In Vitro Study of Intestinal Transport of Inorganic and Methylated

Article pubs.acs.org/crt

In Vitro Study of Intestinal Transport of Inorganic and Methylated Arsenic Species by Caco-2/HT29-MTX Cocultures Marta Calatayud, Marta Vázquez, Vicenta Devesa, and Dinoraz Vélez* Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Avenida Agustín Escardino n° 7, 46980 Paterna (Valencia), Spain ABSTRACT: This study characterizes intestinal absorption of arsenic species using in vitro system Caco-2/HT29-MTX cocultures in various proportions (100/0 to 30/70). The species assayed were As(V), As(III), monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], and dimethylarsinous acid [DMA(III)]. The results show that the apparent permeability (Papp) values of pentavalent species increase significantly in the Caco-2/HT29-MTX cocultures in comparison with the Caco-2 monoculture, probably because of enhancement of paracellular transport. For MMA(III) and DMA(III), Papp decreases in the Caco2/HT29-MTX cell model, and for As(III), there is no change in Papp between the two culture models. Transport studies of arsenic solubilized from cooked foods (rice, garlic, and seaweed) after applying an in vitro gastrointestinal digestion showed that arsenic absorption also varies with the model used, increasing with the incorporation of HT29-MTX in the culture. These results show the importance of choosing a suitable in vitro model when evaluating intestinal arsenic absorption processes.

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INTRODUCTION Arsenic is an element widely distributed in the environment. Drinking water and food are the main source of exposure to this element. The risk associated with exposure is affected by the arsenic species ingested. From a toxicological viewpoint, the species of greatest interest are inorganic arsenic and the monomethylated and dimethylated arsenic species. Inorganic arsenic is the major species in drinking water and is considered a group 1 carcinogen.1 Numerous epidemiological studies have verified a causal relationship between high exposure to inorganic arsenic and various types of cancer.2 In food, the presence of As(III), As(V), pentavalent methylated species [monomethylarsonic acid, MMA(V), and dimethylarsinic acid, DMA(V)], and other organic species has been shown. So far, the presence of trivalent methylated species [monomethylarsonous acid MMA(III)] has only been reported in carrots grown in an area polluted with arsenic.3 However, it would be necessary to use extraction methods that maintain the oxidation state of arsenic species to be able to evaluate with certainty the presence of MMA(III) and DMA(III) (dimethylarsinous acid) in food. The epithelium of the small intestine is the first physiological barrier that arsenic meets after ingestion; yet, there are very few studies that evaluate the mechanisms of absorption and transport of arsenic or the toxic effects of arsenic species on the epithelium.4−6 In vitro studies are a valuable tool for making approximations of intestinal absorption, and they have economic and ethical advantages over in vivo studies. The human intestinal epithelial cell line Caco-2, originally derived from a human colon carcinoma, has been widely used as an in vitro model of absorptive enterocytes to evaluate absorption of © 2012 American Chemical Society

nutrients and drugs. This cell line is capable of differentiating spontaneously and forming monolayers of absorptive enterocytes with brush border and tight junctions.7 Moreover, Caco2 cells express carriers and receptors for nutrients, macromolecules, and drugs.8−10 This cell model has been used previously to evaluate uptake and intestinal transport of inorganic arsenic [As(III) and As(V)], pentavalent methylated species [MMA(V) and DMA(V)], trivalent methylated species [MMA(III) and DMA(III)], and food (rice and seafood).4−6,11−13 However, for hydrophilic molecules that are transported by the paracellular pathway, as is the case with some arsenic species,5,6 the permeability of Caco-2 monolayers is up to 100 times lower than that of the human intestine in vivo,14 because these cells form dense intercellular junctional complexes. This may cause an underestimate of uptake of arsenic species when the Caco-2 cell model is used. Cocultures of Caco-2 and mucus-secreting cells (HT29MTX) have also been used as a model of the intestinal epithelium for various purposes: drug and nutrient absorption, the study of bacterial adhesion, and cell−cell interaction.14−21 This model includes the two most abundant intestinal epithelial cell types (absorptive and goblet cells) and mimics physiological conditions,16 because the presence of HT29-MTX in cocultures with Caco-2 modulates the tight junctional geometry and “tightness” and yields a mucus gel covering the whole cell surface. However, there are no previous references for the study of intestinal absorption of arsenic species in these cocultures. Received: July 2, 2012 Published: November 1, 2012 2654

dx.doi.org/10.1021/tx300295n | Chem. Res. Toxicol. 2012, 25, 2654−2662

Chemical Research in Toxicology

Article

min), aliquots were taken from the basal compartment (400 μL) and replaced with an equal volume of fresh medium. Arsenic was then quantified in these aliquots. The apparent permeability coefficients (Papp) were calculated at 120 min, using eq 1

The use of mixed Caco-2/HT29-MTX cell models may provide information that is closer to the in vivo situation, and it incorporates differential characteristics in comparison with Caco-2 alone that may significantly affect intestinal transport of arsenic. The aim of this work was to evaluate cell retention and transport of inorganic arsenic and pentavalent and trivalent methylated species in various cocultures of the two cell lines.

