before and after paprika application. A: ZO-1 staining on untreated HCT-8. B: staining after application of paprika. Bound antibody was detected by FITC-labeled anti-rat Ig antibody. Note interruptions and granular appearance of tight junctions in B.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

We examined whether extracts of spices have direct effects on the permeability of intestinal epithelia. Spices produced contrasting effects on permeability in our model: Solanaceae spices (paprika, cayenne pepper, chili pepper) decreased TER significantly. This effect was accompanied by significant increases of permeability for macromolecules of 10-40 kDa and discontinuities of ZO-1 staining in immunofluorescence. The second group of spices (black and green pepper, nutmeg, bay leaf) clearly induced an increase of TER, but paracellular permeability of macromolecules was unchanged and ZO-1 staining of tight junctions was unaffected.

With regard to Solanaceae spices, capsaicin represented a candidate producing the observed effects. However, for producing comparable changes of TER, an ~100 times higher concentration was necessary than what was effective in crude spice extract. Thus a potentiating effect of several compounds present in paprika, cayenne pepper and chili pepper may account for the significant decreases of TER. Decreases in TER clearly were paralleled by increased permeability for macromolecules, which was most prominent for smaller molecules (10 and 20 kDa). Interestingly, also the passage of 40-kDa molecules was significantly enhanced, but passage of molecules of 70 kDa remained unaffected. Thus Solanaceae spices may indeed enhance the passage of molecules smaller than 70 kDa through epithelial barriers by increasing the paracellular permeability, whereas transcytosis appeared not unaffected.

Accordingly, discontinuities of intercellular junctions as shown by immunofluorescence using an anti-ZO-1 antibody were observed. (ZO-1 is an excellent marker for intact cell-to-cell contacts (Madara et al. 1988). Fragmentation of the tight junctions might explain the increases in permeability after paprika application. Similar phenomena were observed when the perijunctional actin microfilament ring was affected by cytochalasin B or D (Ma et al. 1995, Madara 1987, Madara et al. 1988, Stevenson and Begg 1994). Active contraction of the intestinal epithelial cells may be one mechanism for regulating epithelial barrier functions.

The opposite effects, namely significant increases of TER were observed with pepper of the botanical family Piperaceae, with nutmeg (Myristicaceae) and bay leaf (Myrticaceae) extract. This effect could be due to cell swelling resulting in narrowing of the intercellular clefts. These contribute (in addition to the tight junctions) to the resistance for paracellular ion flow. Accordingly, increases of TER have been observed at low temperature (Cereijido et al. 1993) or in inflamed ileum (Hyun et al. 1995). TER increases produced by spices could be mimicked by piperine. The presence of piperine in chili pepper extract could therefore explain why initial decreases of TER were followed by increases above the basal level, atypical for the patterns observed with other spices from the botanical family Solanaceae. In the transport studies using FITC dextrans, no differences between permeability of unstimulated and black pepper-stimulated confluent monolayers were seen. Therefore, black pepper extracts influenced neither the paracellular transport nor the transcytosois of molecules between 10 and 70 kDa during the time course of the experiment.

Since studies were done on an epithelial cell line, the observed effects are independent of secondary effects that may be elicited in intact intestinum by release of neuropeptides from C-fibers or of other inflammatory mediators. Spices either may increase epithelial permeability through loosening cell contacts (paprika, Cayenne pepper, chili pepper) or decrease permeability (black pepper, nutmeg, bay leaf), possibly by cell swelling.

We may stress the fact that direct interaction of spices with epithelial cells affects the transport of ions and macromolecules. These effects may be important in explaining digestive properties of spices. Moreover, spices may modulate the absorption of low molecular weight food constituents that are involved in the pathogenesis of food allergy or intolerance. Important allergenic proteins in plant-derived food stuff exhibit molecular weights of 14 and 17 kDa (Breiteneder et al. 1995, Ebner et al. 1991 and 1995, Vanek-Krebitz et al. 1995, Van Ree et al. 1992).

	FOOTNOTES

Manuscript received 19 June 1997. Initial reviews completed 12 September 1997. Revision accepted 6 November 1997.

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