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Papp = (dC /dt )(Vr /ACo)

(1)

where dC/dt is the flow (μM/s) determined by the linear slope of the equation that governs the variation in the arsenic concentrations, corrected by dilution, against time; Vr is the volume of the acceptor compartment (basal, 2 mL); A is the surface occupied by the cell monolayer (4.67 cm2); and Co is the initial arsenic concentration in the donor compartment. At the end of the assay, the culture media were recovered, and the cells were washed three times with Dulbecco's phosphate buffered solution (DPBS, PAA), scraped, and lysed with 1% Triton X-100 (Merck, Germany). Arsenic was quantified in medium and in cells. Arsenic retention and transport percentages were calculated with respect to the initial quantity of arsenic added to the Caco-2 cell cultures. Control cells were used throughout every assay. During the tests, cell monolayer integrity was evaluated by measuring (a) TEER at various points in the study, including the start and end times of the experiment, and (b) the Papp of the paracellular transport marker Lucifer Yellow (LY), added at a concentration of 100 μM to the apical compartment in the control wells and the wells treated with arsenic. The fluorescence of the LY transported to the basal side was measured with a fluorescence microplate reader (PolarSTAR OPTIMA, BMG-Labtech, Germany) at excitation/emission wavelengths of 485/520 nm. To evaluate possible interactions of LY with uptake and transport of arsenic species, parallel experiments were performed with and without paracellular marker, which demonstrated the absence of interference. Estimation of Pore Radius of Tight Junctions in Cocultures. The pore radius of tight junctions in the various cocultures was calculated using the equation R = 2.96 × 106 × PLY + 4.94,22 where R is the pore radius of tight junctions and PLY is the Papp of LY (10−6 cm/ s). Influence of Mucus on Intracellular Accumulation of MMA(III) and DMA(III) in the HT29-MTX Cell Line. The influence of the presence of the mucus monolayer on arsenic retention was studied with differentiated HT29-MTX cells (15 days postseeding). The cells were seeded in six-well plates and after differentiation were exposed for 2 h to 1 μM MMA (III) and 1 μM DMA (III). After exposure, the medium was recovered, and the mucus layer was separated from the cell monolayer, using the method described by Mahler et al.19 with some modifications. Briefly, the monolayer was washed with 1.5 mL of 10 mM N-acetylcysteine (NAC) in HBSS (PAA) at 37 °C with agitation (135 rpm) for 1 h. Subsequently, medium (HBSS + NAC) was recovered to quantify arsenic contents in mucus. Mucus removal was verified by alcian blue staining of the monolayer after the washing procedure. In Vitro Gastrointestinal Digestion and Arsenic Uptake from Foods. Samples of rice (Oriza sativa) and seaweed (Hizikia f usiforme) cooked with Milli-Q water and purple garlic (Allium sativum) cooked with an aqueous standard of As(V) (1 mg/L) were digested using an in vitro simulated gastrointestinal digestion.11 Briefly, after the gastric step (0.02 g pepsin/g sample, pH 2, 2 h, and 37 °C) and the intestinal step (0.0005 g of pancreatin/g sample and 0.003 g of bile extract/g sample, pH 6, 2 h, and 37 °C), samples were centrifuged at 26891g for 30 min at 4 °C to separate the soluble (bioaccessible) fraction. The total arsenic and arsenic species (inorganic arsenic, MMA, and DMA) were analyzed in the bioaccessible fractions. Enzymes employed for the gastrointestinal digestion were purchased from Sigma. For the uptake (retention and transport) assay with cells, the bioaccessible fraction was heated for 4 min at 100 °C to inhibit sample proteases and then cooled by immersion in an ice bath. Glucose (Sigma, 5 mM final concentration) and HEPES (50 mM final concentration) were then added to facilitate cell viability. Water or NaCl (Panreac) was added to adjust the osmolarity to 310 ± 10 mOsm/kg using a freezing point osmometer (Osmomat 030, Berlin,

MATERIALS AND METHODS

Arsenical Species. The standard of As(V) (1000 mg/L) was purchased from Merck (Merck, Darmstadt, Germany). The standard of As(III) (1000 mg/L) was prepared by dissolving 1.320 g of As2O3 (Riedel de Haën, Germany) in 25 mL of 20% (w/v) KOH, neutralizing with 20% (v/v) H2SO4, and diluting to 1 L with 1% (v/v) H2SO4. The standard solutions of MMA(V) and DMA(V) were prepared by dissolving in water the appropriate amount of CH3AsO(ONa)2·6H2O (Carlo Erba, Italy) or (CH3)2AsNaO2·3H2O (Fluka Chemika Biochemika, Spain), respectively. The standard solutions of MMA(III) and DMA(III) were prepared from CH3AsI2 and (CH3)2AsI (Argus Chemicals, Vernio, Italy), respectively. Cell Culture. The Caco-2 cells were acquired from the European Collection of Cell Cultures (ECACC, number 86010202, Salisbury, United Kingdom). Cells were used between passages 5 and 25. Caco-2 cells were routinely grown in 75 cm2 flasks in Dulbecco's modified Eagle's medium (DMEM) at pH 7.4 containing glucose (4.5 g/L) and L-glutamine (0.6 g/L) and supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acids, 10 mM HEPES (N-2hidroxyethylpiperazine-N′-2-ethanosulfonic acid), 100 U/mL of penicillin, 0.1 mg/mL of streptomycin, 0.0025 mg/mL of fungizone, and 1 mM sodium pyruvate (DMEMc). The HT29-MTX cells were kindly provided by Dr. Thécla Lesuffleur (Institut National de la Santé et de la Recherche Médicale, INSERM UMR S938, France) in passage 15, and they were used in this study between passages 16 and 25. Routine maintenance of the cells was carried out in 25 cm2 flasks to which 5 mL of medium was added, consisting of DMEM at pH 7.4 containing glucose (4.5 g/L) and L-glutamine (0.6 g/L) and supplemented with 10% (v/v) fetal bovine serum, 100 U/mL of penicillin, 0.1 mg/mL of streptomycin, 0.0025 mg/mL of fungizone, and 1 mM sodium pyruvate (HTDMEMc). Both cell lines were incubated at 37 °C, in a humidified atmosphere of 95% air and 5% CO2, and the medium was changed every 2−3 days. When the cell monolayer reached 80% confluence, the cells were detached with a solution of trypsin (0.5 mg/L) and ethylene diamine tetraacetic acid (EDTA) (0.22 g/L), followed by reseeding at a density of 4 × 104 cells/cm2. All of the reagents used were purchased from PAA Laboratories GmbH (Germany). Cell Retention, Transport, and Permeability Tests. The cell retention, transport, and permeability tests were performed in twochamber wells with polyester membranes (diameter, 24 mm; pore sizem 0.4 μm; Transwell, Costar Corp., NY). In this system, the cells are kept on a porous support that separates the well into two compartments: apical and basal. During the period of growth and differentiation of the cultures on the Transwell membranes, the cell monolayer integrity was monitored every 2−3 days by measuring the transepithelial electrical resistance (TEER) with a Millicell-ERS (Millipore Corporation, Madrid, Spain). For the preparation of the cocultures, the cells were seeded at a density of 6.5 × 104 cells/cm2 in HT-DMEMc in the following Caco2/HT29-MTX proportions: 100/0, 90/10, 50/50, and 30/70. The cells were incubated routinely until they reached cell differentiation (13−14 days postseeding). Then, the cell monolayers were conditioned with Hank's buffered solution salts (HBSS) (PAA) supplemented with 10 mM HEPES (pH 7.2) for 15 min. The standard solutions of arsenic species prepared in HBSS were then added to the apical medium. For As(V), DMA(V), MMA(V), and As(III), the concentrations assayed were 67 μM, equivalent to 5 mg/L as arsenic. DMA(III) and MMA(III) were assayed at 1 μM (0.25 mg/L as arsenic). At the stipulated test times (5, 15, 30, 45, 60, 90, and 120 2655



Article

Germany). Then, 1.5 mL of this bioaccessible fraction together with 100 μM LY was added to the apical chamber of cocultures of Caco-2/ HT29-MTX in the proportions 100/0, 90/10, and 50/50. HBSS (2 mL) was added to the basal side. The cell culture was incubated for 2 h at 37 °C, 5% CO2, and 95% relative humidity. At the end of this period, the cells and apical and basal media were recovered, and arsenic was quantified. Arsenic retention and transport percentages were calculated with respect to the initial quantity of arsenic added to the cell cultures. Control cells were used throughout every assay. TEER values and transport of LY were the tests used to evaluate cell monolayer integrity during the assay time. Cell Viability. The number of viable cells at the end of each assay was quantified using the trypan blue exclusion technique (Trypan Blue Solution, 0.4% v/v, Sigma). Determination of Arsenic and Chemical Species. The total arsenic was quantified using atomic absorption spectrometry with Zeeman graphite chamber (GF-AAS) (AAnalyst 600, Perkin-Elmer Hispania, S.A., Spain), in accordance with the method described by Calatayud et al.5 The arsenic species were analyzed by hydride generation-cold trap-atomic absorption spectrometry (model 3300, Perkin-Elmer Hispania).23 The cells were treated with 200 μL of Triton X-100 (Merck) at 1% (v/v) and two or three freezing and defreezing cycles before the analysis. Pretreatment in the apical and basal media was not necessary. Statistical Analysis. All of the assays were performed at least in triplicate in independent cultures. The results were analyzed statistically by a one-factor analysis of variance (ANOVA) (SPSS, version 15.0). Differences were considered significant for p < 0.05.

In the differentiated monolayers (14 days postseeding), the apparent permeability coefficient of LY, a molecule that is mainly transported via the paracellular route, increased significantly (p < 0.05) with the increase in the proportion of HT29-MTX in the coculture (Figure 2). The pore radius values estimated with the equation of Saitoh et al.22 for the various monolayers ranged between 5 and 8 Å, increasing as the proportion of HT29-MTX in the coculture increased (Figure 2). The pore radius values correlated negatively with the TEER values (r2 = 0.96). Apparent Permeability Coefficient of Arsenic Species. The trivalent methylated species, MMA(III) and DMA(III), are much more toxic than As(III) and the pentavalent species. That is why they were assayed at much lower concentrations (1 μM) than the other species (67 μM). The apparent permeability coefficient (Papp) is a parameter that indicates the rate at which a compound passes through the intestinal epithelium. Papp was calculated for the pentavalent species [As(V), MMA(V), and DMA(V)] and trivalent inorganic arsenic [As(III)] at 120 min for all of the Caco-2/HT29-MTX proportions (100/0, 90/10, 50/50, and 30/70). During this time, the values of TEER, LY Papp, and cell viability stayed within the limits established for considering that the monolayer remains intact (TEER variation 0.05) between the Papp values obtained for the various Caco-2/HT29-MTX proportions (mean = 3 ± 0.2 × 10−6 cm/s). The Papp value of the methylated species decreased significantly (p < 0.05) as the proportion of HT29-MTX in the coculture increased. For MMA(III), Papp decreased from 8.6 × 10−6 cm/s in Caco-2 alone to 4.4 × 10−6 cm/s in Caco-2/HT29-MTX 50/50, and for DMA(III) in the same Caco-2/HT29-MTX proportions, there was a reduction of the same order (from 11.6 × 10−6 cm/ s to 6.5 × 10−6 cm/s). Cell Retention and Transport of Arsenic Species. In all of the assays conducted, a mass balance was performed, comparing the total arsenic added to the cells initially and the total arsenic obtained after analysis of the cells and medium. The mass balance showed recoveries ranging between 85 and 100%. The results for transport of arsenic species indicate behavior similar to that obtained in the calculation of apparent permeability, as shown in Tables 1 (pentavalent species) and 2 (trivalent species). In the pentavalent species, the incorporation of HT29-MTX in the culture increased transport to the basal side, reaching the highest percentages in the 30/70 coculture

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RESULTS Characterization of Caco-2/HT29-MTX Cocultures. An increase in TEER values during cell growth indicates the development of tight junctions between polarized differentiated cells. The TEER values found in the various cocultures during the growth period are shown in Figure 1. In the Caco-2

Figure 1. TEER values obtained during the culture of the various Caco-2/HT29-MTX proportions assayed: 100/0 (circles), 90/10 (triangles), 50/50 (squares), and 30/70 (diamonds). Results expressed in Ohms cm2 (mean ± standard deviation; n = 10).

monoculture, the TEER value increased progressively during the 14 days postseeding, reaching a maximum of 446 ± 30 Ohms cm2. In the Caco-2/HT29-MTX cocultures, the TEER value remained stable until day 5 postseeding (140 ± 20 Ohms cm2) and thereafter only increased significantly in the 90/10 coculture. The TEER values obtained in the cocultures at 14 days postseeding (164−363 Ohms cm2) were less than those found in Caco-2, especially in the 50/50 and 30/70 cocultures. 2656



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Figure 2. Characterization of Caco-2/HT29-MTX differentiated cocultures. Data represented are as follows: TEER values [Ohms cm2 (black line, right axis)], apparent permeability of LY [10−6 cm/s; 120 min (white bars, left axis)], and estimated pore radius (Å) (gray bars, left axis) for the cell monolayers 14 days postseeding, with different proportions of Caco-2/HT29-MTX. Data represent means ± standard deviations (n = 10).

Figure 3. Apparent permeability coefficients (Papp) for As(V), MMA(V), and DMA(V) in the various Caco-2/HT29-MTX proportions assayed: 100/0 (black bars), 90/10 (light gray bars), 50/50 (dark gray bars), and 30/70 (white bars). Values expressed as means ± standard deviations of at least four replicas of each arsenic species. An asterisk marks statistically significant differences (p < 0.05) with respect to the Caco-2/HT29-MTX proportion 100/0.

Figure 4. Apparent permeability coefficients (Papp) for As(III), MMA(III), and DMA(III) in the various Caco-2/HT29-MTX proportions assayed: 100/0 (black bars), 90/10 (light gray bars), 50/50 (dark gray bars), and 30/70 (white bars). Values expressed as means ± standard deviations of at least four replicas of each arsenic species. An asterisk marks statistically significant differences (p < 0.05) with respect to the Caco-2/HT29-MTX proportion 100/0.

[As(V), 54%; MMA(V), 47%; and DMA(V), 25%]. For the trivalent species, the introduction of HT29-MTX in the culture did not affect all of the species equally; it did not alter basal transport of As(III), and it reduced transport of MMA(III) and DMA(III), as was observed in the permeability study. Cellular retention of the pentavalent species was low (≤0.5%), similar in all of them, and did not alter with the percentage of HT29-MTX in the culture. Similar behavior was observed for As(III), although the retention (≈1.4%) was slightly higher than that of its pentavalent analogue. However, cell retention of MMA(III) and DMA(III) (27−46%) was much higher than that of the other arsenic species studied, increasing as the proportion of HT29-MTX in the culture increased. Both MMA(III) and DMA(III) were highly retained by the mucus secreted by the HT29-MTX cells, in much higher percentages than those found inside these cells [MMA(III): 33

± 8% in mucus vs 7 ± 2% in cells; DMA(III): 65 ± 2% in mucus vs 2.9 ± 0.6% in cells]. Arsenic Absorption from Foods. After the study with arsenic species standards, we evaluated the effect of the incorporation of HT29-MTX in the culture on the transport and cell retention of the bioaccessible fraction obtained after in vitro gastrointestinal digestion of food samples: raw rice and seaweed and garlic boiled in water with As(V) (1 mg/L). The analysis of the bioaccessible fractions obtained after in vitro gastrointestinal digestion of the food samples shows that in the rice and garlic, all of the solubilized arsenic was inorganic arsenic. In the seaweed, however, neither inorganic arsenic nor MMA was detected, and DMA represented only 1% of total arsenic (16 ng), so that the rest of the arsenic may be attributed to species that do not generate hydrides in the analysis conditions employed, and it is possible that they are arsenosugars.24 2657


uptake (basal + cells)

cells

In the two rice samples assayed, the quantity of arsenic added to the cell culture was very similar (≈50 ng). When the Caco2/HT29-MTX coculture was used in the 50/50 proportion, there was a notable increase in transport to the basal side (≈27%), in comparison with the transport found in the 90/10 proportion (≈12%) and in Caco-2 cells (≈13%). Cell retention, however, was much higher in the cocultures, irrespective of the proportion of HT29-MTX that they contained (Caco-2 cells ≈ 7%; cocultures, 22−34%). For the seaweed and garlic samples, the quantity of arsenic added to the culture was about 20 times greater than that added to the rice (1187 and 890 ng, respectively). In both samples, the introduction of HT29-MTX in the coculture produced a notable increase in basal transport of arsenic, especially high in garlic (33% transport in the 50/50 proportion). The cell retention in these two samples ( seaweed. The order for cell retention (rice ≫ garlic ≈ seaweed) also did not vary between the various cell cultures.

The table shows the quantity of arsenic added to the cells (t = 0) and the quantity present in the basal medium and inside the cells (t = 120 min). The values of total uptake (cell content + basal transport) are also shown. Values expressed in ng arsenic (mean ± standard deviation of at least four replicas of each arsenic species) and arsenic percentage (in parentheses).

84 8 1) 7 0.1) 26 1) 124 29 0.4) 2 0.1) 27 0.4)

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DISCUSSION Inorganic arsenic exposure via drinking water affects more than 140 million people worldwide.25 In areas without high arsenic levels in drinking water, cereal and cereal products are considered the primary source of inorganic arsenic in the human diet.2 It has been observed that between 40 and 60% of arsenic ingested is excreted in urine,26−28 but the real bioavailability of arsenic ingested may be higher than what is indicated by urinary arsenic excretion.28 Because of the difficulty of conducting arsenic bioavailability studies in humans, studies have been conducted in laboratory animals, and more rapid and low cost in vitro methods have been introduced. Although there have been previous studies with the Caco-2 cell line,5,6,12 there has been no study of the behavior of arsenic species when goblet cells are incorporated in the cell model. In view of the fact that the use of cocultures rather than Caco-2 cells alone makes it possible to mimic the resistance values present in human intestinal epithelia (20−100 Ohms cm2)16 and that HT29-MTX provides the mucus layer present in the intestine, this model should be explored as a possible alternative for in vitro studies of intestinal transport of arsenic species from food. In the present work, the incorporation of HT29-MTX in the cell culture produced a significant decrease in TEER value and an increase in LY permeability in comparison with the Caco-2 monoculture (Figure 2). Previous studies have shown changes in both parameters when mucus-secreting cells were seeded with Caco-2.14,16,29,30 However, there is high interlaboratory variability in TEER values because the tight junctions in monolayers (mono- or cocultures) are influenced both by culture conditions (medium, passage number, and culture time) and by the composition of the cell subpopulation.30−32 The changes observed in the TEER values and paracellular marker permeability may be due to an increase in the mean size of the pore radius of the tight junctions and to differences in the nature of the junctional complexes between Caco-2 and HT29MTX.33 Previously, Knipp et al.34 indicated that the pore radius of tight junctions has a major influence on drug permeation through the paracellular pathway. Saitoh et al.22 showed that for

a

79 107 3) 4 0.1) 110 4) ± ± ± ± ± ± ±

30/70

4212 1041 (25 19 (0.5 1057 (25 119 49 1) 2 0.1) 51 1) ± ± ± ± ± ± ±

50/50

4530 1270 (28 12 (0.3 1284 (28 25 120 4) 3 0.1) 125 4) ± ± ± ± ± ± ±

90/10

4841 856 (18 10 (0.2 867 (18 ± ± ± ± ± ± ±

71 65 1) 3 0.1) 71 1)

100/0

± ± ± ± ± ± ±

5015 312 (6 11 (0.2 334 (6

30/70

4821 2240 (47 22 (0.5 2281 (50 27 195 4) 2 0.1) 15 4) ± ± ± ± ± ± ±

50/50

4517 1330 (30 13 (0.3 1345 (30 205 103 3) 3 0.1) 105 3) ± ± ± ± ± ± ±

90/10

4994 750 (15 15 (0.3 771 (15 141 43 2) 2 0.1) 51 3) ± ± ± ± ± ± ±

100/0

4719 603 (13 17 (0.4 624 (13 275 264 5) 3 0.1) 217 5) ± ± ± ± ± ± ±

30/70

3971 2140 (54 15 (0.4 2191 (55 39 50 2) 2 0.1) 12 2) ± ± ± ± ± ± ±

50/50

4067 530 (13 14 (0.3 539 (13 ± ± ± ± ± ± ±

211 15 0.5) 3 0.1) 12 1)

90/10

± ± ± ± ± ± ±

4439 164 (4.0 14 (0.3 171 (4

100/0

4250 126 (3.0 13 (0.3 139 (3.0 ng ng (%) ng (%) ng (%)

% Caco-2/HT29MTX

addition basal

67 μM DMA(V) 67 μM MMA(V) 67 μM As(V)

Table 1. Cell Retention and Transport of As(V), MMA(V), and DMA(V) after 120 min for the Various Caco-2/HT29-MTX Proportions Assayed (100/0, 90/10, 50/50, and 30/70)a


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Table 2. Cell Retention and Transport of As(III), MMA(III), and DMA(III) after 120 min for the Various Caco-2/HT29-MTX Proportions Assayed (100/0, 90/10, and 50/50)a 67 μM As(III) % Caco-2/HT29-MTX addition basal cells uptake (basal + cells)

ng ng (%) ng (%) ng (%)

100/0 3250 758 (23 46 (1.4 804 (24

± ± ± ± ± ± ±

105 94 5) 3 0.2) 95 5)

90/10 3700 701 (19 48 (1.5 749 (21

± ± ± ± ± ± ±

91 36 3) 5 0.2) 27 4)

1 μM MMA(III) 50/50 3550 967 (26 46 (1.4 1013 (28

± ± ± ± ± ± ±

201 56 5) 2 0.1) 60 6)

100/0 190 55 (28 55 (28 110 (56

± ± ± ± ± ± ±

11 15 7) 1 1) 15 8)

90/10 126 24 (22 65 (37 89 (59

± ± ± ± ± ± ±

1 μM DMA(III) 50/50

14 3 1) 6 1) 9 6)

141 28 (17 47 (46 75 (64

± ± ± ± ± ± ±

20 2 2) 2 4) 4 3)

100/0 153 56 (47 32 (27 88 (74

± ± ± ± ± ± ±

17 10 4) 7 5) 12 4)

90/10 124 38 (35 40 (37 78 (73

± ± ± ± ± ± ±

16 5 4) 6 3) 9 4)

50/50 142 30 (26 50 (43 80 (70

± ± ± ± ± ± ±

25 6 1) 5 4) 10 5)

a The table shows the quantity of arsenic added to the cells (t = 0) and the quantity present in the basal medium and inside the cells (t = 120 min). The values of total uptake (cell content + basal transport) are also shown. Values expressed in ng arsenic (mean ± standard deviation of at least four replicas of each arsenic species) and arsenic percentage (in parentheses).

significantly when HT29-MTX was incorporated in the culture, indicating that the presence of mucus may affect cellular uptake. The mucus secreted by HT29-MTX constitutes a barrier to the diffusion of nutrients, drugs, ions, toxins, heavy metals, and macromolecules.38 Structurally, mucin glycoproteins consist of a protein backbone with a large number of O-linked carbohydrate side chains. Characteristic secretory mucins include MUC2 and MUC5AC, which structurally contain cysteine-rich regions and participate in the formation of extracellular gels in the gastrointestinal tract.39,40 Studies conducted in MMA(III) and DMA(III) show that these two species are highly retained by the mucus layer, and their penetration into the HT29-MTX cell is much less. They may interact with residues of free cysteine in the mucin, in view of the reports of considerable bonding of trivalent arsenic species to thiolated residues of proteins and peptides.41 This retention by mucus might be the cause of the decrease in Papp of DMA(III). As for MMA(III), its paracellular transport, which should increase in the Caco-2/HT29-MTX cocultures, would be countered by the retention of these species in the mucus layer. With regard to As(III), further studies are needed to clarify why its Papp does not change with the incorporation of HT29-MTX, given that substantial retention by mucus has not been observed. Retention of the trivalent species by mucus might be considered beneficial, because it represents a barrier to the entry of these highly reactive toxic species in the bloodstream. However, it is necessary to evaluate whether these species might affect the production of mucus, which would lead to gut barrier dysfunction, or they might produce other toxic effects on intestinal cells, which have been reported previously by other authors.4,12 The in vivo interpretation of the changes in Papp values obtained in vitro for the various cell cultures assayed can be evaluated by comparing them with the criteria proposed by Yee,42 who indicated that in vitro permeabilities 70%). For all of the pentavalent species, the classification changes as a result of the introduction of HT29MTX in the culture, changing from low absorption in Caco-2 to moderate absorption in the case of the Caco-2/HT29-MTX 50/50 proportion. MMA(V) becomes a highly absorbable compound in the 30/70 proportion. Of the trivalent species, only DMA(III) changes its classification, from highly

Caco-2 monolayers, the optimal pore radius of tight junctions for predicting the absorbable fraction of various drugs in humans is 7 Å, which is in agreement with the pore radius of 6.7−7.9 Å in the human jejunum.35,36 In our study (Figure 2), the pore radius calculated for the cocultures at the various proportions (6−8 Å) is closer to the physiological values than the radius obtained for Caco-2 cells (5 Å). More recent studies indicate that the lower permeability in Caco-2 of compounds that are transported via the paracellular route is due to the lower number of paracellular pores per cm2 in the Caco-2 monoculture as compared with cocultures with HT29.37 Moreover, Wikman-Lared and Artursson14 observed that although a specific proportion of each of the cell types, Caco-2 and HT29-H, was seeded, during the first days of culture, there was greater growth of HT29-H cells, so that when they reached the cell differentiation stage, there was a high proportion of HT29-H cells in the coculture. This gave a greater number of pores per cm2 and a higher paracellular transport value. It is possible that this occurred in the Caco-2/ HT29-MTX cocultures used in this study and that the increase in Papp of LY permeability was due both to the greater number of paracellular pores per cm2 and to leakier tight junctions between the two cell types. This opening of tight junctions and/or increase in porosity affects the transport and Papp of the pentavalent arsenic species, which increase significantly with the proportion of HT29-MTX cells in the coculture (Figure 3 and Table 1). This is indicative of participation of the paracellular pathway in their transport, as described by Calatayud et al.5 The Papp values obtained for the cocultures, however, are higher than the values found by Calatayud et al.5 for these arsenic species using Caco-2 cells in which the opening of the cell junctions was achieved by elimination of extracellular calcium with EDTA. This difference supports the view that not only tight junction opening but also increase in porosity participate in the increases in paracellular transport. The behavior of the trivalent species (Figure 4 and Table 2) was different from the behavior observed for their pentavalent analogs. For As(III), the incorporation of HT29-MTX did not produce any alteration in transport, Papp, or cellular retention, and for the trivalent methylated species, there was a decrease in Papp and transport to the basolateral side. These results seem to indicate very little participation of the paracellular pathway in their transport, although earlier studies conducted in Caco-26 showed significant increases in the Papp of As(III) and MMA(III) after opening of tight junctions. Moreover, cell retention of MMA(III) and DMA(III) (Table 2) increased 2659



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Table 3. Arsenic Retention and Transport for the Various Caco-2/HT29-MTX Proportions Assayed (100/0, 90/10, and 50/50) from the Bioaccessible Fraction of Four Samples of Fooda % Caco-2/HT29-MTX addition (ng As) O. sativa (sample 1)

53 ± 6

O. sativa (sample 2)

49 ± 4

H. fusiforme Allium sativa cooked with As(V)

1187 ± 71 890 ± 57

100/0 basal transport

ng (%) ng (%) ng (%) ng (%)

7.0 (14 6 (13 12 (1.0 97 (11.0

± ± ± ± ± ± ± ±

0.2 2) 1 2) 2 0.1) 3 0.4)

90/10

cell retention 4.0 (7.0 5.0 (9 7 (0.6 8 (2.0

± ± ± ± ± ± ± ±

0.3 0.5) 0.4 1) 1 0.1) 1 0.1)

basal transport 8 (15 6.0 (12.0 67 (6.0 217 (24

± ± ± ± ± ± ± ±

2 4) 0.3 0.5) 4 0.3) 30 3)

50/50

cell retention 10 (28 9.0 (34 14 (1.0 13 (1.0

± ± ± ± ± ± ± ±

1 2) 0.4 2) 2 0.2) 1 0.1)

basal transport 15 (27 14 (29 129 (11 293 (33

± ± ± ± ± ± ± ±

cell retention

2 1) 3 2) 13 1) 17 2)

12 (22 11 (22 21 (2.0 14 (2

± ± ± ± ± ± ± ±

2 1) 3 4) 4 0.3) 1 0.2)

a The table shows the quantity of arsenic added to the cells (t = 0) and the quantity present in the basal medium and inside the cell after 120 min of exposure. Values expressed in ng arsenic (mean ± standard deviation of at least four replicas of each arsenic species) and arsenic percentage (in parentheses).

clear that the presence of the mucus layer and the opening of tight junctions contributed by the HT29-MTX cells to the coculture with Caco-2 has a marked effect on in vitro intestinal absorption of arsenic species. In view of the high exposure to inorganic arsenic through drinking water and food that is found in many rural populations in Asia and Latin America, further research should be devoted to obtaining greater knowledge about intestinal absorption and the establishment of models for the conducting of these studies.

absorbable in the Caco-2 model and the 90/10 coculture to moderately absorbable in the 50/50 coculture. In vivo studies of intestinal absorption of arsenic species standards show high absorption of As(V) and As(III) in chick,43 rat,44 and swine,45 in many cases reaching completing absorption. The pentavalent organic species have lower absorption [33−50% DMA(V) and 17% MMA(V)45,46]. Thus, there are differences with respect to the results obtained with cell cocultures, especially for the inorganic forms. This may possibly be because the gastrointestinal tract is a complex system with numerous variables that are not reproduced in the in vitro models (absorption surface, peristaltic movements, neuroendocrine regulation, and presence of chyme components such as bile salts or GSH). However, the incorporation of HT29-MTX produces a reduction, with respect to the Caco-2 monolayer, in the differences observed between the in vitro and the in vivo models, because it provides the model with more physiological characteristics than Caco-2 alone. A further point is that the effect of the food matrix on intestinal absorption of arsenic may be considerable, and there are few data in this respect. The data obtained in the present work for various foods (Table 3) show that transport to the basolateral side increases with the incorporation of HT29-MTX to the Caco-2 culture, which suggests that the paracellular pathway might be involved in transport of species present in food. Moreover, for a given Caco-2/HT29-MTX proportion, the total uptake percentage (cell retention + basal transport) follows the order rice > garlic ≫ seaweed, which may be related with the arsenic species present in each of the bioaccessible fractions (inorganic arsenic for rice and garlic; arsenosugars and DMA for seaweed) and also with the effect of other components of the matrix. In vivo studies conducted by Juhasz et al.45 on juvenile swine fed with rice showed that the bioavailability differs considerably, depending on whether the samples contain mainly DMA or As(V) (33 vs 100%). With regard to the effect of the other components, the authors just cited postulate that the presence of nondigestible polysaccharides may reduce absorption of inorganic As, making the bioavailability in chard and lettuce (≈50%) less than in rice, mung beans, and radish (77−100%).47 Although there is a need to study a greater number of samples to establish the possible effects of the food matrix, and also to evaluate transport with the same arsenic contents added to the culture, the present work indicates the importance of the food matrix and speciation in the absorption of arsenic. In addition to these factors, there is the influence of the in vitro model that is employed, and it is

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+34)963 900 022. Fax: (+34)963 636 301. E-mail: [email protected]. Funding

This work was supported by projects of the Spanish Ministry of Science and Innovation (AGL2009-10100), for which the authors are deeply indebted. M.C. and M.V. received a Personnel Training Grant from the Spanish Ministry of Education to carry out this study. Notes

The authors declare no competing financial interest.

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REFERENCES

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In Vitro Study of Intestinal Transport of Inorganic and Methylated

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